U.S. patent application number 17/125870 was filed with the patent office on 2021-11-25 for fret-based analytes detection and related methods and systems.
This patent application is currently assigned to California Institute of Technology. The applicant listed for this patent is California Institute of Technology. Invention is credited to Mark D. Goldberg, Emil P. Kartalov, Aditya Rajagopal, Axel Scherer.
Application Number | 20210363581 17/125870 |
Document ID | / |
Family ID | 1000005756335 |
Filed Date | 2021-11-25 |
United States Patent
Application |
20210363581 |
Kind Code |
A1 |
Kartalov; Emil P. ; et
al. |
November 25, 2021 |
FRET-BASED ANALYTES DETECTION AND RELATED METHODS AND SYSTEMS
Abstract
FRET-based analytes detection and related methods and systems
are described where a pair of FRET labeled primers and/or
oligonucleotides are used that are specific for target sequences
located at a distance up to four time the Forster distance of the
FRET chromophores presented on the FRET labeled primers and/or
oligonucleotides one with respect to the other in one or more
polynucleotide analyte; in particular the pair of FRET labeled
primers and/or oligonucleotides is combined with a sample and
subjected to one or more polynucleotide amplification reactions
before measuring FRET signals from at least one FRET
chromophore.
Inventors: |
Kartalov; Emil P.;
(Pasadena, CA) ; Rajagopal; Aditya; (Irvine,
CA) ; Scherer; Axel; (Barnard, VT) ; Goldberg;
Mark D.; (Alta Loma, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
California Institute of Technology |
Pasadena |
CA |
US |
|
|
Assignee: |
California Institute of
Technology
Pasadena
CA
|
Family ID: |
1000005756335 |
Appl. No.: |
17/125870 |
Filed: |
December 17, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16102583 |
Aug 13, 2018 |
10889863 |
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17125870 |
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14633094 |
Feb 26, 2015 |
10077475 |
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16102583 |
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14162725 |
Jan 23, 2014 |
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14633094 |
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61944856 |
Feb 26, 2014 |
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61756343 |
Jan 24, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6883 20130101;
Y10T 436/143333 20150115; C12Q 2600/156 20130101; C12Q 1/703
20130101; C12Q 1/701 20130101; C12Q 1/6851 20130101; C12Q 1/68
20130101 |
International
Class: |
C12Q 1/6883 20060101
C12Q001/6883; C12Q 1/68 20060101 C12Q001/68; C12Q 1/6851 20060101
C12Q001/6851; C12Q 1/70 20060101 C12Q001/70 |
Claims
1-38. (canceled)
39. A kit to detect at least one polypeptide analyte in a sample,
the kit comprising at least one primer pair formed by a forward
primer attaching a first FRET chromophore and a reverse primer
attaching a second FRET chromophore, wherein the first FRET
chromophore and the second FRET chromophore are selected to provide
an energy transfer from one to another when located at a Forster
distance one with respect to the another thus forming a FRET
donor-acceptor chromophore pair; the forward primer has a sequence
specific for a first single strand target polypeptide within the at
least one polypeptide analyte the reverse primer has a sequence
specific for a second single strand target polypeptide within the
at least one polypeptide analyte the first single strand target
polypeptide and the second single strand target polypeptide are
located within the at least only polypeptide analyte so that upon
specific binding of the forward primer with the first target
polypeptide and specific binding of the reverse primer with the
second target polypeptide, the first FRET chromophore and the
second FRET chromophore are located at a distance up to four time
the Forster distance one with respect to the other.
40. The kit of claim 39, wherein each of the first single strand
target polypeptide and the second single strand target polypeptide
encompass a recognition sequence for the polypeptide analyte or for
a variation thereof.
41. The kit of claim 40, wherein the forward primer comprises a
recognition region at a 3' end of the forward primer, the
recognition region of the forward primer complementary and capable
of specifically binding the recognition sequence on the first
single strand target polypeptide; and the reverse primer comprises
a recognition region at a 3' end of the reverse primer, the
recognition region of the reverse primer complementary and capable
of specifically binding the recognition sequence on the second
single strand target polypeptide.
42. The kit of claim 41, wherein at least a portion of the
recognition sequence on the first single strand target polypeptide
is complementary to a corresponding portion of the recognition
sequence of the second single strand target polypeptide.
43. The kit of claim 42, wherein each of the at least a portion the
recognition sequence on the first single strand target polypeptide,
and the corresponding portion of the recognition sequence of the
sequence of the second single strand target polypeptide is equal to
or less than 20 bases and each of the recognition region of the
forward primer and reverse primer is within 20 bases from the
respective 3' terminus
44. The kit of claim 43, wherein the at least a portion the
recognition sequence on the first single strand target polypeptide
is I base, and each of the recognition region of the forward primer
and reverse primer is within I to 3 bases from the respective 3'
terminus.
45. The kit of claim 44, wherein the recognition sequence is for a
genetic variation selected from a substitution, an addition, a
deletion or a translocation.
46. The kit of claim 45, wherein the recognition sequence is for a
genetic variation selected from a substitution, an addition, a
deletion or a translocation.
47. The kit of claim 46, wherein the genetic variation is a
single-nucleotide polymorphism (SNP).
48. The kit of claim 39, wherein the forward primer and the reverse
primer have a length of approximately, 25-30 bases each.
49. The kit of claim 48, wherein the first target sequence and the
second target sequence comprise a 1 base recognition sequence for a
single-nucleotide polymorphism (SNP), the forward primer comprises
a 1 base recognition region located on a 3' terminus of the forward
primer or within 3 bases therefrom, the recognition region of the
forward primer complementary and capable of specifically binding
the recognition sequence on the first single strand target
polypeptide; and the reverse primer comprises a 1 base recognition
region located on a 3' terminus of the reverse primer or within 3
bases therefrom, the recognition region of the reverse primer
complementary and capable of specifically binding the recognition
sequence on the second single strand target polypeptide.
50. The kit of claim 39, wherein at least one or both the forward
primer and the reverse primer attach the first FRET chromophore at
a 5' terminus of the primer.
51. A method of detecting at least one polypeptide analyte m a
sample, the method comprising: combining the sample with the at
least one primer pair formed by the forward primer attaching the
first FRET chromophore and the reverse primer attaching the second
FRET chromophore of the kit according to claim 39; performing at
least one polypeptide amplification reaction with the forward
primer and the reverse primer of the at least one primer pair the
at least one polypeptide amplification reaction comprising
annealing of the forward primer and the reverse primer; and
measuring at least one FRET signal generated by the first FRET
chromophore and/or second FRET chromophores to detect the at least
one polypeptide analyte in the sample following the annealing.
52. The method of claim 51, wherein the at least one FRET signal
measured following the annealing is a FRET acceptor signal the FRET
acceptor signal measured alone or in combination with a FRET donor
signal.
53. The method of claim 51, wherein the performing is carried out
by performing a plurality of polypeptide amplification reactions
with the forward primer and the reverse primer of the at least one
primer pair, and the measuring is performed following the annealing
of each the plurality of polypeptide amplification reactions.
54. The method of claim 51, further comprising providing a
signature profile based on the FRET signal measured following the
annealing of each of the plurality of the polypeptide amplification
reactions
55. The method of claim 51, wherein the performing is performed by
polymerase chain reaction or an isothermal reaction.
56. A kit to detect at least one polypeptide analyte in a sample,
the kit comprising a plurality of primer pairs attaching a
plurality of FRET chromophores wherein each primer pair is formed
by a forward primer and a reverse primer each attaching a FRET
chromophore; the FRET chromophore attached to the forward primer
and the FRET chromophore attached to the reverse primer of each
primer pair are capable of providing an energy transfer from one to
another when located at a Forster distance one with respect to the
another, thus forming a FRET donor-acceptor chromophore pair; the
forward primer of each primer pair has a sequence specific for a
corresponding single stranded target polypeptide specific for the
forward primer within the at least one polypeptide analyte to be
detected, the reverse primer of each primer pair has a sequence
specific for a corresponding single stranded target polypeptide
specific for the reverse primer within the at least one polypeptide
analyte to be detected, the target polypeptide specific for the
forward primer and the target polypeptide specific for the reverse
primer are located within the at least one polypeptide analyte to
be detected so that upon specific binding of the forward primer
with the single stranded target polypeptide specific for the
forward primer and specific binding of the reverser primer with the
single stranded target polypeptide specific for the reverse primer,
the FRET chromophore attached to the forward primer and the FRET
chromophore attached to the reverse primer are located up to four
time the Forster distance one with respect to the other.
57. The kit of claim 56, wherein the single strand target
polypeptide specific for the forward primer of each primer pair and
the single strand target polypeptide specific for the reverse
primer of each primer pair encompass a recognition sequence for the
polypeptide analyte or for a variation thereof.
58. The kit of claim 57, wherein the forward primer of each primer
pair comprises a recognition region at a 3' end of the forward
primer, the recognition region of the forward primer complementary
and capable of specifically binding the recognition sequence on the
corresponding single strand target polypeptide specific for the
forward primer; and the reverse primer of each primer pair
comprises a recognition region at a 3' end of the reverse primer,
the recognition region of the reverse primer complementary and
capable of specifically binding the recognition sequence on the
corresponding single strand target polypeptide specific for the
reverse primer.
59. The kit of claim 58, wherein at least a portion of the
recognition sequence on the single strand target polypeptide
specific for the forward primer of each primer pair is
complementary to a corresponding portion on the recognition
sequence of the single strand target polypeptide specific for the
reverse primer of each primer pair.
60. The kit of claim 59, wherein each of the at least a portion the
recognition sequence on the single strand target polypeptide
specific for the forward primer, and the corresponding portion of
the recognition sequence of the single strand target polypeptide
specific for the reverse primer is equal to or less than 20 bases
and each of the recognition region of the forward primer and
reverse primer is within 20 bases from the respective 3'
terminus.
61. The kit of claim 60, wherein each of the at least a portion the
recognition sequence on the single strand target polypeptide
specific for the forward primer, and the corresponding portion of
the recognition sequence of the single strand target polypeptide
specific for the reverse primer is 1 base, and each of the
recognition region of the forward primer and reverse primer is
within 1 to 3 bases from the respective 3' terminus.
62. The kit of claim 61, the recognition sequence of the single
strand target polypeptide specific for the forward primer of each
primer pair and the recognition sequence of the single strand
target polypeptide specific for the reverse primer of each primer
pair, are for a same genetic variation selected from a
substitution, an addition, a deletion or a translocation, each
primer pair of the plurality of primer pairs specific for a
different genetic variation with respect to another primer pair of
the plurality of primer pairs.
63. The kit of claim 62, the recognition sequence of the single
strand target polypeptide specific for the forward primer of each
primer pair and the recognition sequence of the single strand
target polypeptide specific for the reverse primer of each primer
pair, are for a same genetic variation selected from a
substitution, an addition, a deletion or a translocation, each
primer pair of the plurality of primer pairs specific for a
different genetic variation with respect to another primer pair of
the plurality of primer pairs.
64. The kit of claim 63, wherein the genetic variation is a
single-nucleotide polymorphism (SNP).
65. The kit of claim 64, wherein the single-nucleotide polymorphism
is in a KRAS gene.
66. The kit of claim 56, wherein the forward primer and the reverse
primer have a length of approximately, 25-30 bases each.
67. The kit of claim 66, wherein the target sequence specific for
the forward primer and the target sequence specific for the reverse
primer of each primer pair comprise a recognition sequence for a
single-nucleotide polymorphism (SNP). the forward primer comprises
a 1 base recognition region located on a 3' terminus of the forward
primer or within 3 bases therefrom, the recognition region of the
forward primer complementary and capable of specifically binding
the recognition sequence on the first single strand target
polypeptide; and the reverse primer comprises a 1 base recognition
region located on a 3' terminus of the reverse primer or within 3
bases therefrom, the recognition region of the reverse primer
complementary and capable of specifically binding the recognition
sequence on the second single strand target polypeptide.
68. The kit of claim 39, wherein at least one or both the forward
primer and the reverse primer attach the first FRET chromophore at
a 5' terminus of the primer.
69. A method of detecting at least one polypeptide analyte m a
sample, the method comprising: combining the sample with the
plurality of primer pairs of the kit according to claim 56;
performing at least one polypeptide amplification reaction with the
plurality of primer pairs the at least one polypeptide
amplification reaction comprising annealing of the forward primer
and the reverse primer of each of the plurality of primer pairs;
and measuring at least one FRET signal generated by a FRET
chromophores of the plurality of the FRET chromophores attached to
the plurality of primer pairs to detect the at least one
polypeptide analyte in the sample following the annealing of each
of the forward primer and the reverse primer of the plurality of
primer pairs.
70. The method of claim 69, wherein each primer pair of the
plurality of primer pair is specific for a different variation of
the at least one polypeptide analyte, with respect to another
primer pair of the plurality of primer pairs.
71. The method of claim 70, wherein the at least one FRET signal
measured following the annealing is a plurality of FRET acceptor
signals each from a FRET acceptor chromophore of each from a primer
pair of the plurality of primer pairs, plurality of FRET acceptor
signals measured alone or in combination with a plurality of FRET
donor signals each from a FRET donor chromophore corresponding to
the FRET acceptor chromophore of each primer pair of the plurality
of primer pairs.
72. The method of claim 71, wherein the performing is carried out
by performing a plurality of polypeptide amplification reactions
with the forward primer and the reverse primer of the plurality of
primer pairs, and the measuring is performed following the
annealing of each the plurality of polypeptide amplification
reactions.
73. The method of claim 72, wherein the forward primer and the
reverse primer of each primer pair of the plurality of primer pair
attaches a same FRET donor-acceptor chromophore pair and combining
the sample with the plurality of primer pairs is performed by
providing each primer pairs of the plurality of primer pairs at
different concentrations.
74. The method of claim 73, further comprising providing a
signature profile for the at least one polypeptide and/or one or
more variation thereof based on the FRET signals measured following
the annealing of each of the plurality of the polypeptide
amplification reactions
75. The method of claim 74, wherein the forward primer and the
reverse primer of each primer pair of the plurality of primer pair
attaches a different FRET donor-acceptor chromophore pair from
another primer pair of the plurality of primer pair and combining
the sample with the plurality of primer pairs is performed by
providing each primer pairs of the plurality of primer pairs at a
same or different concentrations.
76. The method of claim 75, further comprising providing a
signature profile for the at least one polypeptide and/or one or
more variation thereof based on the FRET signals measured following
the annealing of each of the plurality of the polypeptide
amplification reactions.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to provisional application
Ser. 61/944,856 entitled "FRETplex PCR assays" filed on Feb. 26,
2014 with docket number CIT6828-P, the disclosure of which is
incorporated by reference in its entirety. This application is a
continuation in part and claims priority to U.S. application Ser.
No. 14/162,725 entitled "Chromophore based characterization and
Detection methods" filed on Jan. 23, 2014, which in turn claims
priority of U.S. provisional application 61/756,343, filed Jan. 24,
2013, the disclosures of which are incorporated herein by reference
in their entirety.
FIELD
[0002] The present disclosure relates to analyte detection and in
particular to a FRET based analytes detection and/or
characterization and related methods and systems.
BACKGROUND
[0003] Nucleic acid analyte identification is a critical procedure
in a variety of biomedical applications, such as in research and
clinical diagnostic environments. Identification of an analyte is
primarily done by sequencing or by amplification-based detection.
For example, in the latter scheme, the polymerase chain reaction is
often used to increase the quantity of the nucleic acid analyte
present.
[0004] Then, the nucleic acid analytes are discriminated using one
of several additional techniques including fluorescence intensity
measurement (e.g., fluorescent probes or intercalating dyes),
length discrimination (e.g., using gel electrophoresis or melt
curve analysis), or chromatography (e.g., haptin-based nucleic acid
capture). Thus, current amplification based detection technology
indirectly detects analytes and requires a secondary technique
(such as gel electrophoresis or mass spectroscopy) for analyte
detection. Amplification-based techniques that directly detect
analytes would improve efficiency, time and cost.
SUMMARY
[0005] Disclosed herein are methods, compositions, and kits for
detecting analytes, particularly polynucleotides and/or
polypeptides. The methods generally involve using oligonucleotides
(e.g., primers, probes) attached to FRET chromophores in
amplification or polymerization reactions in order to detect a
polynucleotide analyte through detection of at least one FRET
signal and in particular of at least one FRET donor and/or FRET
acceptor signals from FRET chromophores used in the methods,
compositions and kits of the disclosure in FRET donor-acceptor
chromophores pairs.
[0006] In some embodiments, provided herein are methods of
detecting at least one polynucleotide analyte in a sample,
comprising: (a) combining the sample with a first primer and a
first oligonucleotide, wherein a first FRET chromophore is attached
to the first primer, a second FRET chromophore is attached to the
first oligonucleotide, the first primer and the first
oligonucleotide are specific for a first polynucleotide analyte and
the first FRET chromophore is different from the second FRET
chromophore; (b) optionally measuring at least a first FRET signal
and in particular a first donor signal generated by a FRET donor of
the first and second FRET chromophores; (c) performing at least one
polymerization reaction with the first primer using the first
polynucleotide analyte as a template; and (d) measuring at least a
second FRET signal and in particular a second acceptor signal
generated by a FRET acceptor of the first and second FRET
chromophores; wherein the first and second signals are used to
detect the first polynucleotide analyte. In the method, the first
FRET chromophore and the second FRET chromophore are capable to
provide an energy transfer from one to another when located at a
Forster distance one with respect to the another, thus forming a
FRET donor-acceptor chromophores pair. In the method the first
primer and first oligonucleotide specifically bind to target
sequences located in the polynucleotide analyte so that upon
specific binding of the first primer with the target sequence
specific for the first primer, and upon specific binding of the
first oligonucleotide with the target sequence specific for the
first oligonucleotide, the first FRET chromophore and the second
FRET chromophore are located within a distance up to four times the
Forster distance one with respect to the other, preferably within
three times the Forster distance one with respect to the other,
more preferably within two times the Forster distance one with
respect to the other, even more preferably within or at the Forster
distance one with respect to the other In the method measuring a
signal from the second FRET chromophore attached to the first
oligonucleotide is performed following binding of the first
oligonucleotide with the first polynucleotide analyte before and/or
after (c) performing at least one polymerization reaction.
[0007] In some cases, the first FRET chromophore is attached to the
5' end of the first primer. In some cases, the first FRET
chromophore is an inorganic or organic dye, a fluorophore or
another chromophore. In some cases, the second FRET chromophore is
attached to the 5' end of the first oligonucleotide. In some cases,
the second chromophore is an inorganic or organic dye. In some
cases, the first FRET chromophore and/or the second FRET
chromophore is a fluorophore. In some cases, the fluorophore is 6-F
AM (Fluorescein), 6-F AM (NHS Ester), Fluorescein dT, HEX, JOE
(NETS Ester), MAX, TET, ROX, TAMRA, TARMA (NHS Ester), TEX615, ATTO
488, ATTO 532, ATTO 550, ATTO 565, ATTO Rho l Ol, ATTO 590, ATTO
633, ATTO 647N, Cy3, Cy5, TYE 563, TYE 665 or TYE 705.
[0008] In some cases, the first oligonucleotide is a second primer
and (c) performing at least one polymerization reaction is
performed by performing at least one polymerization reaction with
the first primer and with the second primer using the first
polynucleotide analyte as a template. In some of those cases the
second primer forms a primer pair with the first primer, and
performing at least one polymerization reaction with the first
primer using the first polynucleotide analyte as a template is
performed using the first primer and the second primer as forward
and reverse primer of the primer pair.
[0009] In some cases methods of detecting a polynucleotide analyte
with at least one primer pair are described, in which a first
primer is a forward primer and a second primer is a reverse primer.
In some of those embodiments the method comprises at least: (a)
combining the sample with the at least one primer pair formed by
the forward primer attaching a first FRET chromophore and the
reverse primer attaching a second FRET chromophore (c) performing
at least one polynucleotide amplification reaction with the forward
primer and the reverse primer of the at least one primer pair; and
(d) measuring at least one FRET signal, and in particular an
acceptor signal, generated by the first FRET chromophore and/or
second FRET chromophores to detect the at least one polynucleotide
analyte in the sample following the performing. In the method, the
first FRET chromophore and the second FRET chromophore are capable
of providing an energy transfer from one to another when located at
a Forster distance one with respect to the another, thus forming a
FRET donor-acceptor chromophores pair. In the method the first
primer and second primer specifically bind to target sequences
located in the polynucleotide analyte so that upon specific binding
of the first primer with the target sequence specific for the first
primer, and upon specific binding of the second primer with the
target sequence specific for the first oligonucleotide, the first
FRET chromophore and the second FRET chromophore are located within
a distance up to four times the Forster distance one with respect
to the other, preferably within three times the Forster distance
one with respect to the other, more preferably within two times the
Forster distance one with respect to the other, even more
preferably within or at the Forster distance one with respect to
the other In some cases, the method can comprise (b) measuring at
least one FRET signal, and in particular a donor signal, generated
by the first FRET chromophore and/or second FRET chromophores
before the performing; and detecting the at least one
polynucleotide analyte by the FRET signal and in particular the
acceptor signal measured following the performing in combination
with the FRET signal and in particular the donor signal measured
before the performing.
[0010] In some cases the measuring (b) of methods herein described
can be performed by measuring the first donor signal generated by
the FRET donor of the first and second FRET chromophore and a first
acceptor signal generated by the acceptor of the first and second
FRET chromophores. In some of those embodiments the measuring (d)
can be performed by measuring the second acceptor signal generated
by the FRET acceptor of the first and second FRET chromophore and a
second donor signal generated by the donor of the first and second
FRET chromophores. In some of those cases, the methods described
herein can further comprise comparing the first donor signal and
the second donor signal generated by the FRET donor of the first
and second FRET chromophores and/or the first acceptor signal and
the second acceptor signal generated by the FRET acceptor of the
first and second FRET chromophores, wherein a change in the first
and second donor signals and/or a change in the first and second
acceptor signals indicates the presence of the first polynucleotide
analyte. In some cases, the change is an increase in intensity of
the second acceptor signal compared to the first acceptor first
signal. In some cases, the increase in fluorescent intensity of the
second signal is at least about a 30% in signal (or at least about
10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 75%). In some
cases, the decrease in fluorescent intensity is a decrease of the
second donor signal compared to the first donor signal, and can be
at least about a 30% in signal (or at least about 10%, 20%, 30%,
35%, 40%, 45%, 50%, 55%, 60%, or 75%).
[0011] In some cases, the second acceptor and/or donor signals are
measured after a second polymerization reaction. In some cases
performing at least one polymerization reaction comprises
performing a plurality of polymerase chain reactions, one or more
polymerase chain reaction possibly followed by measuring an
acceptor and/or donor signal from the first and second FRET. In
some of those cases, the method can further comprises comparing the
donor and/or acceptor signals generated before and after the one or
more polymerase chain reactions.
[0012] In some cases, the polymerization reaction is a polymerase
chain reaction process or an isothermal process. In some cases, the
polymerase chain reaction process is an end-point polymerase chain
reaction process, a real-time polymerase chain reaction process, a
digital polymerase chain reaction process, a droplet digital
polymerase chain reaction process, or a quantitative polymerase
chain reaction process. In some cases, the first primer is a
forward primer and the second primer is a reverse primer. In some
cases, the first primer is a reverse primer and the second primer
is a forward primer. The first and second FRET chromophores
interact through an electron-transfer process as will be understood
by a skilled person. In some cases, the first polynucleotide
analyte is from about 10 to about 500 nucleotides in length. In
some cases, the concentration of the first polynucleotide analyte
is from about 10 .mu.M to about 10 aM. In some cases, the first
polynucleotide analyte is a DNA polynucleotide analyte. In some
cases, the first polynucleotide analyte is an RNA polynucleotide
analyte.
[0013] In some cases, the first polynucleotide analyte comprises a
variation of the polynucleotide sequence or of a portion thereof
which can be a genetic variation. In some cases, the genetic
variation comprises a substitution, an addition, a deletion or a
translocation. In some cases, the genetic variation comprises a
single-nucleotide polymorphism (SNP). In some cases, the at least
one polynucleotide analyte is from a source selected from a human,
a non-human mammal, a plant, a bacteria, a fungus, an archaea, a
parasite, or a virus. In some cases, the virus is a human
immunodeficiency virus, an influenza type A virus, an influenza
type B virus, a respiratory syncytial virus type A (RsvA), a
respiratory syncytial virus type B virus (RsvB), a human rhinovirus
(Hrv), a human metapneumovirus (Hmpv) or a human parainfluenza
virus type 3 (PN-3). In some cases, the sample is a forensic
sample, a clinical sample, a food sample, an environmental sample,
a pharmaceutical sample, or a sample from a consumer product.
[0014] Also disclosed herein are methods of detecting a
polynucleotide analyte and in particular at least one variation,
e.g. a genetic variation, in a polynucleotide analyte comprising:
(a) combining a first polynucleotide analyte with a first primer
and a second primer, wherein a first FRET chromophore is attached
to the first primer, a second FRET chromophore is attached to the
second primer, at least one of the first and the second primers are
specific for a first genetic variation in the first polynucleotide
analyte and the first FRET chromophore is different from the second
FRET chromophore; (b) measuring a first donor signal generated by a
FRET donor of the first and second FRET chromophores and/or a first
acceptor signal generated by a FRET acceptor of the first and
second FRET chromophore; (c) performing at least one polymerization
reaction with the first primer and the second primer using the
first analyte as a template; and (d) measuring a second acceptor
signal generated by a FRET acceptor of the first and second FRET
chromophores and/or a second donor signal generated by the FRET
donor of the first and second FRET chromophore; wherein the first
and second signals are used to detect the first genetic variation
in the first analyte. In methods herein described, the first FRET
chromophore and the second FRET chromophore are capable of
providing an energy transfer from one to another when located at a
Forster distance one with respect to the another, thus forming a
FRET donor-acceptor chromophores pair. In the method the first
primer and second primer specifically bind to target sequences
located in the polynucleotide analyte in a region possibly
including the first genetic variation such that upon specific
binding of the first primer with the target sequence specific for
the first primer and upon specific binding of the second primer
with the target sequence specific for the second primer the first
FRET chromophore and the second FRET chromophore are located within
a distance up to four times the Forster distance one with respect
to the other, preferably within three times the Forster distance
one with respect to the other, more preferably within two times the
Forster distance one with respect to the other, even more
preferably within or at the Forster distance one with respect to
the other.
[0015] In some cases, the first and second donor signals are a
fluorescence emission signal generated by the FRET donor of the
first and second chromophores and the first and second acceptor
signal are another fluorescence emission signal generated by the
FRET acceptor of the first and second chromophores. In some cases
the first primer and the second primer form a primer pair. In some
preferred cases, the first primer and the second primer are both
specific for the first genetic variation in the first analyte. In
other cases, one of the first and second primers is specific for
the first genetic variation in the first analyte and the other
primer is a common primer that is specific for the wild-type of the
first analyte. In some cases the first primer is specific for the
first genetic variation in the first analyte and the second primer
is specific for a second genetic variation in the first
analyte.
[0016] In some cases, the methods described herein further comprise
comparing the first and second donor and/or acceptor signals,
wherein a change in the donor and/or acceptor signals indicates the
presence of the genetic variation in the first analyte.
[0017] In some cases, the analyte is a polynucleotide analyte. In
some cases, the genetic variation comprises a substitution, an
addition, a deletion or a translocation. In some cases, the genetic
variation comprises a single-nucleotide polymorphism (SNP). In some
cases, the first primer comprises a sequence encoding the SNP or
the first primer binds to a region of the analyte encoding the SNP.
In some cases, the second primer comprises a sequence not encoding
the SNP or the second primer comprises a sequence complementary to
a region of the analyte not encoding the SNP. In some cases, the
first primer encodes a region of the analyte less than 500 base
pairs apart from a region of the analyte encoded by the second
primer. In some cases, the change in signal is distinct for at
least two of the mismatched base pairs selected from the group
consisting of UU, UT, UG, UC, UA, AA, TI, GG, CC, AG, AC, TG and
TC. In some cases, the change in signal from a mismatched base pair
is distinct from a change in signal from a complementary base
pair.
[0018] In some cases, step (a) of the methods described herein
further comprises combining the first analyte with a third primer
and a fourth primer, wherein a third FRET chromophore is attached
to the third primer, a fourth FRET chromophore is attached to the
fourth primer, the third and the fourth primers are specific for a
second genetic variation in the first analyte and the third FRET
chromophore is different from the forth FRET chromophore; step (b)
further comprises measuring a third donor signal generated by a
FRET donor of the third and fourth FRET chromophores and/or a third
acceptor signal generated by a FRET acceptor of the third and
fourth FRET chromophore; step (d) further comprises measuring a
fourth acceptor signal generated by a FRET acceptor of the third
and fourth FRET chromophores and/or a fourth donor signal generated
by a FRET donor of the third and fourth FRET chromophore; and the
method further comprises comparing the third and fourth donor
and/or acceptor signals; wherein a change in the third and fourth
donor and/or acceptor signals indicates the presence of the second
single genetic variation in the first analyte. In those cases the
third and fourth FRET chromophore are capable of providing an
energy transfer from one to another when located at a Forster
distance one with respect to the another thus forming a FRET
donor-acceptor chromophores pair. In the method the third primer
and fourth primer specifically bind to target sequences in a region
possibly including the second genetic variation the target
sequences located in the polynucleotide analyte so that upon
specific binding of the third primer with the target sequence
specific for the third primer and specific binding of the fourth
primer with the target sequence specific for the fourth primer the
third FRET chromophore and the fourth FRET chromophore are located
within a distance up to four times the Forster distance one with
respect to the other.
[0019] In some cases, step (a) of the methods above further
comprises combining a second polynucleotide analyte with a third
primer and a fourth primer, wherein a third FRET chromophore is
attached to the third primer, a fourth FRET chromophore is attached
to the fourth primer comprises, the third and the fourth primers
are specific for a second genetic variation in the second analyte
and the third chromophore is different from the fourth chromophore;
step (b) further comprises measuring a third donor signal generated
by a FRET donor of the third and fourth FRET chromophores and/or a
third acceptor signal generated by a FRET acceptor of the third and
fourth FRET chromophore; step (d) further comprises measuring a
fourth acceptor signal generated by a FRET acceptor of the third
and fourth FRET chromophores and/or a fourth donor signal generated
by a FRET donor of the third and fourth FRET chromophore; and the
method further comprises comparing the third and fourth donor
and/or acceptor signals; wherein a change in the third and fourth
donor and/or acceptor signals indicates the presence of the second
single genetic variation in the second analyte. In the method the
third FRET chromophore and the fourth FRET chromophore capable of
providing an energy transfer from one to another when located at a
Forster distance one with respect to the another, thus forming a
FRET donor-acceptor chromophores pair. In the method the third
primer and fourth primer specifically bind to target sequences
located in the second polynucleotide analyte in a region possibly
including the second genetic variation so that upon specific
binding of the third primer with the target sequence specific for
the third primer and specific binding of the fourth primer with the
target sequence specific for the fourth primer the third FRET
chromophore and the fourth FRET chromophore are located within a
distance up to four times the Forster distance one with respect to
the other.
[0020] In some cases, step (a) of the methods above further
comprises combining a second polynucleotide analyte with a third
primer and a fourth primer, wherein the first FRET chromophore is
attached to the third primer, the second FRET chromophore is
attached to the fourth primer, and the third and the fourth primers
are specific for a second genetic variation in the second analyte.
In the method the first FRET chromophore and the second FRET
chromophore capable of providing an energy transfer from one to
another when located at a Forster distance one with respect to the
another, thus forming a FRET donor-acceptor chromophores pair. In
the method the third primer and fourth primer specifically bind to
target sequences located in the second polynucleotide analyte in a
region possibly including the genetic variation so that upon
specific binding of the third primer with the target sequence
specific for the third primer and specific binding of the fourth
primer with the target sequence specific for the fourth primer, the
third FRET chromophore and the fourth FRET chromophore are located
within a distance up to four times the Forster distance one with
respect to the other. In some cases, the polymerization reaction is
a PCR process or an isothermal reaction. In some cases, the PCR
process is an end-point PCR process, a digital PCR process, a
real-time PCR process, a droplet digital PCR process, or a
quantitative PCR process. In some cases, the polymerization
reaction is a quantitative PCR process. In some cases, the
detecting comprises a quantitative PCR method. In some cases, the
measuring comprises a quantitative PCR method and a second method.
In some cases, the second method is a digital PCR process. In some
cases, at least one SNP is detected in a gene. In some cases, at
least one SNP is associated with a disease. In some cases, the
disease is a genetic disorder, an autoimmune disease, a
neurological disease, a cardiovascular disease, or a cancer.
[0021] Also disclosed herein are methods of detecting a plurality
of analytes, comprising: (a) combining the plurality of analytes
with a plurality of primer pairs, wherein each primer pair is
specific to a single analyte and each primer of the primer pair is
attached to a FRET chromophore of a plurality of FRET chromophore
pairs each FRET chromophore pair comprising a FRET donor and a FRET
acceptor; (b) measuring a first set of donor signals generated by
FRET donors of the plurality of FRET chromophores pairs attached to
the plurality of primer pairs and/or a first set of acceptor
signals generated by FRET acceptors of the FRET chromophores
plurality of FRET chromophores pairs attached to the plurality of
primer pairs; (c) performing at least one polymerization reaction
with the plurality of primer pairs using the plurality of analytes
as templates; and (d) measuring a second set of acceptor signals
generated by the FRET acceptors of the plurality of FRET
chromophores pairs attached to the plurality of primer pairs and/or
a second set of donor signals generated by the FRET donors of the
plurality of FRET chromophores pairs attached to the plurality of
primer pairs; wherein the first and second set of donor and/or
signals are used to detect each analyte of the plurality of
analytes. In some cases, the method described herein further
comprises: (e) repeating step (c) and (d) at least once; and (f)
generating a set of signature profiles; wherein the presence of
each analyte of the plurality of analytes is detected by comparing
the set of signature profiles to a control set of signature
profiles. In the method the FRET chromophores attached to a primer
pair are capable of providing an energy transfer from one to
another when located at a Forster distance one with respect to the
another, thus forming a FRET chromophores pair. In the method the
forward primer and reverse primer of each primer pair specifically
bind to target sequences located in the respective polynucleotide
analyte so that upon specific binding of the forward primer with
the target sequence specific for the forward primer and upon
specific binding of the reverse primer with the target sequence
specific for the reverse primer the FRET chromophore attached to
the forward primer and the FRET chromophore attached to the reverse
forward primer are located within a distance up to four times the
respective Forster distance one with respect to the other.
[0022] In some cases FRET chromophores of different primer pairs
are also selected to provide an energy transfer from one to another
when located at a Forster distance one with respect to the another,
thus forming a plurality of FRET chromophore pairs attached to a
same or different primer pairs. In some preferred cases, the first
primer and the second primer are both specific for the detection of
the particular analyte. In some cases, the signature profile for
each analyte of the plurality of analytes is a graph having the
x-axis represented by the number of PCR cycles and the y-axis
represented by the emission intensity generated by FRET acceptor of
at least one FRET chromophore pair attached to the primer pair that
is specific for that particular analyte. In other cases, only one
primer of the primer pair is specific for the detection of a
particular analyte and the other primer is a common primer that is
not specific for the detection of the particular analyte.
[0023] Also disclosed herein are methods of generating a signature
curve profile for a polynucleotide analyte, comprising: (a)
contacting the polynucleotide analyte with a first primer and a
second primer, wherein a first FRET chromophore is attached to the
first primer and a second FRET chromophore is attached to the
second primer, the first primer and the second primer are specific
for the polynucleotide analyte and the first FRET chromophore is
different from the second FRET chromophore; (b) measuring a first
donor signal generated by a FRET donor of the first and second FRET
chromophores at a first temperature and/or a first acceptor signal
generated by a FRET acceptor of the first and second FRET
chromophores; (c) performing at least one polymerization reaction
with the first primer and the second primer using the
polynucleotide analyte as a template; (d) measuring a second
acceptor signal generated by a FRET acceptor of the first and
second FRET chromophores at the first temperature and/or a second
donor signal generated by the FRET donor of the first and second
FRET chromophores; and (e) repeating step (c) and (d) at least
once; wherein the signals create the signature curve profile of the
polynucleotide analyte. In some cases, the method disclosed herein
further comprises: (f) changing the temperature; (g) measuring a
third donor and/or acceptor signal generated by a FRET donor and/or
a FRET acceptor of the first and second FRET chromophores at a
second temperature; and (h) repeating steps f and g at least once;
wherein the signals create the signature curve profile of the
polynucleotide analyte. In the method the first FRET chromophore
and the second FRET chromophore are selected in order to provide an
energy transfer from one to another when located at a Forster
distance one with respect to the another, thus forming a FRET
donor-acceptor chromophores pair. In the method the first primer
and second primer specifically bind to target sequences located in
the polynucleotide analyte so that upon specific binding of the
first primer with the target sequence specific for the first primer
and upon specific binding of the second primer with the target
sequence specific for the second primer the first FRET chromophore
and the second FRET chromophore are located within a distance up to
four times the Forster distance one with respect to the other.
[0024] In some cases, the signature curve is a length curve, a
morphology curve, a melt curve, or a SNP curve. In some cases, the
polymerization reaction is a polymerase chain reaction process. In
some cases, the polymerase chain reaction process is an end-point
polymerase chain reaction process, a real-time polymerase chain
reaction process, a digital polymerase chain reaction process or a
quantitative polymerase chain reaction process. In some cases, the
first FRET chromophore is an inorganic or organic dye, or a
fluorophore. In some cases, the first FRET chromophore is a
fluorophore. In some cases, the fluorophore is 6-F AM
(Fluorescein), 6-F AM (NETS Ester), Fluorescein dT, HEX, JOE (NHS
Ester), MAX, TET, ROX, TAMRA, T ARMA (NHS Ester), TEX 615, ATTO
488, ATIO 532, ATIO 550, ATTO 565, ATTO Rho101, ATTO 590, ATIO 633,
ATTO 647N, TYE 563, Cy3, Cy5, Alexa Fluor family, TYE 665 or TYE
705. In some cases, the second FRET chromophore is an inorganic or
organic dye, or a fluorophore. In some cases, the polynucleotide
analyte is a DNA polynucleotide analyte. In some cases, the
polynucleotide analyte is an RNA polynucleotide analyte. In some
cases, the polynucleotide analyte is from a source selected from
the group consisting of a human, a non-human mammal, a plant, a
bacteria, an archaea, a fungus, a parasite, and a virus. In some
cases, the virus is a human immunodeficiency vims, an influenza
type A virus, an influenza type B virus, a respiratory syncytial
virus type A (RsvA), a respiratory syncytial virus type B virus
(RsvB), a human rhinovirus (Hrv), a human metapneumovirus (Hmpv) or
a human parainfluenza virus type 3 (PTV-3).
[0025] Also disclosed herein are methods of detecting a
polynucleotide analyte comprising: (a) combining the polynucleotide
analyte with at least two FRET chromophores, wherein the at least
two FRET chromophores are each attached to a separate
polynucleotide that is complementary to a region within the
polynucleotide analyte; (b) performing at least one polymerization
reaction to incorporate the at least two FRET chromophores into
products of the polymerization reaction; and (c) detecting a
fluorescent intensity from the at least two FRET chromophores at a
first timepoint and a second timepoint, wherein the second
timepoint is later than the first timepoint and wherein a change
(particularly an increase for acceptor FRET chromophores and a
decrease for a donor FRET chromophore) in fluorescent intensity at
the second timepoint relative to the first timepoint is indicative
of the presence of the polynucleotide analyte. In the method the at
least two FRET chromophores are selected to provide an energy
transfer from one to another when located at a Forster distance one
with respect to the another, thus forming at least one FRET
chromophore pair. In some cases, the polymerization reaction is a
polymerase chain reaction. In some cases, the first timepoint is
after step (a) and the second timepoint is after step (b). In some
cases, the products of the at least one polymerization reaction
each comprise a first polynucleotide strand and a second
polynucleotide strand, wherein the first polynucleotide strand and
the second polynucleotide strand are complementary. In some cases,
the at least two FRET chromophores are different. In some cases,
the at least two FRET chromophores comprise a fluorophore. In some
cases, the fluorophore is incorporated into the first
polynucleotide strand and a second fluorophore or chromophore is
incorporated into the second polynucleotide strand. In some cases,
the fluorophore is incorporated at the 5' end of first
polynucleotide strand and the second fluorophore or other
chromophore is incorporated at the 5' end of the second
polynucleotide strand.
[0026] Also disclosed herein are compositions and kits,
particularly for detecting polynucleotide or polypeptide analytes.
In some cases, the kits comprise oligonucleotides (e.g., primers,
probes, etc.) attached to a FRET chromophore (e.g., fluorophore).
In the kit the FRET chromophores are selected to provide at least
one FRET pair each pair formed by a FRET donor chromophore and a
FRET acceptor chromophore providing an energy transfer from one to
another when located at a Forster distance one with respect to the
another. In some cases, the kit comprises: (a) a first primer or
probe attached to a first FRET chromophore; and (b) a second primer
or probe attached to a second FRET chromophore wherein the first
FRET chromophore and the second FRET chromophore are selected from
to form a FRET donor-acceptor chromophore pair. In the kits the
first primer and second primer specifically bind to target
sequences located in one or more polynucleotide analytes so that
upon specific binding of the first primer with the target sequence
specific for the first primer and upon specific binding of the
second primer with the target sequence specific for the second
primer the first FRET chromophore and the second FRET chromophore
are located within a distance up to four times the Forster distance
one with respect to the other, preferably within three times the
Forster distance one with respect to the other, more preferably
within two times the Forster distance one with respect to the
other, even more preferably within or at the Forster distance one
with respect to the other
[0027] In some cases, the kit comprises: (a) a first primer or
probe attached to one first FRET chromophore, wherein the first
FRET chromophore is a fluorophore; and (b) a second primer or probe
attached to one second FRET chromophore, wherein the first FRET
chromophore is different from the second FRET chromophore and
wherein the first FRET chromophore and the second FRET chromophore
are selected from to form a FRET chromophore pair formed by a FRET
donor and a FRET acceptor. In some cases, the second FRET
chromophore is another fluorophore.
[0028] In some cases the first primer and second primer are forward
and reverse primers of a primer pair specific for, and therefore
target, one or more polynucleotide analyte or a portion thereof. In
particular, in some of those cases, at least one of the first and
second primer, preferably both first and second primer are specific
for a first genetic variation in at least one polynucleotide
analyte. In some of those embodiments, the first primer and second
primer specifically bind to target sequences located in the
polynucleotide analyte in a region possibly including the first
genetic variation such that upon specific binding of the first
primer with the target sequence specific for the first primer and
upon specific binding of the second primer with the target sequence
specific for the second primer the first FRET chromophore and the
second FRET chromophore are located within a distance up to four
times the Forster distance one with respect to the other.
[0029] In some cases the kit further comprises (c) a third primer
or probe attached to a third FRET chromophore, and (d) a fourth
primer or probe attached to a fourth FRET chromophore, wherein the
third FRET chromophore and the third FRET chromophore are selected
from to form a FRET chromophore pair formed by a FRET donor and a
FRET acceptor. In some cases the third and the fourth primers are
specific for one or more polynucleotide analyte or a portion
thereof different from the one or more polynucleotide analyte or a
portion thereof targeted by the first primer and second primer. In
particular in some cases the third and fourth primer are specific
for at least one polynucleotide that is different from the at least
one polynucleotide analyte targeted by the first and second
primers. In some cases, the third and fourth primer are specific
for a second genetic variation different from the first genetic
variation. In some cases, the second genetic variation can be in
the same at least one polynucleotide analyte targeted by the first
primer and the second primer. In some cases the second genetic
variation can be in a different at least one polynucleotide analyte
targeted by the first primer and the second primer.
[0030] In some cases, the kit comprises: a plurality of primer
pairs each primer pair of the plurality of primer pairs comprising
a plurality of primer pairs attaching a plurality of FRET
chromophores wherein each primer pair is formed by a forward primer
and a reverse primer each attaching a FRET chromophore. In each
primer pair the FRET chromophore attached to the forward primer and
the FRET chromophore attached to the reverse primer are selected to
provide an energy transfer from one to another when located at a
Forster distance one with respect to the another thus forming a
FRET donor-acceptor chromophore pair. In each primer pair the
forward primer has a sequence specific for a target polynucleotide
specific for the forward primer within the at least one
polynucleotide analyte the reverse primer has a sequence specific
for a target polynucleotide specific for the reverse primer within
the at least one polynucleotide analyte, the target polynucleotide
specific for the forward primer and the target polynucleotide
specific for the reverse primer are located within the at least
only polynucleotide analyte so that upon specific binding of the
forward primer with the target polynucleotide specific for the
forward primer and specific binding of the reverser primer with the
target polynucleotide specific for the reverse primer, the FRET
chromophore attached to the forward primer and the FRET chromophore
attached to the reverse primer are located within a distance up to
four times the Forster distance one with respect to the other.
[0031] In some cases, at least one of the FRET chromophores
attached to the forward and reverse primers of a primer pairs is a
fluorophore. In some cases, a primer pair of the plurality of
primer pairs is specific for a same or different one or more
polynucleotide analytes targeted by another primer pair of the
plurality of primer pairs. In some cases a primer pair of the
plurality of primer pairs is specific for a different genetic
variation targeted by another primer pair of the plurality of
primer pairs. In some of those cases the genetic variations
targeted by primer pairs of the plurality of primers are in a same
polynucleotide analytes. In some cases the genetic variation
targeted by primer pairs of the plurality of primer pairs are in
different polynucleotide analyte.
[0032] In some cases, the fluorophore is 6-F AM (Fluorescein), 6-F
AM (NHS Ester), Fluorescein dT, HEX, JOE (NETS Ester), MAX, TET,
ROX, TAMRA, T ARMA (NHS Ester), TEX 615, ATTO 488, ATTO 532, ATTO
550, ATTO 565, ATTO Rho101, ATTO 590, ATTO 633, ATTO 647N, TYE 563,
Cy3, Cy5, Alexa Fluor family, TYE 665 or TYE 705. In some cases, a
forward primer comprises an oligonucleotide sequence that is
specific to a genetic variation. In some of those cases, a
corresponding reverse primer comprises an oligonucleotide sequence
that is specific to the same genetic variation targeted by the
first primer. In some cases, the kit comprises at least three
primers, wherein each primer is attached to a different FRET
chromophore. In some cases, the kit comprises a set of primers
wherein each primer is attached to a FRET donor and acceptor
chromophore, respectively. In some cases, the kit does not contain
a probe of any kind.
[0033] The details of one or more embodiments of the disclosure are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages will be apparent from the
description and drawings, and from the claims
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] Various aspects of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0035] FIG. 1 shows a Jablonski diagram showing Forster resonance
energy transfer with typical timescales indicated.
[0036] FIG. 2 shows a diagram illustrating spectra of various FRET
donor and acceptors where the normalized intensity is plotted
versus the wavelength lambda.
[0037] FIG. 3 shows a schematic diagram of the directions of
transitions dipoles in energy transfer between FRET donor and FRET
acceptor chromophores.
[0038] FIG. 4 shows a schematic representation exemplifying
amplification of a nucleic acid analyte using FRET labeled-primers
(e.g. a FRET donor-attached forward primer and a FRET
acceptor-attached to reverse primer). S1 illustrate the
denaturation of a double-stranded polynucleotide analyte. S2
illustrate the annealing of the FRET-labeled-primers (indicated in
the figure by arrows) to opposite strands of the analyte. S3 and S4
illustrate the elongation process in which polymerase extends the
primers during a PCR reaction until providing a complete double
stranded polynucleotide. S5 illustrates the formation of a
double-stranded PCR product containing both a donor and a receptor
which leads to the generation of a fluorescence signal following
repeated cycles.
[0039] FIG. 5 illustrates a diagnostic protocol and treatment
method for use with detection methods described herein.
[0040] FIG. 6 illustrates a conceptual schematic of an exemplary
computer server to be used for processing methods described
herein.
[0041] FIG. 7 shows a chart reporting the wild type concentration
calibration experiments using FRET-chromophore-labeled primers with
qPCR. Each multiplexed assay contains 20 uL of Taq 5.times.
mastermix, 8 uL of 10 uM for each primer 216C, 216A and 216T and
the WT sequence at different concentrations (10 uM, 100 uM, 1000
uM, 10,000 uM and 100,000 uM). 10 uL was used for each
concentration. The y-axis shows averaged relative FRET emission
intensities obtained from 22 qPCR replica experiments. For each
qPCR experiment, the relative FRET emission intensity (delta value)
is calculated by subtracting the maximum FRET emission intensity
with the minimum FRET emission intensity.
[0042] FIG. 8 shows a chart reporting detection of 216T mutant
sequence from KRAS gene using FRET-chromophore-labeled primers with
quantitative PCR (qPCR). The assay contains 2 uL of 10 uM forward
and reverse primer concentration, 20 uL of Taq 5.times. mastermix,
10 uL of 10 uM 216T mutant sequence and 10 uL of 100 uM wild type
sequence. The forward primer is labeled with Cy3 as a FRET donor
chromophore and the reverse primer is labeled with Cy5 as a FRET
acceptor chromophore. The chart shows an averaged FRET emission
signal intensity of 10 qPCR experiments (y-axis) plotted as a
function of PCR cycles (x-axis) with a standard deviation of
1.sigma.. The FRET emission signal intensity is generated by Cy5
(y-axis) and detected after each annealing step. The delta value
calculated by subtracting the maximum FRET signal recorded during
the PCR saturation period and the minimum FRET signal recorded at
the beginning of the PCR cycles is 0.27 A.U. The PCR reaction
system reaches saturation at cycle 18 and the exponential growth
begins at cycle 7.
[0043] FIG. 9 shows a chart reporting detection of 216A mutant
sequence from KRAS gene using FRET-chromophore-labeled primers with
quantitative PCR (qPCR). The assay contains 8 uL of 10 uM forward
and reverse primer concentration, 20 uL of Taq 5.times. mastermix,
10 uL of 10 uM 216A mutant sequence and 10 uL of 100 uM wild type
sequence. The forward primer is labeled with Cy3 as a FRET donor
chromophore and the reverse primer is labeled with Cy5 as a FRET
acceptor chromophore. The chart shows an averaged FRET emission
signal intensity of 10 qPCR replica experiments (y-axis) plotted as
a function of PCR cycles (x-axis) with a standard deviation of
1.sigma.. The FRET emission signal intensity is generated by Cy5
(y-axis) and detected after each annealing step. The delta value
calculated by subtracting the maximum FRET signal recorded during
the PCR saturation period and the minimum FRET signal recorded at
the beginning of the PCR cycles is 0.35 A.U. The system appears to
already be in the amplification cycle at the start of the
experiment and reaches saturation at cycle 25.
[0044] FIG. 10 shows a chart reporting detection of 216C mutant
sequence from KRAS gene using FRET-chromophore-labeled primers with
quantitative PCR (qPCR). The assay contains 4 uL of 10 uM forward
and reverse primer concentration, 20 uL of Taq 5.times. mastermix,
10 uL of 10 uM 216C mutant sequence and 10 uL of 100 uM wild type
sequence. The forward primer is labeled with Cy3 as a FRET donor
chromophore and the reverse primer is labeled with Cy5 as a FRET
acceptor chromophore. The chart shows an averaged FRET emission
signal intensity of 10 qPCR replica experiments (y-axis) plotted as
a function of PCR cycles (x-axis) with a standard deviation of
1.sigma.. The FRET emission signal intensity is generated by Cy5
(y-axis) and detected after each annealing step. The delta value
calculated by subtracting the maximum FRET signal recorded during
the PCR saturation period and the minimum FRET signal recorded at
the beginning of the PCR cycles is 0.6 A.U. The PCR reaction system
reaches saturation at cycle 20 and the exponential growth begins at
cycle 5.
[0045] FIG. 11 shows a chart reporting detection of double mutation
216T and 216A from KRAS gene using FRET-chromophore-labeled primers
with quantitative PCR (qPCR). The assay contains 8 uL of 10 uM
forward and reverse primer concentration for 216A, 2 uL of 10 uM
forward and reverse primer concentration for 216T, 20 uL of Taq
5.times. mastermix, 10 uL of 10 uM for each 216A and 216T mutant
and 10 uL of 100 uM wild type sequence. The forward primer is
labeled with Cy3 as a FRET donor chromophore and the reverse primer
is labeled with Cy5 as a FRET acceptor chromophore. The chart shows
an averaged FRET emission signal intensity of 10 qPCR replica
experiments (y-axis) plotted as a function of PCR cycles (x-axis)
with a standard deviation of 1.sigma.. The FRET emission signal
intensity is generated by Cy5 (y-axis) and detected after each
annealing step. The delta value calculated by subtracting the
maximum FRET signal recorded during the PCR saturation period and
the minimum FRET signal recorded at the beginning of the PCR cycles
is 2.0 A.U. The PCR reaction system reaches saturation at cycle 20
and the exponential growth begins at cycle 5.
[0046] FIG. 12 shows a chart reporting detection of double mutation
216T and 216C from KRAS gene using FRET-chromophore-labeled primers
with quantitative PCR (qPCR). The assay contains 4 uL of 10 uM
forward and reverse primer concentration for 216C, 2 uL of 10 uM
forward and reverse primer concentration for 216T, 20 uL of Taq
5.times. mastermix, 10 uL of 10 uM for each 216C and 216T mutant
and 10 uL of 100 uM wild type sequence. The forward primer is
labeled with Cy3 as a FRET donor chromophore and the reverse primer
is labeled with Cy5 as a FRET acceptor chromophore. The chart shows
an averaged FRET emission signal intensity of 10 qPCR replica
experiments (y-axis) plotted as a function of PCR cycles (x-axis)
with a standard deviation of 1.sigma.. The FRET emission signal
intensity is generated by Cy5 (y-axis) and detected after each
annealing step. The delta value calculated by subtracting the
maximum FRET signal recorded during the PCR saturation period and
the minimum FRET signal recorded at the beginning of the PCR cycles
is 1.6 A.U. The PCR reaction system reaches saturation at cycle 25
and the exponential growth begins at cycle 6.
[0047] FIG. 13 shows a chart reporting detection of double mutation
216A and 216C from KRAS gene using FRET-chromophore-labeled primers
with quantitative PCR (qPCR). The assay contains 4 uL of 10 uM
forward and reverse primer concentration for 216C, 8 uL of 10 uM
forward and reverse primer concentration for 216A, 20 uL of Taq
5.times. mastermix, 10 uL of 10 uM for each 216A and 216C mutant
and 10 uL of 100 uM wild type sequence. The forward primer is
labeled with Cy3 as a FRET donor chromophore and the reverse primer
is labeled with Cy5 as a FRET acceptor chromophore. The chart shows
an averaged FRET emission signal intensity of 10 qPCR replica
experiments (y-axis) plotted as a function of PCR cycles (x-axis)
with a standard deviation of 1.sigma.. The FRET emission signal
intensity is generated by Cy5 (y-axis) and detected after each
annealing step. The delta value calculated by subtracting the
maximum FRET signal recorded during the PCR saturation period and
the minimum FRET signal recorded at the beginning of the PCR cycles
is 1.9 A.U. The PCR reaction system reaches saturation at cycle 30
and the exponential growth begins at cycle 5.
[0048] FIG. 14 shows a chart reporting detection of triple mutation
216A, 216T and 216C from KRAS gene using FRET-chromophore-labeled
primers with quantitative PCR (qPCR). The assay contains 4 uL of 10
uM forward and reverse primer concentration for 216C, 8 uL of 10 uM
forward and reverse primer concentration for 216A, 2 uL of 10 uM
forward and reverse primer concentration for 216T, 20 uL of Taq
5.times. mastermix, 10 uL of 10 uM for each 216A, 216T and 216C
mutant and 10 uL of 100 uM wild type sequence. The forward primer
is labeled with Cy3 as a FRET donor chromophore and the reverse
primer is labeled with Cy5 as a FRET acceptor chromophore. The
chart shows an averaged FRET emission signal intensity of 10 qPCR
replica experiments (y-axis) plotted as a function of PCR cycles
(x-axis) with a standard deviation of 1.sigma.. The FRET emission
signal intensity is generated by Cy5 (y-axis) and detected after
each annealing step. The delta value calculated by subtracting the
maximum FRET signal recorded during the PCR saturation period and
the minimum FRET signal recorded at the beginning of the PCR cycles
is 2.2 A.U. The PCR reaction system reaches saturation at cycle 25
and the exponential growth begins at cycle 5.
[0049] FIG. 15 shows a chart of relative FRET emission intensities
(delta values) obtained from FIGS. 7-13 plotted as a function of
the accumulated primer concentration ratio (x-axis) with a standard
deviation of 1.sigma.. Each delta value was the average of FRET
emission signal intensity of 24 qPCR replica experiments for each
concentration value. Statistical analysis was used to remove
outliners from the data set.
[0050] FIG. 16 shows a chart reporting the detection of the
presence or absence of a KRAS mutation 216A, 216T and 216C using
FRETplexing with qPCR. Each multiplexed assay contains 20 uL of Taq
5.times. matermix, 10 uL of 10 uM for a given mutant sequence, 8 uL
of 10 uM primer concentration for each of three primer pairs. The
forward primer is labeled with Cy3 as a FRET donor chromophore and
the reverse primer is labeled with Cy5 as a FRET acceptor
chromophore. The chart shows averaged relative FRET emission
intensities (delta values at y-axis) of 12 qPCR replica experiments
plotted for each mutation also in comparison with wild type assay
at two different concentrations: 10 uL of 100 uM wild type in one
experiment and 10 uL of 1000 uM wild type in the other
experiment.
[0051] FIG. 17 shows a chart reporting detection of 216C mutant
sequence from KRAS gene using quenchiplexing with qPCR. The
multiplexed assay contains 0.5 uL of 10 uM forward and reverse
primer concentration, 20 uL of Taq 5.times. matermix and 10 uL of
10 uM mutant 216C. The forward primer is labeled with Cy3 as a
fluorophore and the reverse primer is labeled with iABkFQ as a
quencher. The chart shows averaged fluorescence emission signal of
12 qPCR replica experiments (at y-axis) plotted as a function of
PCR cycles with a standard deviation of 1.sigma.. The amplification
phase starts at cycle 8 and the saturation phase at cycle 18.
[0052] FIG. 18 shows a chart reporting detection of 216T mutant
sequence from KRAS gene using quenchiplexing with qPCR. The
multiplexed assay contains 1 uL of 10 uM forward and reverse primer
concentration, 20 uL of Taq 5.times. matermix and 10 uL of 10 uM
mutant 216T. The forward primer is labeled with Cy3 as a
fluorophore and the reverse primer is labeled with iABkFQ as a
quencher. The chart shows averaged fluorescence emission signal of
12 qPCR replica experiments (at y-axis) plotted as a function of
PCR cycles with a standard deviation of 1.sigma.. The amplification
phase starts at cycle 8 and the saturation phase at cycle 20
[0053] FIG. 19 shows a chart reporting detection of 216A mutant
sequence from KRAS gene using quenchiplexing with qPCR. The
multiplexed assay contains 2 uL of 10 uM forward and reverse primer
concentration, 20 uL of Taq 5.times. matermix and 10 uL of 10 uM
mutant 216A. The forward primer is labeled with Cy3 as a
fluorophore and the reverse primer is labeled with iABkFQ as a
quencher. The chart shows averaged fluorescence emission signal of
12 qPCR replica experiments (at y-axis) plotted as a function of
PCR cycles with a standard deviation of 1.sigma.. The amplification
phase starts at cycle 8 and the saturation phase at cycle 32.
[0054] FIG. 20 shows a chart reporting detection of 216C and 216T
mutant sequence from K-RAS gene using quenchiplexing with qPCR. The
multiplexed assay contains 0.5 uL of 10 uM forward and reverse
primer concentration for 216C, 1 uL of 10 uM forward and reverse
primer concentration for 216T, 20 uL of Taq 5.times. matermix, 10
uL of 10 uM mutant 216C and 10 uL of 10 uM 216T. The forward primer
is labeled with Cy3 as a fluorophore and the reverse primer is
labeled with iABkFQ as a quencher. The chart shows averaged
fluorescence emission signal of 12 qPCR replica experiments (at
y-axis) plotted as a function of PCR cycles with a standard
deviation of 1.sigma.. The amplification phase starts at cycle 9
and the saturation phase at cycle 35.
[0055] FIG. 21 shows a chart reporting detection of 216C and 216A
mutant sequence from K-RAS gene using quenchiplexing with qPCR. The
multiplexed assay contains 0.5 uL of 10 uM forward and reverse
primer concentration for 216A, 2 uL of 10 uM forward and reverse
primer concentration for 216T, 20 uL of Taq 5.times. matermix, 10
uL of 10 uM mutant 216C and 10 uL of 10 uM 216A. The forward primer
is labeled with Cy3 as a fluorophore and the reverse primer is
labeled with iABkFQ as a quencher. The chart shows averaged
fluorescence emission signal of 12 qPCR replica experiments (at
y-axis) plotted as a function of PCR cycles with a standard
deviation of 1.sigma.. The amplification phase starts at cycle 11
and the saturation phase at cycle 36.
[0056] FIG. 22 shows a chart reporting detection of 216T and 216A
mutant sequence from K-RAS gene using quenchiplexing with qPCR. The
multiplexed assay contains 1 uL of 10 uM forward and reverse primer
concentration for 216T, 2 uL of 10 uM forward and reverse primer
concentration for 216A, 20 uL of Taq 5.times. matermix and 10 uL of
10 uM mutant 216T and 10 uL of 10 uM 216A. The forward primer is
labeled with Cy3 as a fluorophore and the reverse primer is labeled
with iABkFQ as a quencher. The chart shows averaged fluorescence
emission signal of 12 qPCR replica experiments (at y-axis) plotted
as a function of PCR cycles with a standard deviation of 1.sigma..
The amplification phase starts at cycle 11 and the saturation phase
at cycle 38.
[0057] FIG. 23 shows a chart reporting detection of 216C, 216T and
216A mutant sequence from KRAS gene using quenchiplexing with qPCR.
The multiplexed assay contains 0.5 uL of 10 uM forward and reverse
primer concentration for 216C, 1 uL of 10 uM forward and reverse
primer concentration for 216T, 2 uL of 10 uM forward and reverse
primer concentration for 216A, 20 uL of Taq 5.times. matermix, and
10 uL of 10 uM for each mutant 216C, 216T and 216A. The forward
primer is labeled with Cy3 as a fluorophore and the reverse primer
is labeled with iABkFQ as a quencher. The chart shows averaged
fluorescence emission signal of 12 qPCR replica experiments (at
y-axis) plotted as a function of PCR cycles with a standard
deviation of 1.sigma.. The amplification phase starts at cycle 11
and the saturation phase at cycle 36.
DETAILED DESCRIPTION
[0058] Provided herein are methods, systems, compositions, and kits
for the detection of one or more analytes using oligonucleotides
(e.g., primers and/or probes) specific for an analyte attaching
FRET chromophores. For example, the incorporation of FRET
chromophores into an amplification product (e.g., through the use
of forward and reverse primers that are attached to different
chromophores as illustrated, e.g., in FIG. 1) provides a number of
advantages.
[0059] For example, methods described herein can provide for the
direct detection of one or more analytes in a single reaction. In
addition, the direct incorporation of FRET chromophores into the
amplification product (e.g., the analyte) allows for more accurate
quantification of the analyte and for the real-time monitoring of
the progression of an amplification reaction.
[0060] Methods described herein are particularly suitable for
detecting genetic variations, such as single nucleotide
polymorphisms (SNPs) or other qualitative information of an
analyte. In some cases, a single base-pair mismatch between a FRET
chromophore-labeled primer and the analyte can be detected upon
amplification of the analyte. For example, a change in signal may
occur if there is a disruption in a contiguous double-stranded DNA
sequence upon amplification of the analyte when a SNP is present. A
single base pair misalignment (e.g. internal misalignment (such as
a SNP) or terminal overhangs) results in significant decrease in
signal compared to the signal generated upon amplification of the
analyte without a base pair misalignment due to a disruption in
electron transport between chromophores incorporated into an
analyte containing a base pair misalignment.
[0061] Also described herein are methods which allow detection of
analytes present in low concentrations. In some cases, the
sensitivity of the methods described herein can detect analytes at
concentrations of about 10 uM to about 1 aM. In some cases, the
methods provided herein can be combined with a digital
amplification process (e.g. droplet digital PCR), to further
enhance the detection. In some cases, the methods provided herein
can be used to detect analytes that are present at a trace
concentration in a sample (e.g. a rare SNP).
[0062] Further described herein are methods to detect of multiple
analytes in a single reaction or experiment, without the need to
resort to additional experiments. Thereby, the disclosed methods
can reduce or eliminate the associated cost of additional reagents
or materials and increase time and efficiency.
[0063] The methods and system herein described by using FRET
attached to at least one primer in target amplification allows use
of a mutation-specific oligonucleotide reducing thus detection that
can lead to false positives by mispriming or unrelated hydrolysis.
In particular, when both the first and second primers are specific
to a genetic variation, a second FRET signal, and in particular a
second FRET acceptor signal, is generated only if both primers
amplify, which leads to higher specificity compared to traditional
allele-specific assays. The methods also avoid the false signal
from the excitation light scattering off vessels or samples. In
some embodiments herein described the positive identification of a
mutation can be simultaneously monitored as an increase in one FRET
signal, such as the fluorescence emission signal generated by a
FRET acceptor chromophore, and a decrease in another FRET signal,
such as the fluorescence emission signal generated by a FRET donor
chromophore, thereby increasing reliability and eliminating certain
sources of false positives. Additionally, the currently described
FRET-based methods and systems are also inherently compatible with
commercially available blockers to the wild type, which can be
incorporated to work to full effect without adversely affecting the
detection and characterization of analytes using FRET-chromophore
labeled primers.
I. Certain Terminology
[0064] It is to be understood that the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not restrictive of any subject matter
claimed. In this application, the use of the singular includes the
plural unless specifically stated otherwise. It must be noted that,
as used in the specification and the appended claims, the singular
forms "a," "an" and "the" include plural referents unless the
context clearly dictates otherwise. In this application, the use of
"or" means "and/or" unless stated otherwise. Furthermore, use of
the term "including" as well as other forms, such as "include",
"includes," and "included," is not limiting.
[0065] The term "about," as used herein, generally refers to a
range that is 15% greater than or less than a stated numerical
value within the context of the particular usage. For example,
"about 10" would include a range from 8.5 to 11.5.
[0066] The term "primer", as used herein, generally refers to a
short linear oligonucleotide that hybridizes to a target nucleic
acid sequence (e.g., a DNA template to be amplified) to prime a
nucleic acid synthesis reaction. The primer can be an RNA
oligonucleotide, a DNA oligonucleotide, or a chimeric sequence. The
primer may contain natural, synthetic, or nucleotide analogues
(e.g., those that increase T.sub.m). Both the upper and lower
limits of the length of the primer are empirically determined. The
lower limit on primer length is the minimum length that is required
to form a stable duplex upon hybridization with the target nucleic
acid under nucleic acid amplification reaction conditions. Very
short primers (usually less than 3 nucleotides long) do not form
thermodynamically stable duplexes with target nucleic acid under
such hybridization conditions. The upper limit is often determined
by the possibility of having a duplex formation in a region other
than the pre-determined nucleic acid sequence in the target nucleic
acid. Generally, suitable primer lengths are in the range of about
3 nucleotides long to about 40 nucleotides long. The "primers" used
in the methods of amplification of a target nucleic acid described
herein will be of a length appropriate for a particular set of
experimental conditions. The determination of primer length is well
within the routine capabilities of those of skill in the art.
[0067] The terms "polynucleotide," "oligonucleotide," or "nucleic
acid," as used herein, are used herein to refer to biological
molecules comprising a plurality of nucleotides. Exemplary
polynucleotides include deoxyribonucleic acids, ribonucleic acids,
and synthetic analogues thereof, including peptide nucleic acids.
Polynucleotides can typically be provided in single-stranded form
or double-stranded form (herein also duplex form, or duplex).
[0068] Additional terminology will be defined in connection with
the description of various embodiments of the methods, systems,
compositions and kit herein described.
II. Methods of Detection
[0069] Described herein are methods for detecting at least one
polynucleotide analyte in a sample.
[0070] The term "sample" as used herein indicates a limited
quantity of something that is indicative of a larger quantity of
that something, including but not limited to fluids from a
biological environment, specimen, cultures, tissues, synthetic
compounds or portions thereof.
[0071] The terms "detect" or "detection" as used herein indicates
the determination of the existence, presence, absence or features
of a target in a limited portion of space, including but not
limited to a sample, a reaction mixture, a molecular complex and a
substrate. The "detect" or "detection" as used herein can comprise
determination of chemical and/or biological properties of the
target, including but not limited to ability to interact, and in
particular bind, other compounds, ability to activate another
compound and additional properties identifiable by a skilled person
upon reading of the present disclosure. The detection can be
quantitative or qualitative. A detection is "quantitative" when it
refers, relates to, or involves the measurement of quantity or
amount of the target or signal (also referred as quantitation),
which includes but is not limited to any analysis designed to
determine the amounts or proportions of the target or signal, or to
describes distinctive nature, features, or characteristics of the
target or signal. Quantitative detection is also referred to as
"characterization". A detection is "qualitative" when it refers,
relates to, or involves identification of a quality or kind of the
target or signal in terms of relative abundance to another target
or signal, or the presence or absence of a particular feature,
which is not quantified.
[0072] In particular, in methods and systems herein described and
related compositions and kits, detection and/or characterization of
analytes and in particular polynucleotide analytes is performed
using FRET-based signal detection.
[0073] The term "FRET" refers to Forster Resonance Energy Transfer,
a mechanism that describes energy transfer between two light
sensitive molecules, such as a donor chromophore and an acceptor
chromophore. A donor chromophore initially in its electronic
excited state may transfer energy to an acceptor chromophore
through non-radiative dipole-dipole coupling. The efficiency of
this energy transfer is inversely proportional to the distance
between the donor and acceptor, allowing FRET sensitive to small
change in distance between the donor and the acceptor
chromophores.
[0074] The term "FRET donor chromophore", "donor chromophore" or
"donor" refers to a chromophore or dye molecule that initially
absorbs the energy. The term "FRET acceptor chromophore", "acceptor
chromophore" or "acceptor" refers to a chromophore or dye molecule
to which the energy is subsequently transferred. The transfer of
energy leads to a reduction in the donor's fluorescence intensity
and an increase in the acceptor's emission intensity. A pair of
chromophores that interact in such a manner that FRET occurs is
referred to as a FRET donor-acceptor chromophore pair. Examples of
FRET donor and FRET acceptor chromophore include Indocarbocyanine
(Cy3)-Indodicarbocyanine (Cy5), Green Fluorescent Protein
(GFP)-Yellow Fluorescent Protein (YFP), Yellow Fluorescent Protein
(YFP)-Red Fluorescent Protein (RFP) and additional FRET donors and
acceptor pairs identifiable by a skilled person.
[0075] In embodiments herein described oligonucleotides are labeled
with FRET chromophore and are combined with the sample for a time
and under conditions to allow hybridization of the FRET labeled
oligonucleotides with target sequences located within at least one
polynucleotide analyte, in which the hybridization is performed in
combinations designed so that emission of a signal by the FRET
chromophores or change in such signal provides detection related to
at least one analyte polynucleotide based on the following
equation:
E = k ET k f + k ET + .SIGMA. .times. .times. k i ( 1 )
##EQU00001##
where E is the efficiency of energy transfer k.sub.ET is the rate
of FRET k.sub.f is the rate of radiative relaxation (fluorescence)
k.sub.i are the non-radiative relaxation rates (e.g., internal
conversion, intersystem crossing, external conversion and
additional rates identifiable by a skilled person). In particular,
the FRET efficiency (E) is the quantum yield of the energy transfer
transition, i.e. the fraction of energy transfer event occurring
per donor excitation event.
[0076] Within a point dipole-dipole approximation, the FRET
efficiency can be related to the donar-acceptor distance via
E = 1 1 + ( r / R 0 ) 6 ( 2 ) ##EQU00002##
where r is the separation distance between the donor and the
acceptor (FIG. 1) and R.sub.0 is the characteristic distance (the
Forster distance or Forster radius) at which the energy transfer
efficiency is 50%. The FRET efficiency E depends on the
donor-to-acceptor separation distance r with an inverse 6th power
law due to the dipole-dipole coupling mechanism.
[0077] To enhance the FRET efficiency, the donor group should have
good abilities to absorb photons and emit photons. That means the
donor group should have a high extinction coefficient and a high
quantum yield. The overlap of emission spectrum of the donor and
absorption spectrum of the acceptor means that the energy lost from
excited donor to ground state could excite the acceptor group. The
energy matching is called the resonance phenomenon. Thus, the more
overlap of spectra, the better a donor can transfer energy to the
acceptor. The overlap integral, J(.lamda.), between the donor and
the acceptor stands for the overlap of spectra, as shown in FIG.
2.
[0078] In the illustration of FIG. 2, the overlap integral is given
by
J=.intg.F.sub.D(.lamda.) .sub.A(.lamda.).lamda..sup.4d.lamda.
(3)
where F.sub.D(.lamda.) is the normalized emission spectrum of the
donor. .epsilon..sub.A standards for the molar absorption
coefficient of the acceptor. .lamda. is the wavelength.
[0079] The resonance energy transfer mechanism is also affected by
the orientations of the emission transition dipole of the donor and
the absorption dipole of the acceptor. The orientation parameter
.kappa..sup.2 gives the quantitative value of interaction between
two dipole moments. .kappa..sup.2 can theoretically be values from
0 (when dipoles are perpendicular to each other) to 4 (when dipoles
are collinear). .kappa..sup.2 is equal to 1 when these two
transition dipoles are parallel. The orientation of transition
dipoles is shown in FIG. 3. For a freely rotational donor and
acceptor group, the average .kappa..sup.2 is treated 2/3.
[0080] In FRET based detection, a long Forster distance R.sub.0 can
cause a high FRET efficiency as will be understood by a skilled
person. Based on Forster's analysis, R.sub.0 is described by the
following equation
R 0 6 = 9 .times. Q 0 .function. ( ln .times. .times. 10 ) .times.
.kappa. 2 .times. J 128 .times. .times. .pi. 5 .times. n 4 .times.
N A ( 4 ) ##EQU00003##
where Q.sub.0 is the fluorescence quantum yield of the donor in the
absence of the acceptor, .kappa..sup.2 is the dipole orientation
factor, n is the refractive index of the medium, N.sub.A is
Avogadro's number, and J is the spectral overlap integral.
[0081] Accordingly, in methods and systems herein described and
related compositions and kits, FRET chromophore are typically
selected in FRET donor-acceptor chromophore pair in which a first
FRET chromophore and a second corresponding FRET chromophore are
capable of providing an energy transfer from one to another at 50%
efficiency when located at a Forster distances one with respect to
the another so that one of the first FRET chromophore and second
FRET chromophore is the FRET donor of the FRET donor-acceptor
chromophore pair and the other of the first FRET chromophore and
second FRET chromophore is the FRET acceptor of the FRET
donor-acceptor chromophore pair. FRET donor and acceptor
chromophore within a same FRET donor-acceptor chromophore pair are
herein also indicated as corresponding FRET donor chromophore (or
FRET donor) and FRET acceptor chromophore (or FRET acceptor).
[0082] Exemplary corresponding FRET donor and acceptors of a FRET
donor-acceptor chromophore pair and related Forster distances of is
reported in Table 1 below.
TABLE-US-00001 TABLE 1 Forster distance FRET Donor Corresponding
FRET Acceptor (R.sub.0, nm) Naphthalene Dansyl 2.2 LY TNP-ATP 3.5
Dansyl ODR 4.3 LY EM 5.3 FITC EM 6.0 BPE CY5 7.2 Cy3 Cy5 5.6
Fluorescein TMR 5.5 Fluorescein QSY 7 and QSY 9 dyes 6.1 ATTO488
ATTO633 5.3 ATTO532 ATTO550 6.8 Abbreviation: BPE, B-phycoerythrin;
CY5, carboxymethylindocyanine; Dansyl, just dansyl group; EM, eosin
maleimide; FITC, fluorscein-5-isothiocyanate; LY, Lucifer yellow;
ODR, octadecylrhodamine; TNP-ATP, trinitrophenyl-ATP. TMR:
Tetramethylrhodamine ATTO488* ATTO532*:, ATTO550*: ATTO633*
*related ATTO-TEC webpage at the time of filing
[0083] In particular, the Forster distance indicated in Table 1 for
the exemplary FRET donor-acceptor chromophore pairs of Table 1 is
one of the distances were the energy transference from the FRET
donor to the FRET acceptor. Occurs. In particular, energy transfer
can occur between corresponding FRET donor and acceptor at
distances greater than the Forster distance and in particular at a
distance up to four times the Forster distance for a particular
FRET donor-acceptor chromophore pair, preferably three times the
Forster distance, more preferably two times the Forster distance
and most preferably at the Forster distance or within the Forster
distance.
[0084] Accordingly additional distances where energy transfer from
the FRET donor to the FRET acceptor of FRET donor-acceptor
chromophore pairs of Table 1 or other FRET donor-acceptor
chromophore pairs can be identified by a skilled person and include
distances up to about 28 nm, preferably equal to or lower than
about 20 nm, more preferably equal or lower than about 15 nm and
most preferably between about 5 nm and about 15 nm or lower than
about 5 nm depending on the FRET donor-acceptor chromophore pair as
will be understood by a skilled person.
[0085] Additional, corresponding FRET donors and acceptors forming
additional FRET donor-acceptor chromophore pairs are identifiable
by a skilled person.
[0086] In some embodiments, detection methods provided herein may
use an amplification technique (e.g., polymerase chain reaction
(PCR)) to incorporate FRET chromophores directly onto the product
(e.g., an amplicon) based on a template polynucleotide. In some of
those embodiments at least one primer used for the amplification,
is a FRET labeled oligonucleotide herein described.
[0087] In particular, in some embodiments, the method comprises:
combining the sample with the at least one pair of primers
comprising a forward primer attaching a first FRET chromophore and
the reverse primer attaching a second FRET chromophore. In the
method, the first FRET chromophore and the second FRET chromophore
are selected to be corresponding FRET donor and acceptor
chromophores thus forming a FRET donor-acceptor chromophores pair.
The method further comprises performing at least one polynucleotide
amplification reaction with the forward primer and the reverse
primer of the at least one pair of primers. The method also
comprises detecting a FRET signal from the sample generated the
first FRET chromophore and the second FRET chromophore following
the performing. In the method the first primer and second primer
specifically bind to target sequences located in the polynucleotide
analyte so that upon specific binding of the first primer with the
target sequence specific for the first primer, and upon specific
binding of the second primer with the target sequence specific for
the first oligonucleotide, the first FRET chromophore and the
second FRET chromophore are located within four times the Forster
distance one with respect to the other, preferably within three
times the Forster distance one with respect to the other, more
preferably within two times the Forster distance one with respect
to the other, even more preferably within or at the Forster
distance one with respect to the other.
[0088] In particular, in some cases the location of the target
sequences can be selected to provide a distance between
corresponding FRET donor acceptor chromophore following binding
equal to their Forster distance.+-.0.25 nm to 0.5 nm, or 0.5 nm to
1 nm or 1 nm to 2 nm, depending on the specific FRET donor acceptor
chromophore pair as will be understood by a skilled person. In some
cases the distance between corresponding FRET donor acceptor
chromophore following binding of the related primer pair on
respective target sequence is up 30 nm, or equal to or lower than
20 nm, equal or lower than 15 nm, or 10 nm or 5 nm or lower than 5
nm depending on the FRET donor-acceptor chromophore pair and the
experimental design.
[0089] The conversion between chromophore distance and
corresponding base pairs on the target sequences can be performed
based on information concerning persistence length of single
stranded and double stranded polynucleotide identifiable by a
skilled person. For example for DNA this value may be directly
measured using an atomic force microscope to directly image DNA
molecules of various lengths, or by other techniques such as
molecular combing, optical tweezers and additional techniques
identifiable by a skilled person. In an aqueous solution, the
average persistence length of double strand DNA is 46-50 nm or
140-150 base pairs (the diameter of DNA is 2 nm), although can vary
significantly. In comparison, single stranded DNA is known to have
persistence length of about 4 nm (see Tinland et al.
Macromolecules, 1997, 30 (19), pp 5763-5765).
[0090] An exemplary illustration of such detection method is shown
in FIG. 4, in which a pair of primers, each labeled with either a
FRET-donor chromophore or an FRET-acceptor chromophore at its 5'
end respectively, is used to detect an analyte by amplification.
During the initial amplification cycle, a duplex DNA separates,
allowing primers to bind to specific regions of the individual
template strands. A polymerase (e.g. Taq polymerase) can be used to
extend the primers along the template strand (FIG. 4.S3). A change
in signal can be observed after the initial cycle (FIG. 4.S4) in
particular when a number of a same template is present in the
sample. The intensity of the signal increases as the PCR progresses
and the quantity of the amplicons formed following the elongation
increases (FIG. 4. S5). This change in signal indicates the
presence of the amplified product defined by the primers (e.g. the
analyte).
[0091] In particular, in some cases exemplified in FIG. 4, primers
used in embodiments herein described are formed by a forward primer
and a reverse primer used in combination to form a pair of primers
or primers pair. The term "forward primer" is a primer that is
complementary and anneals to the 5' end of the 5'->3' strand of
a double-stranded polynucleotide analyte. The term "reverse primer"
is a primer that is complementary and anneals to the 5' end of the
complementary 3'->5' strand of the double-stranded
polynucleotide analyte.
[0092] The term "complementary" as used herein indicates a property
of single stranded polynucleotides in which the sequence of the
constituent monomers on one strand chemically matches the sequence
on another other strand to form a double stranded polynucleotide.
Accordingly two polynucleotides having chemically matching
sequences are herein also indicated as "self-complementary".
[0093] A "single-stranded polynucleotide" refers to an individual
string of monomers linked together through an alternating sugar
phosphate backbone. In particular, the sugar of one nucleotide is
bond to the phosphate of the next adjacent nucleotide by a
phosphodiester bond. Depending on the sequence of the nucleotides,
a single-stranded polynucleotide can have various secondary
structures, such as the stem-loop or hairpin structure, through
intramolecular self-base-paring. A hairpin loop or stem loop
structure occurs when two regions of the same strand, usually
complementary in nucleotide sequence when read in opposite
directions, base-pairs to form a double helix that ends in an
unpaired loop. The resulting lollipop-shaped structure is a key
building block of many RNA secondary structures. The term "small
hairpin RNA" or "short hairpin RNA" or "shRNA" as used herein
indicate a sequence of RNA that makes a tight hairpin turn and can
be used to silence gene expression via RNAi.
[0094] A "double-stranded polynucleotide" refers to two
single-stranded polynucleotides bound to each other through
complementarily binding. The duplex typically has a helical
structure, such as double-stranded DNA (dsDNA) molecule, is
maintained largely by non-covalent bonding of base pairs between
the strands, and by base stacking interactions.
[0095] Chemical matching between complementary single strand
polynucleotides indicates that the base pairs between the monomers
of a single strand polynucleotide can be non-covalently connected
via two or three hydrogen bonds with corresponding monomers in the
complementary single strand polynucleotide. In particular, in this
application, when two polynucleotide strands, sequences or segments
are noted to be complementary, this indicates that they have a
sufficient number of complementary bases to form a
thermodynamically stable double-stranded duplex. Double stranded of
complementary single stranded polynucleotides include dsDNA, dsRNA,
DNA: RNA duplexes as well as intramolecular base paring duplexes
formed by complementary sequences of a single polynucleotide strand
(e.g. hairpin loop) complementarily binding one with another.
[0096] The term "complementarily bind", "complementary bind", as
used herein with respect to nucleic acids indicates the two
nucleotides on opposite polynucleotide strands or sequences that
are connected via hydrogen bonds to form a "base pair", a
"complementary base pair". For example, in the canonical
Watson-Crick DNA base pairing, adenine (A) forms a base pair with
thymine (T) and guanine (G) forms a base pair with cytosine (C). In
RNA base paring, adenine (A) forms a base pair with uracil (U) and
guanine (G) forms a base pair with cytosine (C). Accordingly, the
term "base pairing" as used herein indicates formation of hydrogen
bonds between base pairs on opposite complementary polynucleotide
strands or sequences following the Watson-Crick base pairing rule
as will be applied by a skilled person to provide duplex
polynucleotides. Accordingly, when two polynucleotide strands,
sequences or segments are noted to be binding to each other through
complementarily binding or complementarily bind to each other, this
indicate that a sufficient number of bases pairs forms between the
two strands, sequences or segments to form a thermodynamically
stable double-stranded duplex, although the duplex can contain
mismatches, bulges and/or wobble base pairs as will be understood
by a skilled person.
[0097] The term "thermodynamic stability" as used herein indicates
a lowest energy state of a chemical system. Thermodynamic stability
can be used in connection with description of two chemical entities
(e.g. two molecules or portions thereof) to compare the relative
energies of the chemical entities. For example, when a chemical
entity is a polynucleotide, thermodynamic stability can be used in
absolute terms to indicate a conformation that is at a lowest
energy state, or in relative terms to describe conformations of the
polynucleotide or portions thereof to identify the prevailing
conformation as a result of the prevailing conformation being in a
lower energy state. Thermodynamic stability can be detected using
methods and techniques identifiable by a skilled person. For
example, for polynucleotides thermodynamic stability can be
determined based on measurement of melting temperature T.sub.m,
among other methods, wherein a higher T.sub.m can be associated
with a more thermodynamically stable chemical entity as will be
understood by a skilled person. Contributors to thermodynamic
stability can comprise chemical compositions, base compositions,
neighboring chemical compositions, and geometry of the chemical
entity.
[0098] In particular, in embodiments herein described the forward
primer and a reverse primer used in combination in methods herein
described are designed to specifically bind target sequences within
the at least one polynucleotide analyte.
[0099] The wording "specific" "specifically" or "specificity" as
used herein with reference to the binding of a first molecule to
second molecule refers to the recognition, contact and formation of
a stable complex between the first molecule and the second
molecule, together with substantially less to no recognition,
contact and formation of a stable complex between each of the first
molecule and the second molecule with other molecules that may be
present. Exemplary specific bindings are antibody-antigen
interaction, cellular receptor-ligand interactions, polynucleotide
hybridization, enzyme substrate interactions etc. The term
"specific" as used herein with reference to a molecular component
of a complex, refers to the unique association of that component to
the specific complex which the component is part of. The term
"specific" as used herein with reference to a sequence of a
polynucleotide refers to the unique association of the sequence
with a single polynucleotide which is the complementary sequence.
By "stable complex" is meant a complex that is detectable and does
not require any arbitrary level of stability, although greater
stability is generally preferred.
[0100] In embodiments herein described oligonucleotides and in
particular primers of primer pairs are designed to attach a donor
and acceptor of a FRET donor-acceptor chromophore pair, and to
specifically bind to target sequences within one or more target
polynucleotide analyte selected so that when the primers
specifically bind to the corresponding target sequence, the primers
present the FRET donor and acceptor at a distance up to four times
the Forster distance for that FRET donor-acceptor chromophore
pair.
[0101] As used herein, the term "corresponding" in connection with
molecules refers to the binding a molecule to another molecule. The
term "corresponding" used in connection with sequences refers to
the complementarity of a sequence with respect to another and the
related ability to complementarily bind. Thus, polynucleotides that
complementarily bind one to the other are indicated as
corresponding. Also the sequences of polynucleotides complementary
one to the other are indicated as corresponding sequences and can
be provided in corresponding polynucleotides. The term
"corresponding" in connection with FRET chromophores indicates FRET
donor and acceptor chromophore capable of providing an energy
transfer from the FRET donor to the FRET acceptor when located at
Forster distance one with respect to the other.
[0102] The term "present" as used herein with reference to a
compound or functional group indicates attachment performed to
maintain the chemical reactivity of the compound or functional
group as attached. Accordingly, a functional group presented on a
ligand, is able to perform under the appropriate conditions the one
or more chemical reactions that chemically characterize the
functional group.
[0103] Accordingly, following selection of target sequences in one
or more polynucleotide analytes to have a distance up to four times
the Forster distance upon binding of primers in accordance with an
experimental design, the primers (and/or other oligonucleotides)
are built to specifically bind to the target sequences with a
similar thermodynamic stability (e.g. with a Tm within 5.degree.
C., preferably between lower than 5.degree. C., more preferably
within 1-2.degree. C. and most preferably within 0.5 and 1.degree.
C.).
[0104] In particular, in some cases, a primer and/or other
oligonucleotides herein described are specific for at least one
polynucleotide analyte and/or a variation thereof. In those cases,
the primer and/or other oligonucleotides are complementary and
specifically bind a corresponding recognition sequence in single
stranded target polynucleotides within the at least one
polynucleotide analyte.
[0105] A "recognition sequence" is a sequence that is configured to
set apart or identify a polynucleotide or sequence thereof from
others in a sample. Therefore a recognition sequence for a
polynucleotide or for a variation thereof indicates a sequence that
provides a characteristic mark, an identifier capable to mark the
polynucleotide as unique and set the item apart from others, in a
particular sample.
[0106] In particular, the primer and/or other oligonucleotide
typically comprises a recognition region complementary and capable
of specifically binding the recognition sequence on the
corresponding single strand target polynucleotide. The recognition
region in cases herein described is preferably located on the 3'
end of the primer or other oligonucleotide, in particular when the
FRET chromophore is located at the 5' end of the primer or other
oligonucleotide
[0107] The terms "5' end" and "3' end" of a polynucleotide indicate
the two ends of the polynucleotide encompassing the terminal
residues of the polynucleotides and are distinguished based on the
nature of the free group on each extremity. The 5'-end designates a
portion of the polynucleotide strand that has the fifth carbon in
the sugar-ring of the deoxyribose or ribose at its terminus (5'
terminus). The 3'-end of a strand designates a portion of the
polynucleotide strand terminating at the hydroxyl group of the
third carbon in the sugar-ring of the nucleotide or nucleoside at
its terminus (3' terminus). The 5' end and 3' end and in particular
the 5' terminus and 3' terminus in various cases can be modified
chemically or biologically e.g. by the addition of functional
groups or other compounds as will be understood by the skilled
person. In some cases the 5' end encompasses a portion from the 5'
terminus to a residue approximately in the middle of the primer or
oligonucleotide (e.g. approximately 10 bases in a primer of 20
bases) In some cases the 3' end encompasses a portion from the 3'
terminus to a residue approximately in the middle of the primer or
oligonucleotide (e.g. approximately 12 bases in a primer of 25
bases).
[0108] In embodiments where at least one primer pair is used to
detect at least one polynucleotide analyte or a variation thereof
(e.g. a genetic variation) in each primer pair used each of the
forward primer and the reverse primer of the primer pair typically
comprises a recognition region complementary and capable of
specifically binding a corresponding recognition sequence on the
corresponding single strand target polynucleotide to which each of
the forward primer and the reverse primer specifically binds.
[0109] In preferred cases the recognition region is located within
the 3' end of the forward primer and/or the reverse primer.
[0110] In particular, in embodiments where investigation of a
variation of polynucleotide analyte sequences is desired and in
particular a genetic variation such as a SNP or deletion or
insertion is desired, the target sequence is selected in a region
of the target polynucleotide including a recognition sequence for
the variation, and the primer can be selected and designed to
specifically bind to the recognition sequence, preferably through a
recognition region in the terminal portion at the 3' end of the
primer (or of other oligonucleotide).
[0111] In several cases primers can be 20-35 base pair depending on
the sequences of the target the sequence of the primer and the
experimental conditions as will be understood by a skilled
person.
[0112] In embodiments herein describe each of the primers (or other
oligonucleotides) are labeled with the FRET chromophore based on
the specific target sequence and Forster distance for the related
FRET donor-acceptor chromophore in a configuration that allow
presentation of the FRET chromophore following binding of the
primer (or other oligonucleotide) to the target sequence in an
orientation allowing energy transfer with the corresponding FRET
label of the FRET donor-acceptor chromophore pair. In some cases,
each primer attaches a plurality of FRET label of a same type (e.g.
a plurality of donors or a plurality of acceptor) can be used on a
same primer or oligonucleotide.
[0113] The term "attach" or "attached" as used herein, refers to
connecting or uniting by a bond, link, force or tie in order to
keep two or more components together, which encompasses either
direct or indirect attachment where, for example, a first molecule
is directly bound to a second molecule or material, or one or more
intermediate molecules are disposed between the first molecule and
the second molecule or material. In some embodiments, the primer
(or other oligonucleotide) attaches the FRET chromophore by direct
covalent attachment. In some cases, the primer (or other
oligonucleotide) indirectly through binding with other molecule
(e.g. aptamers or other synthetic recognition molecules)
[0114] In several embodiments, the FRET chromophore is attached
within the 5' end of the primer or other oligonucleotide. In
particular, the FRET chromophore can be attached within
approximately 10 bases from the 5' terminus of the primer or other
oligonucleotide, possibly within 5 bases from the 5' terminus of
the primer or other oligonucleotide. Preferably the FRET
chromophore can be attached at the 5' terminus of the primer.
[0115] In methods herein described amplification is then performed
and measuring a signal can be performed before and/or after each
step according to the experimental design. Typically, measuring is
performed at least following annealing of at least one
amplification cycle by measuring at least an acceptor signal from a
FRET acceptor chromophore alone or in combination with measuring of
a FRET donor signal from the corresponding FRET donor. In some
cases the measuring following annealing of the primers can also be
preceded by an optional measuring of the donor and/or acceptor of
the FRET donor-acceptor chromophore before the amplification cycle.
In some cases measuring before amplification cycle is performed by
measuring a donor signal of the FRET donor-acceptor chromophore
pair (or other primer/oligonucleotide pairing herein described) and
possibly an acceptor signal of the FRET donor-acceptor chromophore
pair. In some cases, several amplification cycles can be performed
and the measuring of the FRET signal can be performed before and/or
after annealing of one or more amplification cycles.
[0116] In embodiments where a specific binding of the primer pairs
(or binding of other oligonucleotide) occurs, a successful binding
of the primers to the corresponding target sequences and in
particular to the recognition sequences of through specific binding
of corresponding recognition region, is detectable by a change in
signals detected before and after at least one cycle of
amplification possibly after each of a plurality of amplification
cycles, and/or in by a change in signals detected after each
amplification cycle.
[0117] In some cases, the change in signal is an increase in signal
(e.g., an increased fluorescence emission intensity of the FRET
acceptor chromophore of a FRET donor-acceptor chromophore pair when
a donor chromophore and an acceptor chromophore are incorporated
into the amplified product). In some case, the change in signal is
a decrease in signal (e.g., a decreased fluorescence emission
intensity of the FRET donor chromophore of a FRET donor-acceptor
chromophore pair when a donor chromophore and an acceptor
chromophore are incorporated into the amplified product). In some
cases, the increase in one signal indicates a presence of the
product or analyte. In some cases, the decrease in another signal
indicates a presence of the product or analyte. In some cases, the
lack of a change in signal (e.g., no significant change in
fluorescence intensity) indicates the absence of the product or
analyte. For example, in the cases when the Cy3-Cy5 pair is used as
a FRET donor-acceptor chromophore pair, in which Cy3 is attached to
a first primer as a FRET donor chromophore, Cy5 is attached to a
second primer as a FRET acceptor chromophore, and the first primer
and second primer constitute a primer pair. Before the primers bind
to a template strand, Cy3 emits yellow light and Cy5 produces low
emission signal in red. After the primers bind and are subsequently
extended along the template strand, FRET occurs between Cy3 and
Cy5, resulting in a decrease in the yellow emission by Cy3
accompanied by an increase in the red emission by Cy5. The change
in fluorescence emission signal can be measured at the optimal
excitation and emission wavelength of the donor Cy3 and the
acceptor Cy5, respectively.
[0118] In some cases, the change in signal can be defined by a
percentage change. In some cases, the change in signal can be about
0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or
more. In some cases, the change in signal can be greater than
0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000%.
In some cases, the change in signal can be less than 0.001%, 0.01%,
0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%,
300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000%. In some cases,
the change in signal can be about 50%. In some cases, the change in
signal indicates the presence of the product. In some cases, the
increase in one FRET signal indicates the presence of the product.
In some cases, the decrease in another FRET signal indicates the
presence of the product.
[0119] In some cases one or more primers attaching FRET chromophore
can be used together with oligonucleotides attaching a FRET
chromophore used as probes where the FRET chromophore are selected
to form upon binding a FRET donor-acceptor FRET chromophore pair.
The term "probe" or "hybridization probe", as used herein,
generally refers to a fragment of linear oligonucleotide of
variable length that is used in samples containing DNA, RNA or any
synthetic molecule that resembles DNA or RNA to detect the presence
of target nucleotide sequences that are complementary to the
sequence in the probe.
[0120] In particular in some cases at least one primer attaching a
first FRET chromophore and/or at least one probe attaching a second
FRET chromophore of a FRET chromophore pair can be used in
combinations where the detection is performed following presence,
absence or change of a signal from a FRET chromophore. In some
cases, at least one primer-attaching a FRET chromophore is used
with a probe or a plurality of probes attaching a FRET chromophore.
In some cases, a plurality of primers attaching FRET chromophores
is used with a plurality of probes attaching FRET chromophores. In
some cases, a plurality of primers attaching FRET chromophores is
used with a single probe attaching a FRET chromophore. In some
cases, a primer pair attaching FRET chromophores is used to detect
an analyte. In some cases, a primer pair attaching a FRET
donor-acceptor chromophore pair is used to detect multiple
analytes. In some cases, a plurality of primer pairs attaching a
each attaching a FRET donor acceptor chromophore pair are used to
detect an analyte. In some cases, a primer and a probe is used to
detect an analyte. In some cases, a primer attaching a FRET
chromophore (e.g. a donor) and a probe attaching a corresponding
FRET chromophores (e.g. the acceptor of a same FRET donor-acceptor
chromophore pair of the donor attached to the primer) is used to
detect multiple analytes. In some cases, a combination of primers
attaching FRET chromophores and probes attaching FRET chromophores
is used to detect an analyte. In some cases, a combination of
primers attaching FRET chromophores and probes attaching FRET
chromophores is used to detect multiple analytes with each target
identified by a separate pair of primer-probe. In some cases, a
probe attaching FRET chromophores is used to detect an analyte
presenting another FRET label forming a FRET donor-acceptor
chromophore pair.
[0121] In embodiments where detection of a polynucleotide analyte
and in particular of a genetic variation in the polynucleotide
analyte is desired the target sequences for the primer pair or
primer-probe pair specific for the genetic variation are
complementary for at least a portion of the recognition sequence
for the genetic variation (e.g. in their 3' ends for at least 1
bp). (see Examples 3 and 4).
[0122] In preferred embodiments, at least a portion of the
recognition sequence on the single strand target polynucleotide
specific for a forward primer of a primer pair is complementary to
a corresponding portion of the recognition sequence of the single
strand target polynucleotide specific for the reverse primer on the
same polynucleotide analyte. In those embodiments, the recognition
sequence of the single strand target polynucleotide specific for a
forward primer and the recognition sequence of the single strand
target polynucleotide specific for a reverse primer are at least
partially self-complementary. In those embodiments, the forward and
reverse primer of the primer pair are complementary to
corresponding recognition sequences and are therefore at least in
part self-complementary.
[0123] In some embodiments, the recognition sequence of the single
strand target polynucleotide specific for the forward primer, and
the corresponding portion of the recognition sequence of the
sequence of the single strand target polynucleotide specific for
the reverse prime are self-complementary for a sequence equal to or
less than 20 bases and each of the recognition regions of the
forward primer and reverse primer is within 20 bases from the
respective 3' terminus. In particular, in some cases the
recognition sequences can be for a genetic variation selected from
a substitution, an addition, a deletion or a translocation.
[0124] In some embodiments, the recognition sequence on the single
strand target polynucleotide specific for the forward primer, and
the corresponding portion of the recognition sequence on the single
strand target polynucleotide specific for the reverse prime are
self-complementary for a sequence of 1 base. In some cases, each of
the recognition regions of the forward primer and reverse primer
are possibly within 1 to 3 bases from the respective 3' terminus.
In particular, in some cases the recognition sequences can be for a
genetic variation selected from a substitution, an addition, a
deletion or a translocation and in particular a single-nucleotide
polymorphism (SNP).
[0125] In some cases of genetic variation, one or more bases are
inserted into a target sequence, resulting in a shift in the
reading frame. In such cases, FRET-chromophore-labeled primers can
be designed to include a recognition region at the 3' end specific
for the inserted bases for detecting the presence or absence of
such insertion. Such FRET-based qPCR applications can be used for
inherited mutation detection, such as insertions in p53 family
genes. Each of the recognition region of the forward primer and
reverse primer is preferably located at the respective 3'
terminus.
[0126] In embodiments where detection of a plurality of analytes is
performed possibly comprising a genetic variation a plurality of
FRET labeled primer pairs each attaching a FRET donor-acceptor
chromophore pair can be used with each pair specific for one
polynucleotide analyte and/or a single variation within one or more
polynucleotide analyte.
[0127] In those embodiments, a plurality of primer and/or probe
pairs can be used to perform the detection, each pair attaching
corresponding FRET donor-acceptor chromophores.
[0128] In some cases, a FRET-chromophore-labeled probe is used to
detect multiple analytes presenting one or more FRET labels. In
some cases, multiple FRET-chromophore-labeled probes are used to
detect an analyte. In some cases, a single FRET donor/acceptor
chromophore pair is used to detect an analyte. In some cases, a
single FRET donor/acceptor chromophore pair is used to detect
multiple analytes. In some cases, multiple FRET donor/acceptor
chromophore pairs are used to detect an analyte. In some cases, the
signal is not limited to a signal generated by a FRET
donor/acceptor chromophore pair. In some cases, the signal can be
generated by different chromophores.
[0129] In some cases, a plurality of FRET-chromophore-labeled
primers is used in the detection methods. In some cases, a
FRET-chromophore-labeled primer is used with a
FRET-chromophore-labeled probe or a plurality of
FRET-chromophore-labeled probes. In some cases, a
FRET-chromophore-labeled primer is used without any type of probe.
In some cases, a plurality of FRET-chromophore-labeled primers are
used with a plurality of FRET-chromophore-labeled probes. In some
cases, a plurality of FRET-labeled primers are used with a single
FRET-chromophore-labeled probe. In some cases, a plurality of
FRET-chromophore-labeled primers are used without any type of
probe.
[0130] In some embodiments, a plurality of primer and/or probe
pairs attaching a same FRET donor acceptor chromophore pair are
used in methods herein described wherein the primers are added at
different concentrations and the signal measured after annealing of
at least one, preferably multiple amplification cycles (e.g. an
acceptor signal measured alone or in combination with a donor
signal) can be used to form signature profile for the at least one
polynucleotide and/or at least one variation thereof.
[0131] In some embodiments, a plurality of primer and/or probe
pairs each attaching a different FRET donor acceptor chromophore
pair are used in methods herein described wherein a same or
different concentration of each primer pair is used and the signal
measured after annealing of at least one, preferably multiple
amplification cycles (e.g. an acceptor signal measured alone or in
combination with a donor signal) can be used to form signature
profile for the at least one polynucleotide and/or at least one
variation thereof as will be understood by a skilled person.
[0132] In some embodiments, the measured FRET signals before and/or
after annealing in each cycle can be traced to form a profile (e.g.
a real-time curve) that can be used for detection (e.g.
quantitative measurement) of the target polynucleotide analyte or
portion thereof, and in particular a variation and in particular a
genetic variation thereof.
[0133] In some cases, a signature profile is used to detect the
presence of a SNP in a sample containing a plurality of
polynucleotide analytes. In some cases, the signature profile for
each SNP of the plurality of analytes is a curve having the x-axis
represented by the number of PCR cycles and the y-axis represented
by the emission intensity generated by the FRET acceptor of at
least one FRET chromophore pair attached to the primer pair that is
specific for that particular SNP. The signature profile can be
obtained for each single nucleotide mismatch and/or combinations
thereof.
[0134] In particular in embodiments where different concentration
of primer pairs of a plurality of primers are used, the
concentration can be selected so that a certain concentration
identifies one primer pair or a combination thereof.
[0135] Additional profiles and related applications are described
in the Examples section and can be identified by a skilled person
upon reading of the present disclosure.
[0136] In some cases, the method is used with a second method. In
some cases, the second method is an amplification method. In some
cases, the amplification method is an isothermal reaction method or
a polymerase chain reaction method. In some cases, the polymerase
chain reaction is a multiplex-PCR, a quantitative PCR (qPCR), an
end point PCR or a digital PCR (e.g. droplet digital PCR). In some
cases, the polymerase chain reaction is a droplet digital PCR. In
some cases, the second method is a droplet digital PCR method. In
some cases, the method is used in combination with a second and a
third method. In some cases, the third method is an amplification
method, an electrophoresis (e.g. gel electrophoresis, capillary
electrophoresis), a mass spectroscopy method, a chromatography
method or an assay (e.g. in vitro cell based assay).
A. Detection of Analytes from Intensity-Length Relationship
[0137] Described herein is a method of detecting the presence of
one or more analytes in a sample. Methods provided herein involve,
e.g., the measurement of the change in signal intensity when at
least two chromophores interact with each other. For example, in
the case of a FRET donor and FRET acceptor chromophore interaction,
the closer the donor is from the acceptor, the brighter the
fluorescence signature of a particular nucleic acid analyte will
typically be. So long that the analyte lengths are small, the
persistence length of the nucleic acid analyte typically determines
the intensity of fluorescence. For example, for a given
donor-acceptor pair, the intensity of the fluorescence can be
correlated with the persistence length. Further, the intensity of
fluorescence often indicative of an energy transfer between the
donor and the acceptor. The efficiency of this energy transfer is
described by the following equation:
E = 1 1 + ( r / R 0 ) 6 ( 2 ) ##EQU00004##
where r is the distance between the donor and the acceptor (FIG. 1)
and R.sub.0 is the characteristic distance (the Forster distance or
Forster radius), with a 50% transfer efficiency which is a constant
related to each donor/acceptor pair that can be calculated from
certain parameters of the absorption and emission spectra of each
chromophore. (See Biophysical Chemistry, D. Freifelder, ed., W.H.
Freeman and Company, San Francisco (1976) at page 426-28). Further,
R.sub.0 is described by the following equation:
R 0 6 = 9 .times. Q 0 .function. ( ln .times. .times. 10 ) .times.
.kappa. 2 .times. J 128 .times. .times. .pi. 5 .times. n 4 .times.
N A ( 4 ) ##EQU00005##
where Q.sub.0 is the fluorescence quantum yield of the donor in the
absence of the acceptor, .kappa..sup.2 is the dipole orientation
factor, n is the refractive index of the medium, N.sub.A is
Avogadro's number, and J is the spectral overlap integral.
[0138] Therefore positioning of FRET labels within a distance up to
four times the Forster distance will result in transfer of energy
and emission of a signal by the FRET acceptor. Therefore, the
changes in fluorescence intensity typically vary with the length of
the synthesized strand. In some cases, an increase in fluorescence
intensity is accompanied by the decrease with the length of the
synthesized strand. Utilizing this relationship, the presence of an
analyte can be detected based on the intensity correlated with its
length. For example, a set of fluorescence intensity ladders
reminiscent of molecular weight ladders based on DNA length can be
established as a control. In a sample, multiple pairs of primers
labeled with the same donor/acceptor pair are amplified. Upon
completion of the amplification process, the observed intensities
can be correlated with the controls, thereby detecting a particular
analyte. In some cases, the presence or absence of a particular
analyte can be monitored throughout the amplification process, by
taking measurements during each amplification cycle and comparing
with the control ladder. In some cases, the ladder comprises a
plurality of signals. In some cases, the plurality of signals
generates multiple curves. In some cases, the ladder is represented
by a plurality of curves. In some cases, the ladder comprises
multiple sets of initial and end points. In some cases, each step
of the ladder comprises a plurality of signals. In some cases, the
plurality of signals generates a curve. In some cases, each step of
the ladder is represented by a curve. In some cases, each step of
the ladder comprises a set of initial and end points. In some
cases, the curve represents a signature profile of an analyte based
on its length. In some cases, the set of initial and end points
represents a signature profile of an analyte based on its length.
In some cases, each step of the ladder generates a signature
profile of an analyte based on its length. In some cases, the
ladder comprises multiple steps or multiple signature profiles of
analytes. In some cases, the ladder comprises a single step or a
single signature profile of an analyte. In some cases, the ladder
comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,
55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150,
160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500 or more
steps. In some cases, the ladder comprises more than 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85,
90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250,
300, 350, 400, 450, 500 steps. In some cases, the ladder comprises
less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55,
60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160,
170, 180, 190, 200, 250, 300, 350, 400, 450 or 500 steps. In some
cases, multiple analytes are detected by a single FRET
donor/acceptor pair. In some cases, a single analyte is detected by
a single FRET donor/acceptor pair. In some cases, the signal is not
limited to a signal generated by a donor and acceptor pair. In some
cases, the signal can be generated by different donor/acceptor
chromophores.
B. Detection of Genetic Variations
[0139] Disclosed herein is a method of determining the presence or
absence of a genetic variation, e.g., based on the change in signal
intensity due, e.g., to a disruption in the electron transport
mechanism described herein. Genetic variations include deletion and
insertion of one or more nucleotides, translocations different
nucleotide occurrences (e.g. single point mutations such as SNPs or
a base-pair substitution), or variations in the number of multiple
nucleotide repetitions. For example, to detect the presence of a
single deletion or alteration (e.g. a SNP) in a template (e.g. an
analyte), a first FRET labeled primer is designed to hybridize to a
region encoding the deletion. A second primer comprises a sequence
complementary to the region of the analyte about less than 500 bp
away from the first primer. Upon amplification, a change in signal
is observed. However, since a kink is present in the product
template, an inefficient electron transport results in a decrease
in the change of signal, e.g., when compared to the change in
signal observed for an analyte without the genetic variation.
[0140] In some cases, the genetic variation detected is a different
nucleotide occurrence in the analyte. In some cases, the different
nucleotide occurrence is a single-nucleotide polymorphism (SNP). A
SNP is a DNA sequence variation that occurs when a single
nucleotide (e.g. A, T, C or G) in the genome is altered. In some
cases, this alteration leads to either a presence of disease or is
associated with (or a marker for) the presence of a disease or
diseases. For example, a single nucleotide mutation from GAG to GTG
in the .beta.-globin gene that encodes haemoglobin results in
development of sickle-cell anaemia.
[0141] In general, each individual has many SNPs that create a
unique human DNA pattern. In some cases, a SNP is a common SNP or a
rare SNP. In some cases, a SNP is a common SNP. In some cases, a
common SNP has a minor allele frequency of about 1%, 2%, 3%, 4%,
5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,
19%, 20% or more. In some cases, a common SNP has a minor allele
frequency of greater than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,
11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%. In some cases,
a common SNP has a minor allele frequency of less than 1%, 2%, 3%,
4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,
18%, 19% or 20%. In some cases, a SNP is a rare SNP. In some cases,
a rare SNP has a minor allele frequency of about 1%, 2%, 3%, 4%,
5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,
19%, 20% or more. In some cases, a rare SNP has a minor allele
frequency of greater than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,
11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%. In some cases,
a rare SNP has a minor allele frequency of less than 1%, 2%, 3%,
4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,
18%, 19% or 20%.
[0142] In some cases, provided herein is a method to detect the
presence of a SNP in particular by a FRET labeled primer pairs in
which the forward and reverse primers comprise a 1 base recognition
region specific for the SNP and located at the 3' end, preferably
within 3 bases from the 3' terminus, more preferably at the 3'
terminus. In some cases, the method described herein is used to
detect the presence of a common SNP. In some cases, the method
described herein is used to detect the presence of a rare SNP. In
some cases, the method described herein is used to detect the
presence of a combination of common and rare SNPs.
[0143] In some cases, the method described herein is used to detect
the presence of SNP in a sample. In some cases, the method
described herein is used to detect multiple SNPs in a sample. In
those embodiments a plurality of FRET-chromophore-labeled primer
pairs can be used, each having the forward and reverse primers
comprising a 1 base recognition region specific for the SNP and
located at the 3' end, preferably within 3 bases from the 3'
terminus, more preferably at the 3' terminus. In those embodiments
the plurality of FRET labeled primers pair can attach a same or
different FRET donor-acceptor chromophore pair and can be used at a
same or different volumes and/or concentrations as will be
understood by a skilled person upon reading of the present
disclosure. In some cases, the method described herein is used to
detect multiple common SNPs in a sample. In some cases, the method
described herein is used to detect multiple rare SNPs in a sample.
In some cases, the method described herein is used to detect a
combination of common and rare SNPs in a sample. In some cases, the
method described herein is used to detect a single SNP in a sample.
In some cases, the method described herein is used to detect a
single common SNP in a sample. In some cases, the method described
herein is used to detect a single rare SNP in a sample.
[0144] In some cases, the presence of SNPs correlates directly with
the development of a disease. In some cases, the presence of SNPs
increases the chances of developing a disease. In some cases, the
disease comprises a genetic disorder, an autoimmune disease, a
neurological disease, a cardiovascular disease and cancer.
[0145] In some cases, provided herein is a method to detect the
presence of a genetic variation involving more than 1 base pair by
a FRET-chromophore-labeled primer pairs in which the forward and
reverse primers comprise a up to 20 base recognition region
specific for the variation and located at the 3' end, preferably
including particular deletions, insertions of a single or multiple
bases, substitution or any type of base modifications, possibly
resulting in a shift of the reading frame. In some of these cases
the genetic variation can be associated to cancers such as cancers
associated with insertions in p53 gene.
C. Monitoring an Amplification Reaction
[0146] Disclosed herein is a method for detecting a change in
signal generated by a set of chromophores for monitoring a
reaction. In some cases, the method described herein can be used to
monitor the progress of a PCR reaction. For example, at cycle 1, a
set of fluorescence signals are measured, one measurement at the
denaturing step and one measurement at the annealing step. During
cycle 2, a second set of fluorescence signals are measured at the
denaturing and annealing steps. A change in fluorescence between
the signals taken at the two annealing step indicate an occurrence
of a PCR reaction, while the signals taken during the denaturing
steps are used as a control. In some cases, the change in signal
from the two annealing steps is an increase in one signal. In some
cases, the change is a decrease in another signal. In some cases,
the change in signal is defined by a percentage. In some cases, the
change in signal can be about 0.001%, 0.01%, 0.1%, 1%, 10%, 20%,
30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%,
600%, 700%, 800%, 900%, 1000% or more. In some cases, the change in
signal can be greater than 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%,
700%, 800%, 900% or 1000%. In some cases, the change in signal can
be less than 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%
or 1000%. In some cases, the signals taken at the two annealing
step remain constant. In some cases, the constant signal indicates
the PCR reaction is functioning properly. In some cases, the
signals taken at the two annealing steps indicate a change in
signal. In some cases, this change in signal indicates the PCR
reaction has failed. In some cases, the signals from the annealing
steps serve as a control for each amplification cycle. In some
cases, multiple reactions are monitored. In some cases, multiple
reactions from a single sample are monitored. In some cases,
multiple reactions from multiple samples are monitored. In some
cases, a single reaction is monitored. In some cases, a single
reaction from a single sample is monitored.
D. Detection of Morphological Change
[0147] Disclosed herein is a method for detecting or monitoring a
morphological change in an analyte based on changes in signals. In
some cases, an analyte is a protein, a polynucleotide, a lipid, a
carbohydrate or an antibody. In some cases, an analyte is a
polynucleotide. In some cases, the polynucleotide is a DNA or an
RNA. In some cases, DNA and RNA can adopt different conformations
such as a hairpin, tetraloop or pseudoknot. For example, to detect
the different morphological state of a DNA containing a hairpin, a
donor/acceptor pair can be conjugated to the respective stem of the
hairpin. Since the FRET donor is in close proximity to the FRET
acceptor, a signal can be observed. As the temperature increases,
the DNA hairpin unwinds and a fluorescence signal may be decreased.
In some cases, multiple signals are measured as the DNA unwinds. In
some cases, only an initial and an end-point signals are measured
as the DNA unwinds. In some cases, the multiple signals can
generate a curve. In some cases, the multiple signals are used to
generate a signature profile of a DNA containing a hairpin. In some
cases, the signature profile is a curve. In some cases, an initial
and an end-point signals are used to generate a signature profile.
In some cases, the signature profile obtained from the DNA
denaturation study is used as a control to detect the presence of a
hairpin in a target DNA. In some cases, the method described herein
is used to monitor the stability of a DNA or RNA conformation after
introduction of addition, deletion, substitution or base
modifications (e.g. unnatural bases) within the DNA or RNA. In some
cases, the stability is affected by external factors. In some
cases, the external factors include pH, organic or inorganic agents
(e.g. salt, intercalating dye) or additional analytes. In some
cases, the additional analyte is a DNA, RNA, protein or an
antibody. In some cases, the method described herein is used to
monitor the stability of a DNA or RNA conformation after
introduction of the external factors.
[0148] In some cases, the method described herein is used to
monitor a morphological change of a protein. For example, a protein
residing in a native state can be a folded protein, a partially
folded protein or a disordered protein. Folding or unfolding occurs
due to the presence of binding partners, organic or inorganic
agents, pH, and temperature. For a folded protein, an increase in
temperature induces the protein to undergo an unfolding state. By
labeling proteins with a plurality of donors and/or acceptors, a
change in fluorescence signal can be measured with each iterative
temperature increase and can be compared to the signals taken at
its native state. In some cases, multiple signals are measured as
the protein unfolds. In some cases, only an initial and an
end-point signals are measured as the protein unfolds. In some
cases, multiple signals can generate a curve. In some cases,
multiple signals are used to generate a signature profile of the
protein. In some cases, the signature profile is a curve. In some
cases, an initial and an end-point signals are used to generate a
signature profile. In some cases, the signature profile obtained
from the protein unfolding study is used as a control to detect the
morphology of proteins containing similar folds. In some cases, the
method described herein is used to monitor the stability of a
protein. In some cases, unfolding of the protein can be induced
upon addition of an external factor. In some cases, the external
factors include pH, organic or inorganic agents (e.g. salt,
intercalating dye) or additional analytes. In some cases, the
additional analyte is a DNA, RNA, protein or an antibody.
[0149] In some cases, the method described herein is used to
monitor the morphology of an polynucleotide analyte-polynucleotide
analyte interaction such as a protein-protein, protein-antibody or
protein-polynucleotide (e.g. protein-DNA or protein-RNA)
interactions. For example, during a protein-DNA interaction, a
protein can adopt a different conformation upon binding of the DNA.
In some cases, the change in signal associated with binding can be
used to compare with the protein at its bound or unbound state. In
some cases, multiple signals are measured as the protein-DNA
complex forms. In some cases, only an initial and an end-point
signals are measured as the complex forms. In some cases, the
multiple signals can generate a curve. In some cases, the multiple
signals are used to generate a signature profile of the protein. In
some cases, the signature profile is a curve. In some cases, an
initial and an end-point signals are used to generate a signature
profile. In some cases, the signature profile obtained from the
protein-DNA study is used as a control to detect the formation of
protein complex with additional DNAs. In some cases, the methods
described herein can monitor the stability of the protein complex
with addition of another external factor. In some cases, the
methods described herein can be used to monitor the morphological
change of an analyte with multiple binding partners.
III. Multiplex Detection
[0150] Disclosed herein are examples of determining the presence of
a plurality of analytes using a plurality of FRET donor/acceptor
chromophores to detect polynucleotide analytes or other analytes
associated thereto. For example, a multiplex detection method can
combines the use of color, multiplicity of signal intensity, and/or
mathematical strategies to circumvent degeneracy and ensure an
infinite number of unique codes that can be unambiguously decoded
in any combination of occurrences. For example, in detecting a
sample containing four analytes, each analyte can be assigned a
donor (blue, cyan, yellow and red florescent protein) and a green
fluorescent protein as an acceptor attached to analyte-specific
oligonucleotides (e.g., a forward PCR primer and a reverse PCR
primer). Upon amplification, the presence or absence of an analyte
is determined based on the presence or absence of a signal in that
particular color.
[0151] In some cases, multiple color codes are generated using a
plurality of chromophores. In some cases, multiple color codes are
generated using a plurality of FRET donors and acceptors. In some
cases, multiple color codes are generated using combinations of
FRET donor/acceptor chromophore pairs. In some cases, multiple FRET
chromophore pairs are assigned to multiple analytes. In some cases,
a single FRET chromophore pair is assigned to multiple analytes. In
some cases, a single FRET chromophore pair is assigned to one
analyte.
[0152] In some cases, one color code or FRET chromophore pair is
assigned to one analyte. In some cases, one color code or FRET
chromophore pair is assigned to multiple analytes (e.g., to
discriminate multiple analytes of varying lengths in a single
detection reaction). In some cases, one color code or chromophore
pair is assigned to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, 50, 100, 500, 1000, 5000, 10,000 or 100,000 analytes. In some
cases, multiple color codes or chromophore pairs are assigned to
one or more analytes. In some cases, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, 50, 100, 500, 1000, 5000, or 1000 color codes
or chromophore pairs are assigned to one or more analyte. In some
cases, at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,
50, 100, 500, 1000, 5000, 10,000 or 100,000 color codes or
chromophore pairs are assigned to one analyte.
[0153] In some cases, one color is assigned as a control. In some
cases, the control is a positive control or a negative control. In
some cases, a particular signal multiplicity in a particular color
is assigned as indicating a positive control. In some cases,
multiple colors and/or signal multiplicities are assigned as
positive controls.
A. Multiplex Detection for Genetic Variation
[0154] In some cases, the methods disclosed herein can be used to
detect the presence of multiple genetic variations (e.g., SNPs). In
some cases, an analyte contains a plurality of genetic variations.
In some cases, an analyte contains one genetic variation. In some
cases, one color is assigned to one genetic variation. In some
cases, a sample contains a plurality of genetic variations, wherein
a color code or FRET chromophore pair is assigned to each genetic
variation. In some cases, a sample contains one genetic variation.
In some cases, multiple genetic variations are tested for in the
same sample, and each is assigned a different multiplicity of
signal (FRET signal intensity) of a same FRET chromophore pair or a
same color code, for purposes of unambiguous identification. In
some cases, multiple genetic mutations are tested for in the same
sample, and each is assigned with a different
color-and-multiplicity combination, for the same purposes of
unambiguous identification. In some cases, multiple genetic
mutations are tested for in the same sample, and each is assigned
with a same multiplicity and a same color code, for purposes of
determining how many (if any) mutations on the panel are present in
the sample without identifying them explicitly.
B. Multiplex Detection for SNP
[0155] In some cases, disclosed herein are methods of detecting of
a SNP in an analyte. In some cases, an analyte contains a plurality
of SNPs. In some cases, an analyte contains 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300,
350, 400, 450, 500 or more SNPs. In some cases, an analyte contains
at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55,
60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160,
170, 180, 190, 200, 250, 300, 350, 400, 450, or 500 SNPs. In some
cases, an analyte contains no more than 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,
44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300,
350, 400, 450, or 500 SNPs. In some cases, an analyte contains 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70,
75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180,
190, 200, 250, 300, 350, 400, 450, 500 or more common SNPs. In some
cases, an analyte contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,
110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350,
400, 450, or 500 common SNPs. In some cases, an analyte contains no
more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,
55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150,
160, 170, 180, 190, 200, 250, 300, 350, 400, 450, or 500 common
SNPs. In some cases, an analyte contains 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,
44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300,
350, 400, 450, 500 or more rare SNPs. In some cases, an analyte
contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130,
140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, or 500
rare SNPs. In some cases, an analyte contains no more 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80,
85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,
250, 300, 350, 400, 450, or 500 rare SNPs.
[0156] In some cases, a sample contains a plurality of analytes. In
some cases, multiple SNPs are detected from a plurality of analytes
in the sample. In some cases, multiple common SNPs are detected
from a plurality of analytes in the sample. In some cases, multiple
rare SNPs are detected from a plurality of analytes in the sample.
In some cases, multiple SNPs are detected from an analyte in the
sample. In some cases, multiple common SNPs are detected from an
analyte in the sample. In some cases, multiple rare SNPs are
detected from an analyte in the sample. In some cases, one SNP is
detected from an analyte in the sample. In some cases, one common
SNP is detected from an analyte in the sample. In some cases, one
rare SNP is detected from an analyte in the sample. In some cases,
one SNP is detected in the sample. In some cases, one common SNP is
detected in the sample. In some cases, one rare SNP is detected in
the sample.
[0157] In some cases, a sample contains 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,
44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300,
350, 400, 450, 500, 600, 700, 800, 900, 1000, 5000, 10,000, 50,000,
100,000 or more SNPs. In some cases, a sample contains at least 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70,
75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180,
190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000,
5000, 10,000, 50,000, 100,000 or more SNPs. In some cases, a sample
contains no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130,
140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500,
600, 700, 800, 900, 1000, 5000, 10,000, 50,000, 100,000 or more
SNPs. In some cases, a sample contains 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,
44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300,
350, 400, 450, 500, 600, 700, 800, 900, 1000, 5000, 10,000, 50,000,
100,000 or more common SNPs. In some cases, a sample contains at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55,
60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160,
170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800,
900, 1000, 5000, 10,000, 50,000, 100,000 or more common SNPs. In
some cases, a sample contains no more than 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300,
350, 400, 450, 500, 600, 700, 800, 900, 1000, 5000, 10,000, 50,000,
100,000 or more common SNPs. In some cases, a sample contains 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70,
75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180,
190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000,
5000, 10,000, 50,000, 100,000 or more rare SNPs. In some cases, a
sample contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130,
140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500,
600, 700, 800, 900, 1000, 5000, 10,000, 50,000, 100,000 or more
rare SNPs. In some cases, a sample contains no more than 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75,
80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,
200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 5000,
10,000, 50,000, 100,000 or more rare SNPs.
[0158] In some cases, the method of detection utilizes an
amplification method. In some cases, an amplification method
comprises a polymerase chain reaction (PCR) method and an
isothermal reaction method. In some cases, a PCR comprises a
multiplex PCR, a real-time PCR, a quantitative PCR and a digital
PCR (e.g. droplet digital PCR). In some cases, the method of
detection utilizes a quantitative PCR method. In some cases, the
quantitative PCR method is used in combination with a second
method. In some cases, the second method is a digital PCR method.
In some cases, the second method is a droplet digital PCR
method.
[0159] In some cases, a signature profile is used to detect the
presence of a SNP. In some cases, a signature profile is used to
pinpoint the nucleotide mutation. In some cases, a signature
profile is unique for each nucleotide mismatch, UU, UT, UG, UC, UA,
AA, TT, GG, CC, AG, AC, TC, TC, and distinct from the wild-type. In
some cases, a signature profile of a nucleotide mismatch is
compared to that of a wild-type. In some cases, the signature
profile of a SNP is compared to that of a wild-type. The signature
profile of the wild-type can be generated separately in one or more
experiments independent of the detection of the SNP. In some cases,
a fluorescence signal of a SNP is compared to a fluorescence signal
of a wild-type. In some cases, a change in fluorescence signal is
detected between the signals of a SNP and a wild-type. In some
cases, the change in signal can be calculated as a percentage. In
some cases, the percentage of signal change is 0.01, 0.1, 0.2, 0.5,
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,
37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65,
70, 75, 80, 85, 90, 95 or 100%. In some cases, the percentage of
signal change is about 0.01, 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95
or 100%. In some cases, a change in signal is detected between the
fluorescence signals of an AG mismatch and a wild-type. In some
cases, the change in signal is calculated as a percentage. In some
cases, the percentage of signal change is 0.01, 0.1, 0.2, 0.5, 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70,
75, 80, 85, 90, 95 or 100%. In some cases, the percentage of signal
change is about 0.01, 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100%.
In some cases, a change in signal is detected between the
fluorescence signals of an AC mismatch and a wild-type. In some
cases, the change in signal is calculated as a percentage. In some
cases, the percentage of signal change is 0.01, 0.1, 0.2, 0.5, 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70,
75, 80, 85, 90, 95 or 100%. In some cases, the percentage of signal
change is about 0.01, 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100%.
In some cases, a change in signal is detected between the
fluorescence signals of a TG mismatch and a wild-type. In some
cases, the change in signal is calculated as a percentage. In some
cases, the percentage of signal change is 0.01, 0.1, 0.2, 0.5, 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70,
75, 80, 85, 90, 95 or 100%. In some cases, the percentage of signal
change is about 0.01, 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100%.
In some cases, a change in signal is detected between the
fluorescence signals of a TC mismatch and a wild-type. In some
cases, the change in signal is calculated as a percentage. In some
cases, the percentage of signal change is 0.01, 0.1, 0.2, 0.5, 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70,
75, 80, 85, 90, 95 or 100%. In some cases, the percentage of signal
change is about 0.01, 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or
100%.
[0160] In some cases, a pair of primers is utilized to detect a
SNP. In some cases, the first primer comprises a sequence encoding
the SNP. In some cases, the first primer hybridizes to a region of
the analyte encoding the SNP. In some cases, the second primer
comprises a sequence not encoding the SNP. In some cases, the first
and second primers both comprise a sequence encoding the SNP. In
some cases, the second primer comprises a sequence complementary to
a region of the analyte not encoding the SNP. In some cases, the
first primer encodes a region on the analyte less than 500 base
pairs apart from a region encoded by the second primer. In some
cases, the first primer encodes a region on the analyte less than
490, 480, 470, 460, 450, 440, 430, 420, 410, 400, 390, 380, 370,
360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240,
230, 220, 210, 200, 195, 190, 185, 180, 175, 170, 165, 160, 155,
150, 145, 140, 135, 130, 125, 120, 115, 110, 105, 100, 99, 98, 97,
96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80,
79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63,
62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46,
45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29,
28, 27, 26, 25, 24, 23, 22, 21, 20 base pairs apart from a region
encoded by the second primer. In some cases, the first primer
encodes a region on the analyte no more than 490, 480, 470, 460,
450, 440, 430, 420, 410, 400, 390, 380, 370, 360, 350, 340, 330,
320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200,
195, 190, 185, 180, 175, 170, 165, 160, 155, 150, 145, 140, 135,
130, 125, 120, 115, 110, 105, 100, 99, 98, 97, 96, 95, 94, 93, 92,
91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75,
74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58,
57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41,
40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24,
23, 22, 21, 20 base pairs apart from a region encoded by the second
primer. In some cases, the first primer encodes a region on the
analyte about 490, 480, 470, 460, 450, 440, 430, 420, 410, 400,
390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, 270,
260, 250, 240, 230, 220, 210, 200, 195, 190, 185, 180, 175, 170,
165, 160, 155, 150, 145, 140, 135, 130, 125, 120, 115, 110, 105,
100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85,
84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68,
67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51,
50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34,
33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20 base pairs
apart from a region encoded by the second primer.
[0161] In some cases, the first primer is a forward primer and the
second primer is a reverse primer. In some cases, the forward
primer hybridizes to a region of the analyte encoding the SNP. In
some cases, the reverse primer comprises a sequence not encoding
the SNP. In some cases, the reverse primer comprises a sequence
complementary to a region of the analyte not encoding the SNP. In
some preferred cases, both the forward primer and the reverse
primer comprise a sequence encoding the SNP.
C. Supermulticolor Detection
[0162] Methods and systems and related compositions and kits herein
described can also be used for simultaneous identification of
multiple mutations in a sample such as a tissue sample. The
identification can be performed in some cases, in a so-called
FRETplexing method comprising allele-specific PCR assays using
FRET-chromophore-labeled primers with supercolor multiplexing
technology. The method produces fluorescence maps of the
distribution of multiple mutations during PCR amplification. In
several embodiments, the method provides results with specificity,
sensitivity and robustness that are increased with respect to
certain methods known in the art (e.g. certain methods using Taqman
probes identifiable by a skilled person). In some cases,
FRETplexing methods herein described allow identification of
multiple genetic variations, such as multiple mutations and
morphological information at reduced cost if compared to other
approaches identifiable by a skilled person.
[0163] The term "supercolor multiplexing" as used herein refers to
methods for multiplexed detection of a plurality of analytes in a
sample described in U.S. Pat. No. 8,838,394. In the supercolor
multiplexing method using one or more colors each color at
different intensities of fluorescence input. Accordingly, multiple
reaction are coded with one output of 1 unit per reaction in
end-point measurement by calibration of starting chromophore
amounts in a base-2 geometric progression, e.g. 1, 2, 4, 8, 16, 32,
etc., which results in unambiguous decoding of the present analytes
in any combination of occurrence. For example, for a two reaction
detection with a same primer pair an output of 3 indicates that
both reactions worked, as 1+2=3. In a same scenario an output of 1
indicates that the first reaction worked but not the second; an
output 2 indicates that the second reaction worked but not the
first. In some cases, the plurality of analytes can be detected in
a single sample volume by acquiring a cumulative measurement or
measurements of at least one quantifiable component of a
signal.
[0164] In an exemplary embodiments in which detection of 3 SNPs is
desired, in methods, and kits herein described a same FRET pair
labeled with a FRET donor-acceptor chromophore pair (e.g. Cy3 on
the forward primer and Cy5 on the reverse primer) can be used. As
primers extend and hybridize, an end-point will be detected for the
related FRET signals, which is cumulative of the FRET end-point
signals for all reactions where successful binding occurred.
Accordingly, a detection of an endpoint 4 will indicate that only
the third SNP is present, a detection of a 5 endpoint will indicate
that 1.sup.st and 3.sup.rd SNP are present as 5=1+4. Each signal
can be used to construct a coding scheme which can then be used to
obtain information concerning the presence or absence of each
analyte of the plurality of analytes or to characterize features
related to each of the plurality of analytes. In some cases, the
each signal can be controlled by attaching fluorophores of
different colors to one or more oligonucleotide primers or probes.
In some cases, the each signal can be controlled by attaching
fluorophores of a single color to one or more oligonucleotide
primers or probes and varying intensities within that color for
different oligonucleotide primers or probes. By utilizing
combinations of different colors and intensities, any number of
analytes can be coded. Detailed description with regard to
encoding, analysis and decoding used in supercolor multiplexing
methods can be referred to U.S. Pat. No. 8,838,394, which is
incorporated by reference in its entirety.
[0165] In some cases, the intensities of the color for different
oligonucleotide primers or probes can be controlled by varying the
concentration of the primer pair. For example, for obtaining an
intensity level at 1.times., the concentration of a first primer
pair can be selected at 2 uL of 10 uM according to Example 9; the
concentration of a second primer pair can be selected at 4 uL of 10
uM (2.times.); the concentration of a third primer pair can be
selected at 8 uL of 10 uM (4.times.), and so on according to the
general concepts of the supercolor multiplexing methods described
above. PCR experiments can be performed for a number of multiplexed
assays containing one or more mutations with one or more
corresponding primer pairs at the selected primer concentrations.
For each multiplexed assay, a real-time PCR curve can be generated
with the x-axis being the number of PCR cycles and the y-axis being
the measured emission intensity of the multiplexed assay in A.U,
such as the graph shown in FIG. 8 for single mutation 216T with the
primer concentration at 1.times.(2 uL of 10 uM) according to
Example 8. A Delta value from each of such PCR amplifications can
be obtained by subtracting the maximum fluorescence signal recorded
at the end of the PCR cycles and the minimum fluorescence signal
recorded at the beginning of the PCR cycles. The delta values can
then be used to chart the relatively output intensity changes that
correspond to that specific mutation or mixture of various
mutations. The plotted chart having an x-axis being the accumulated
intensity level calculated from various combinations of the primer
concentration and the y-axis being the measured emission intensity
of the multiplexed assay in A.U. can then be used as a look-up
table for determining the presence or absence of any particular
mutation in a given sample containing a plurality of analytes
and/or characterizing the sample to obtain additional information
such as which mutation/mutations are present or absent in the
sample.
IV. Characterization of Analytes
[0166] In some embodiments, methods of detection herein described
can be used to characterize one or more analytes. In particular, in
some cases characterization of at least one polynucleotide analyte
can be performed using the FRET based methods and systems herein
described and related kits and compositions.
[0167] The related method comprises selecting at least one
polynucleotide region within the at least one polynucleotide
analyte, the at least one polynucleotide region having at least one
feature affecting biological or chemical characteristics of the at
least one polynucleotide analyte. The method also comprises
selecting at least one pair of primers attaching a FRET chromophore
donor-acceptor primer pairs herein described, having a forward
FRET-chromophore-labeled primer and a reverse
FRET-chromophore-labeled primer specific for a first target
polynucleotide and a second target polynucleotide within the at
least one polynucleotide region.
[0168] The method further comprises performing at least one
polynucleotide amplification reaction with the at least one pair
primers to detect a variation and in particular a genetic variation
in the region of interest. The method also comprises detecting a
FRET signal from the sample generated the first FRET chromophore
and the second FRET chromophore following the performing.
[0169] In some cases, each analyte of the at least one
polynucleotide analyte is encoded by a color and intensity
combination. Alternatively, each analyte of the at least one
polynucleotide analyte is encoded by a single color with varying
intensity. In some cases, the at least one pair of primers are
labeled with an identical FRET donor-acceptor chromophore pair but
with different intensity levels. The intensity levels can be varied
by adjusting the relative concentrations and/or volumes of primers
when comparing two constituent assays that form a multiplexed
assay. Typically, the forward and reverse primer of each primer
pair are at a same concentration and/or volume to ensure efficient
hybridization and maximal FRET signal. For example, to code for
analytes A and B using specific primers FA, RA, FB, RB, where
F=forward, R=reverse, concentrations C.sub.(FA)=C.sub.(RA) and
C.sub.(FB)=C.sub.(RB) are set up in a way so that
C.sub.(FA):C.sub.(FB)=1:2. Then if an accumulated concentration or
FRET signal intensity level is 3.times., both analytes A and B are
present. If an accumulated concentration of FRET signal intensity
level is 2.times., only analyte B is present. If an accumulated
concentration of FRET signal intensity level is 1.times., only
analyte A is present. For example, in FIG. 15 of Example 11, the
relative FRET signal intensities obtained from qPCR experiments are
plotted as a function of accumulated primer concentrations levels.
The intensity level corresponding to the 1.times. primer
concentration is 0.25 and the 2.times. primer concentration yields
an intensity twice that of the 1.times. primer concentration, which
is an intensity of 0.5. When the 1.times. and 2.times. primer
concentrations are combined, a total FRET signal intensity of 0.75
is observed for the 3.times. concentration.
[0170] In some cases, quantitative information about the analytes
can be extracted by comparing the threshold cycles of the resulting
FRET signal real-time PCR curve with the threshold cycles of an
included positive control, in analogy with traditional real-time
qPCR. The signal multiplicity above described can be generally
orthogonal information that is statistically independent of the
information given by the threshold cycles. Thus in those cases both
pieces of information can be extracted from the FRET signal
signature profile for quantitative analysis purposes.
V. Analytes
[0171] An analyte may be any suitable polynucleotide analyte that
can be analyzed using the methods and compositions of the present
disclosure, where the analyte is capable of interacting with a
reagent (e.g., an oligonucleotide such as a primer or probe
attached to a chromophore) in order to generate a signal that can
be measured. An analyte may be naturally-occurring or synthetic. An
analyte may be present in a sample obtained using any methods known
in the art. In some cases, a sample may be processed before
analyzing it for an analyte. The methods and compositions presented
in this disclosure may be used in solution phase assays, without
the need for particles (such as beads) or a solid support.
[0172] In some cases, an analyte may be a polynucleotide, such as
DNA, RNA, peptide nucleic acids, and any hybrid thereof, where the
polynucleotide contains any combination of deoxyribo- and/or
ribo-nucleotides. Polynucleotides may be single stranded or double
stranded, or contain portions of both double stranded or single
stranded sequence. Polynucleotides may contain any combination of
nucleotides or bases, including, for example, uracil, adenine,
thymine, cytosine, guanine, inosine, xanthine, hypoxanthine,
isocytosine, isoguanine and any nucleotide derivative thereof. As
used herein, the term "nucleotide" may include nucleotides and
nucleosides, as well as nucleoside and nucleotide analogs, and
modified nucleotides, including both synthetic and naturally
occurring species. Polynucleotides may be any suitable
polynucleotide for which one or more reagents as described herein
may be produced, including but not limited to cDNA, mitochondrial
DNA (mtDNA), messenger RNA (mRNA), ribosomal RNA (rRNA), transfer
RNA (tRNA), nuclear RNA (nRNA), small interfering RNA (siRNA),
small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), small
Cajal body-specific RNA (scaRNA), microRNA (miRNA), double stranded
(dsRNA), ribozyme, riboswitch or viral RNA. Polynucleotides may be
contained within any suitable vector, such as a plasmid, cosmid,
fragment, chromosome, or genome.
[0173] Genomic DNA may be obtained from naturally occurring or
genetically modified organisms or from artificially or
synthetically created genomes. Analytes comprising genomic DNA may
be obtained from any source and using any methods known in the art.
For example, genomic DNA may be isolated with or without
amplification. Amplification may include PCR amplification,
multiple displacement amplification (MDA), rolling circle
amplification and other amplification methods. Genomic DNA may also
be obtained by cloning or recombinant methods, such as those
involving plasmids and artificial chromosomes or other conventional
methods (see Sambrook and Russell, Molecular Cloning: A Laboratory
Manual., cited supra.) Polynucleotides may be isolated using other
methods known in the art, for example as disclosed in Genome
Analysis: A Laboratory Manual Series (Vols. I-IV) or Molecular
Cloning: A Laboratory Manual. If the isolated polynucleotide is an
mRNA, it may be reverse transcribed into cDNA using conventional
techniques, as described in Sambrook and Russell, Molecular
Cloning: A Laboratory Manual., cited supra.
[0174] In some embodiments, detection performed on one or more
polynucleotide analytes can be indicative of features of another
analyte associated therefore. Such other analyte may be a protein,
polypeptide, lipid, carbohydrate, sugar, small molecule, or any
other suitable molecule that can be detected through detection of
polynucleotide analytes performed with the methods and compositions
provided herein. An analyte may be an enzyme or other protein. An
analyte may be a drug or metabolite (e.g. anti-cancer drug,
chemotherapeutic drug, anti-viral drug, antibiotic drug, or
biologic). An analyte may be any molecule, such as a co-factor,
receptor, receptor ligand, hormone, cytokine, blood factor,
antigen, steroid, or antibody.
[0175] An analyte may be any molecule from any pathogen, such as a
virus, bacteria, parasite, fungus, or prion (e.g., PrP.sup.Sc).
Exemplary viruses include those from the families Adenoviridae,
Flaviviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae,
Papovaviridae, Paramyxoviridae, Picornaviridae, Polyomavirus,
Retroviridae, Rhabdoviridae, and Togaviridae. Specific examples of
viruses include adenovirus, astrovirus, bocavirus, BK virus,
coxsackievirus, cytomegalovirus, dengue virus, Ebola virus,
enterovirus, Epstein-Barr virus, feline leukemia virus, hepatitis
virus, hepatitis A virus, hepatitis B virus, hepatitis C virus,
hepatitis D virus, hepatitis E virus, herpes simplex virus (HSV),
HSV type 1, HSV type 2, human immunodeficiency virus (HIV), HIV
type 1, HIV type 2, human papilloma virus (HPV), HPV type 1, HPV
type 2, HPV type 3, HPV type 4, HPV type 6, HPV type 10, HPV type
11, HPV type 16, HPV type 18, HPV type 26, HPV type 27, HPV type
28, HPV type 29, HPV type 30, HPV type 31, HPV type 33, HPV type
34, HPV type 35, HPV type 39, HPV type 40, HPV type 41, HPV type
42, HPV type 43, HPV type 44, HPV type 45, HPV type 49, HPV type
51, HPV type 52, HPV type 54, HPV type 55, HPV type 56, HPV type
57, HPV type 58, HPV type 59, HPV type 68, HPV type 69, influenza
type A virus, influenza type B virus, JC virus, Marburg virus,
measles virus, metapneumovirus, mumps virus, Norwalk virus,
parovirus, polio virus, rabies virus, respiratory syncytial virus
including type A and type B, retrovirus, rhinovirus, rotavirus,
Rubella virus, smallpox virus, vaccinia virus, West Nile virus,
yellow fever virus, and human parainfluenza virus type 3.
[0176] Exemplary bacteria include those from the genuses
Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia,
Clostridium, Corynebacterium, Enterococcus, Escherichia,
Francisella, Haemophilus, Helicobacter, Legionella, Leptospira,
Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas,
Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus,
Treponema, Vibrio, and Yersinia. Specific examples of bacteria
include Bordetella parapertussis, Bordetella pertussis, Borrelia
burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis,
Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae,
Chlamydia psittaci, Chlamydia trachomatix, Clostridium botulinum,
Clostridium difficile, Clostridium perfringens, Clostridium tetani,
Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus
faecium, Escherichia coli, Francisella tularensis, Haemophilus
influenzae, Helicobacter pylori, Legionella pneumophila, Leptospira
interrogans, Listeria monocytogenes, Mycobacterium leprae,
Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma
pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis,
Pseudomonas aeruginosa, Rickettsia rickettsii, Salmonella
choleraesuis, Salmonella dublin, Salmonella enteritidis, Salmonella
typhi, Salmonella typhimurium, Shigella sonnei, Staphylococcus
aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus,
Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus
pyogenes, Treponema pallidum, Vibrio cholerae, Yersinia pestis, and
Yersinia enterocolitica.
[0177] Exemplary parasites include those from the genuses
Acanthamoeba, Babesia, Balamuthia, Balantidium, Blasocystis,
Cryptosporidium, Dientamoeba, Entamoeba, Giardia, Isospora,
Leishmania, Naegleria, Pediculus, Plasmodium, Rhinosporidium,
Sarcocystis, Schistosoma, Toxoplasma, Trichomonas, and Trypanosoma.
Specific examples of parasites include Babesia divergens, Babesia
bigemina, Babesia equi, Babesia microfti, Babesia duncani,
Balamuthia mandrillaris, Balantidium coli, Dientamoeba fragilis,
Entamoeba histolytica, Giardia lamblia, Isospora belli, Naegleria
fowleri, Pediculus humanus, Plasmodium falciparum, Plasmodium
knowlesi, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax,
Rhinosporidium seeberi, Sarcocystis bovihominis, Sarcocystis
suihominis, Schistosoma mansoni, Toxoplasma gondii, Trichomonas
vaginalis, Trypanosoma brucei, and Trypansoma cruzi.
[0178] Exemplary fungi include those from the genuses
Apophysomyces, Aspergillus, Blastomyces, Candida, Cladosporium,
Coddidioides, Cryptococcos, Exserohilum, Fusarium, Histoplasma,
Pichia, Pneumocystis, Saccharomyces, Sporothrix, Stachybotrys, and
Trichophyton. Specific examples of fungi include Aspergillus
fumigatus, Aspergillus flavus, Aspergillus clavatus, Blastomyces
dermatitidis, Candida albicans, Coccidioides immitis, Crytptococcus
neoformans, Exserohilum rostratum, Fusarium verticillioides,
Histoplasma capsulatum, Pneumocystis jirovecii, Sporothrix
schenckii, Stachybotrys chartarum, and Trichophyton
mentagrophytes.
[0179] In some cases, an analyte may be any molecule derived from
an archaea. Exemplary archaea include those from the genuses
Acidilobus, Acidococcus, Aeropyrum, Archaeoglobus, Caldisphaera,
Caldococcus, Cenarchaeum, Desulfurococcus, Geogemma, Geoglubus,
Haladaptatus, Halomicrobium, Hyperthermus, Ignicoccus, Ignisphaera,
Methanobacterium, Natronococcus, Nitrosopumilus, Picrophilus,
Pyrodictium, Pyrolobus, Staphylothermus, Stetteria,
Sulfophobococcus, Thermodiscus, Thermosphaera and Thermoplasma.
Specific examples of archea include A. aceticus, A. camini, A.
fulgidus, A. infectus, A. lithotrophicus, A. pernix, A. profundus,
A. veneficus, A. saccharovorans, A. sulfurreducens, C. dracosis, C.
lagunensis, C. noboribetus, C. symbiosum, D. amylolyticus, D.
fermentans, D. mobilis, D. mucosus, G. barossii, G. indica, G.
pacifica, H. butylicus, N. maritimus, G. ahangari, H.
paucihalophilus, H. mukohataei, H. katesii, H. zhouii, I.
aggregans, I. islandicus, I. pacificus, I. hospitalis, M.
aarhusense, M. alcaliphilum, M. beijingense, M. bryantii, M.
congolense, M. curvum, M. espanolae, M. formicicum, M. ivanovii, M.
oryzae, M. palustre, M. subterraneum, M. thermaggregans, M.
uliginosum, N. amylolyticus, N. jeotgali, N. occultus, P. abyssi,
P. brockii, P. occultum, P. fumarii, P. oshimae, P. torridus, S.
hellenicus, S. marinus, S. hydrogenophila, S. zilligii, T.
maritimus, T. aggregans, T. acidophilum, T. sp. P61, T. sp. S01, T.
sp. S02, T. sp. XT101, T. sp. XT102, T. sp. XT103, T. sp. XT107 and
T. volcanium.
[0180] In some cases, an analyte may be any molecule derived from a
mammal. In some cases, the mammal is a human, a non-human primate,
mouse, rat, rabbit, goat, dog, cat, or cow. In some embodiments,
the mammal is a human. In some cases, a human is a patient.
[0181] In some cases, an analyte may be any molecule derived from a
plant. In some cases, a plant is any of various photosynthetic,
eukaryotic, multicellular organisms of the kingdom Plantae
characteristically producing embryos, containing chloroplasts,
having cellulose cell walls, and lacking the power of
locomotion.
[0182] In some cases, the methods provided in this disclosure may
be used to detect any one of the analytes described above, or
elsewhere in the specification. In some cases the methods provided
in this disclosure may be used to detect panels of the analytes
described above, or elsewhere in the specification. For example, a
panel may comprise an analyte selected from the group consisting of
any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70,
80, 90, 100, 500, 1000, 5000, 10000 or more analytes described
above or elsewhere in the specification.
[0183] An analyte may be obtained from any suitable location,
including from organisms, whole cells, cell preparations and
cell-free compositions from any organism, tissue, cell, or
environment. Analytes may be obtained from environmental samples,
forensic samples, biopsies, aspirates, formalin fixed embedded
tissues, air, agricultural samples, soil samples, petroleum
samples, water samples, or dust samples. In some instances, an
analyte may be obtained from bodily fluids which may include blood,
urine, feces, serum, lymph, saliva, mucosal secretions,
perspiration, central nervous system fluid, vaginal fluid, or
semen. Analytes may also be obtained from manufactured products,
such as cosmetics, foods, personal care products, and the like.
Analytes may be the products of experimental manipulation
including, recombinant cloning, polynucleotide amplification,
polymerase chain reaction (PCR) amplification, isothermal
amplification, purification methods (such as purification of
genomic DNA or RNA), and synthesis reactions.
[0184] More than one type of analyte may be detected in each
multiplexed assay. For example, a polynucleotide, a protein, a
polypeptide, a lipid, a carbohydrate, a sugar, a small molecule, or
any other suitable molecule may be detected simultaneously in the
same multiplexed assay with the use of suitable reagents. Any
combination of analytes may be detected at the same time.
[0185] Detection of an analyte may be useful for any suitable
application, including research, clinical, diagnostic, prognostic,
forensic, and monitoring applications. Exemplary applications
include detection of hereditary diseases, identification of genetic
fingerprints, diagnosis of infectious diseases, cloning of genes,
paternity testing, criminal identification, phylogeny,
anti-bioterrorism, environmental surveillance, and DNA computing.
For example, an analyte may be indicative of a disease or
condition. An analyte may be used to make a treatment decision, or
to assess the state of a disease. The presence of an analyte may
indicate an infection with a particular pathogen, or any other
disease, such as cancer, autoimmune disease, cardiorespiratory
disease, liver disease, digestive disease, and so on. The methods
provided herein may thus be used to make a diagnosis and to make a
clinical decision based on that diagnosis. For example, a result
that indicates the presence of a bacterial polynucleotide in a
sample taken from a subject may lead to the treatment of the
subject with an antibiotic.
[0186] In some cases the methods and compositions of the present
disclosure may be used to detect at least 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300,
350, 400, 450, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000,
5000, 6000, 7000, 8000, 9000, 10,000, 100,000 or more analytes. In
some cases the methods and compositions of the present disclosure
may be used to detect about 1-10,000, 1-1000, 1-100, 1-50, 1-40,
1-30, 1-20, 1-10, or 1-5 analytes.
[0187] In some cases, this disclosure provides assays that are
capable of unambiguously detecting the presence or absence of each
of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60,
65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170,
180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900,
1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000 or
100,000 analytes, in any combination of presence or absence, in a
single sample volume. In some cases, this disclosure provides
assays that are capable of unambiguously detecting the presence or
absence of each of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,
47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120,
130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450,
500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000,
8000, 9000, 10,000 or 100,000 analytes, in any combination of
presence or absence, in a single sample volume. In some cases, this
disclosure provides assays that are capable of unambiguously
detecting the presence or absence of less than 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90,
95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250,
300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 2000, 3000,
4000, 5000, 6000, 7000, 8000, 9000, 10,000 or 100,000 analytes, in
any combination of presence or absence, in a single sample volume.
In all of the above cases, in addition or in the alternative to
detection of presence or absence of one or more analytes,
quantitative detection of one or more analytes can also be
performed as will be understood by a skilled person upon reading of
the disclosure
A. Distance/Length of polynucleotide analytes
[0188] In one aspect, the methods provided herein may be used to
detect polynucleotide analytes containing 1-500 base pairs (bp),
referred to as the "length" of the analytes. In some cases, the
methods provided herein may be used to detect polynucleotide
analytes containing 10-450 bp, 15-400 bp, 20-350 bp, 25-300 bp,
30-250 bp, 35-200 bp, or 40-190 bp. In some cases, the methods may
be used to detect a polynucleotide analyte containing 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,
60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,
77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,
94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,
108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,
121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,
134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,
147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159,
160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172,
173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,
186, 187, 188, 189, 190 or more base pairs. In some cases, the
methods may be used to detect polynucleotide analyte containing at
least 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,
55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,
89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,
104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116,
117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,
130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,
143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,
156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,
169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181,
182, 183, 184, 185, 186, 187, 188, 189 or 190 base pairs. In some
cases, the methods may be used to detect polynucleotide analyte
containing no more than 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,
51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,
68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,
85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,
101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,
114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,
127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,
140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152,
153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,
166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,
179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189 or 190 base
pairs.
B. Sensitivity
[0189] In some cases, the methods disclosed herein may be used to
detect polynucleotide analyte at concentrations of about 100 uM to
about 1 fM. In some cases, the methods provided herein may be used
to detect a polynucleotide analyte at concentrations of about 10
uM-20 fM, 1 uM-40 fM, 500 nM-60 fM, 100 nM-70 fM, 50 nM-80 fM, 30
nM-90 fM, 10 nM-100 fM. In some cases, the methods may be used to
detect a polynucleotide analyte at a concentration of 10 nM, 9 nM,
8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 950 pM, 900 pM, 850
pM, 800 pM, 750 pM, 700 pM, 650 pM, 600 pM, 550 pM, 500 pM, 450 pM,
400 pM, 350 pM, 300 pM, 250 pM, 200 pM, 180 pM, 160 pM, 140 pM, 120
pM, 100 pM, 95 pM, 90 pM, 85 pM, 80 pM, 75 pM, 70 pM, 65 pM, 60 pM,
55 pM, 50 pM, 45 pM, 40 pM, 35 pM, 30 pM, 25 pM, 20 pM, 18 pM, 16
pM, 14 pM, 12 pM, 10 pM, 8 pM, 6 pM, 4 pM, 2 pM, 1 pM, 900 fM, 800
fM, 700 fM, 600 fM, 500 fM, 400 fM, 300 fM, 200 fM, 100 fM, 50 fM,
10 fM, 1 fM, 100 aM, 10 aM, or 1 aM. In some cases, the methods may
be used to detect a polynucleotide analyte at a concentration of at
least 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM,
950 pM, 900 pM, 850 pM, 800 pM, 750 pM, 700 pM, 650 pM, 600 pM, 550
pM, 500 pM, 450 pM, 400 pM, 350 pM, 300 pM, 250 pM, 200 pM, 180 pM,
160 pM, 140 pM, 120 pM, 100 pM, 95 pM, 90 pM, 85 pM, 80 pM, 75 pM,
70 pM, 65 pM, 60 pM, 55 pM, 50 pM, 45 pM, 40 pM, 35 pM, 30 pM, 25
pM, 20 pM, 18 pM, 16 pM, 14 pM, 12 pM, 10 pM, 8 pM, 6 pM, 4 pM, 2
pM, 1 pM, 900 fM, 800 fM, 700 fM, 600 fM, 500 fM, 400 fM, 300 fM,
200 fM, 100 fM, 50 fM, 10 fM, 1 fM, 100 aM, 10 aM, or 1 aM. In some
cases, the methods may be used to detect a polynucleotide analyte
at a concentration of no more than 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5
nM, 4 nM, 3 nM, 2 nM, 1 nM, 950 pM, 900 pM, 850 pM, 800 pM, 750 pM,
700 pM, 650 pM, 600 pM, 550 pM, 500 pM, 450 pM, 400 pM, 350 pM, 300
pM, 250 pM, 200 pM, 180 pM, 160 pM, 140 pM, 120 pM, 100 pM, 95 pM,
90 pM, 85 pM, 80 pM, 75 pM, 70 pM, 65 pM, 60 pM, 55 pM, 50 pM, 45
pM, 40 pM, 35 pM, 30 pM, 25 pM, 20 pM, 18 pM, 16 pM, 14 pM, 12 pM,
10 pM, 8 pM, 6 pM, 4 pM, 2 pM, 1 pM, 900 fM, 800 fM, 700 fM, 600
fM, 500 fM, 400 fM, 300 fM, 200 fM, 100 fM, 50 fM, 10 fM, 1 fM, 100
aM, 10 aM, or 1 aM.
C. Specificity
[0190] In some methods provided herein, a primer pair may be
specific for one or a plurality of analytes. In some cases, a
primer pair is specific to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,
47, 48, 49, or 50 analytes. In some cases, a primer pair is
specific to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, or 50 analytes. In some cases, a primer pair is specific to
less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50
analytes. In some cases, a primer pair is specific to one analyte.
In some cases, a primer pair is universal to all analytes.
VI. Probes and Primers
[0191] Some of the methods provided in this disclosure utilize a
reagent (e.g. an oligonucleotide such as a primer or a probe that
is attached to a FRET chromophore) that can generate a signal in
the presence of an analyte. Any suitable reagent may be used with
the present disclosure. Generally, a reagent will have an
analyte-specific component and a component that generates a signal
in the presence of the analyte. In some cases, these reagents are
referred to as probes and primers. In some cases, the probes are
hybridization probes. In some cases, the hybridization probes are n
oligonucleotide probes attached to FRET chromophores. In some
cases, the probes are antibodies that detect an analyte, with a
FRET chromophore label of a pair of FRET chromophore labels that
emits signal upon binding of the antibody to an analyte presenting
the other FRET chromophore of a same FRET pair. In some cases, the
reagent is a primer. In some cases, the primer is attached to a
chromophore. In some cases, the primer is attached to a donor
chromophore. In some cases, the primer is attached to an acceptor
chromophore.
[0192] In particular, in various embodiments at least one pair of
FRET-chromophore-labeled reagents (e.g. primers), is designed so
that each pair of the at least one pair has a first
FRET-chromophore-labeled reagent (e.g. a forward
FRET-chromophore-labeled primer) attaching a first FRET chromophore
of a FRET donor-acceptor chromophore pair, and a second
FRET-chromophore-labeled reagent (e.g. a reverse
FRET-chromophore-labeled primer) attaching a second FRET
chromophore of the FRET donor-acceptor chromophore pair, the second
FRET chromophore being different from the first FRET chromophore.
In the embodiments herein described the first FRET chromophore and
the second FRET chromophore are selected in the pair in view of
their capability of providing an energy transfer from one to
another when located at a Forster distance one with respect to the
another.
[0193] In the embodiments herein described, the first
FRET-chromophore-labeled primer and second FRET-chromophore-labeled
primer are specific for a first and second target polynucleotide
respectively within the at least one polynucleotide analyte,
wherein the first target polynucleotide and the second target
polynucleotide are located within the at least one polynucleotide
analyte so that upon specific binding with the first
FRET-chromophore-labeled reagent and the second
FRET-chromophore-labeled reagent, the first FRET chromophore and
the second FRET chromophore are located within a distance up to
four times the Forster distance one with respect to the other. In
embodiments, where the first FRET-chromophore-labeled primer and
the second FRET-chromophore-labeled primer are a
FRET-chromophore-labeled forward and reverse primer, a forward
FRET-chromophore-labeled primer has a sequence specific for a first
target polynucleotide within the at least one polynucleotide
analyte and the reverse FRET-chromophore-labeled primer has a
sequence specific for a second target polynucleotide.
[0194] The methods of the present disclosure may use one or more
reagents (e.g., an oligonucleotide such as a primer or a probe that
is attached to a chromophore) to detect each analyte. For example,
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,
37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more
reagents may be used to detect of each analyte. In some cases, at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50
reagents may be used to detect of each analyte. In some cases,
fewer than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50
reagents may be used to detect of each analyte.
[0195] In some cases, a sample is contacted with 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more reagents to detect
of all analytes. In some cases, a sample is contacted with at least
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,
37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more
reagents to detect of all analytes. In some cases, a sample is
contacted with fewer than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, or 50 reagents to detect all analytes.
[0196] In all of the above cases, in addition or in the alternative
to detection of presence or absence of one or more analytes,
quantitative detection of one or more analytes can also be
performed as will be understood by a skilled person upon reading of
the disclosure.
[0197] In particular primers or other oligonucleotides are designed
to specifically bind to respective target sequences on at least one
polynucleotide analyte or region thereof to be targeted. The target
sequences for each pair of primers or primer/oligonucleotide
attaching a FRET donor-acceptor chromophore pair are selected on
the at least one polynucleotide or region therefore to be targeted
so that upon specific binding of the primer pair or primer/probe
pair with the corresponding target sequence, the primer pair or
primer/probe pair present the FRET donor-acceptor chromophore at a
distance up to four times their Forster distance one with respect
to the other.
[0198] General concepts for PCR primer design are well-known for a
person skilled in the art. In obtaining a balance between
specificity and efficiency of amplification, parameters that need
to be considered for designing primers include primer length,
reasonable GC content and melting temperature (T.sub.m) that
provide a sufficient thermal condition for efficient annealing, the
PCR product length, placement of the primers within the target
sequence, and many other factors identifiable to a skilled person
in the art. In particular, complementarity between primers should
be avoided as undesirable formation of primer-dimer may be formed
as a result of the amplification of the complementary primers
themselves. The selection of PCR primer sets can be performed
manually or using analytic computer software widely available to a
skilled person.
[0199] The FRET-chromophore-labeled primers are designed and
labeled in such a way that resulting amplicons can produce FRET
signal after annealing step. In cases when SNPs are the target of
interest, primes typically have their 3' end opposite to the
targeted point mutation for maximal specificity. In particular
[0200] As described above, primers attached to a FRET donor or a
FRET acceptor chromophore may be used to detect at least an analyte
in a polynucleotide amplification assay. The receptor can emit a
fluorescence signal generated upon excitation by the energy
released from the donor when the donor and acceptor are in close
proximity. The sequence of the primer can be designed to be
complementary to a polynucleotide sequence present in an analyte,
and the primer is capable of hybridizing to the analyte. The
sequence of the primer can also be designed to contain one or more
nucleotide variations in a polynucleotide sequence of an analyte,
and the primer is capable of hybridizing to the analyte. A donor
chromophore can be attached to the 5' end of one of a primer pair.
An acceptor chromophore can be attached to the 5' end of the second
primer of the primer pair. In some cases, one of the primer pair is
specific to the target sequence of the analyte containing one or
more nucleotide variations and the other primer is specific to the
wild-type of the analyte. In some cases, both primers are specific
to the target sequence of the analyte containing one or more
nucleotide variations. In some cases, the 3'-end basepairs of both
primers are specifically designed to match the SNP sequence in the
cases for SNP detection and characterization. Hybridization of the
primers may be performed in a nucleic acid amplification reaction
comprising donor/acceptor attached primers (e.g., a polymerase
chain reaction). Upon extension of the primers by a DNA polymerase,
the donor and acceptor are incorporated in the amplicon or
amplification product (e.g., an analyte). The incorporation of the
donor and the acceptor in the newly generated amplicon can lead to
signal generation (e.g., an increase of fluorescence emission
intensity from the receptor or a decrease of fluorescence emission
intensity from the donor). With each iterative amplification
reaction, the fluorescence intensity is changed by a factor having
a value in a range between 1 and 2, with a maximum value of 2. The
amount of fluorescence emission intensity detected can be used to
directly determine the amount of analyte present. If no analyte is
present, little or no emission will be observed.
[0201] The size of the generated amplicons can depend on the
specific application. In some cases, the generated amplicons that
lead to emission of fluorescence can have a length anywhere between
30-1000 bp. In some cases, the generated amplicons are preferably
within the range of 30-250 bp. In some cases, the generated
amplicons are preferably within the range of 40-60 bp for
generating a preferred signal intensity as FRET signal decrease
over distance. In some embodiments, the generated amplicons are
about 40 bp long.
[0202] In some cases, a sample to be analyzed is contacted with 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more pairs of
primers (e.g. a forward primer and a reverse primer). In some cases
a sample to be analyzed is contacted with at least 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more pairs of primers. In
some cases a sample to be analyzed is contacted with fewer than 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 pairs of
primers. In some cases, the number of pairs of primers is 2-10,
3-15, 4-20, 3-10, 4-10, 5-10, 6-8, or 6-10. In some cases, a sample
to be analyzed is contacted with 1 pair of primers.
[0203] In some cases, a sample may contain one or more analytes. In
some cases, one primer pair may be used to detect each analyte. In
some cases, a sample is contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,
44, 45, 46, 47, 48, 49, 50, 100, 500, 1000, 5000, 10000 or more
different pairs of primers with each primer pair detecting a single
analyte. In some cases, a sample is contacted with at least 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 500, 1000,
5000, 10000 or more different pairs of primers with each primer
pair detecting a single analyte. In some cases, a sample is
contacted with fewer than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 100, 500, 1000, 5000, 10000 or more different pairs of
primers with each primer pair detecting a single analyte. In some
cases, the number of pairs of primers is 2-10, 3-15, 4-20, 3-10,
4-10, 5-10, 6-8, or 6-10. In all of the above cases, in addition or
in the alternative to detection of presence or absence of one or
more analytes, quantitative detection of one or more analytes can
also be performed as will be understood by a skilled person upon
reading of the disclosure.
[0204] In some cases, primers may be specific for a particular
analyte and capable of amplifying a region complementary to a
probe. In some cases, the number of primers used is equivalent to
the number of probes. In other cases, the number of probes used may
exceed the number of primer used. In some cases, the number of
primers and probes is defined by a ratio. In some cases, the ratio
of primer to probe is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800,
900, or 1000. In some cases, the ratio of probe to primer is 0, 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90,
100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000.
[0205] The term "FRET-chromophore-labeled primer" refers to a
primer attached with either a FRET donor chromophore or a FRET
acceptor chromophore. A primer attached with a FRET donor
chromophore is referred to as a FRET-donor-labeled primer and a
primer attached with a FRET acceptor chromophore is referred to as
a FRET-acceptor-labeled primer.
[0206] The FRET chromophore labels of the present disclosure can be
attached to a primer at any location except for the 3' end. In some
cases, a single chromophore is attached to the primer at the 5'
end. In some cases, multiple chromophores are labeled to the primer
with at least one chromophore labeled at the 5' end. When a donor
chromophore is attached to the 5' end of one primer and an acceptor
chromophore is attached to the 5' end of the other primer, the
distance from the 5' end of one primer to the 5' end of the other
primer along the template sequence should be short enough to allow
efficient FRET between the donor and acceptor chromophores. In some
cases, chromophores are not directly attached to the primers.
Instead, the chromophores are attached to aptamers or other
synthetic recognition molecules that are subsequently attached to
the primers. Methods of chromophore labeling are well defined in
the art. See, e.g. Pesce et al, editors, Fluorescence Spectroscopy,
Marcel Dekker, New York, (1971); White et al. Fluorescence
Analysis: A Practical Approach, Marcel Dekker, New York, (1970);
and the like. Further, there is extensive guidance in the
literature for derivatizing donor and acceptor chromophore
molecules for covalent attachment via common reactive groups that
can be added to an oligonucleotide. See, e.g. U.S. Pat. Nos.
3,996,345; and 4,351,760. In examples that utilize chromophore
labels as described herein, any suitable labeling techniques
identifiable by a skilled person may be used.
[0207] Attachment of donors and acceptors to a primer may be
performed in the same reaction or in serial reactions. A series of
reactions may be performed to label probes with at least one donor
or acceptor chromophore and the reaction products may be mixed to
generate a mixture of probes with different donor or acceptor
chromophores.
[0208] In some cases, a single primer pair may be used for each
analyte. In order to utilize supercolor multiplexing, each primer
pair may be labeled with a same donor and acceptor chromophore pair
but at varied pre-determined concentration ratios for different
primer pairs. The concentration ratios ensure that the respective
FRET signals will produce intensities of respective predetermined
ratios as well. Then the measured intensity can be decoded to give
information about the present analyte(s), if any. A positive
control can be set up in the same way to produce a predetermined
intensity, e.g. 1.times.. Then the control becomes part of the same
measurement and coding scheme.
[0209] Although many aspects of the present disclosure are
exemplified using nucleic acid-based probes and primers, one of
ordinary skill in the art will readily recognize that other forms
of probes and primers would work equally well with the examples
described in this disclosure. For example, a binding molecule
specific to an analyte could be used as a probe. Non-limiting
exemplary binding molecules include an antibody recognizing an
analyte, and generating a signal in the presence of an analyte.
VII. Chromophores
[0210] Chromophores are molecules capable of selective light
absorption resulting in the coloration of these molecule containing
compounds. The color arises when a molecule at an excited state
releases energy in the form of light with a defined spectrum. When
two chromophores are selected such that the emission spectrum of
one (e.g. donor) overlaps the excitation spectrum of the other
(e.g. acceptor), the two chromophores can form a FRET pair. A FRET
pair of chromophores when positioned at a Forster distance of each
other can produce FRET signal with 50% energy transfer efficiency
when the donor is excited optically. Exemplary FRET-based
chromophores include, but are not limited to, a fluorochrome, a
non-fluorochrome chromophore, an absorption chromophore, a
fluorophore, any organic or inorganic dye, metal chelate, or any
fluorescent enzyme substrate. In some cases, the chromophore is a
fluorochrome. In some cases, the fluorochrome is a fluorophore.
[0211] Several chromophores are described in the art, e.g. Berlman,
Handbook of Fluorescence Spectra of Aromatic Molecules, 2nd
Edition, Academic Press, New York, (1971). In examples that utilize
fluorescent labels as described herein, any suitable fluorescent
label may be used.
[0212] Exemplary FRET donors suitable for use with the present
disclosure includes rhodamine, rhodol, fluorescein,
thiofluorescein, aminofluorescein, carboxyfluorescein,
chlorofluorescein, methylfluorescein, sulfofluorescein,
aminorhodol, carboxyrhodol, chlororhodol, methylrhodol,
sulforhodol; aminorhodamine, carboxyrhodamine, chlororhodamine,
methylrhodamine, sulforhodamine, and thiorhodamine; cyanine,
indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine,
cyanine 2, cyanine 3, cyanine 3.5, cyanine 5, cyanine 5.5, cyanine
7, oxadiazole derivatives, pyridyloxazole, nitrobenzoxadiazole,
benzoxadiazole, pyrene derivatives, cascade blue, oxazine
derivatives, Nile red, Nile blue, cresyl violet, oxazine 170,
acridine derivatives, proflavin, acridine orange, acridine yellow,
arylmethine derivatives, auramine, crystal violet, malachite green,
tetrapyrrole derivatives, porphin, phtalocyanine, and bilirubin;
1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalene
sulfonate, 2-p-touidinyl-6-naphthalene sulfonate,
3-phenyl-7-isocyanatocoumarin,
N-(p-(2-benzoxazolyl)phenyl)maleimide, stilbenes, pyrenes, and the
like.
[0213] Exemplary FRET acceptors suitable for use with the present
disclosure include Cy5, HEX, ROX, TMR, YFP, and RFP.
[0214] The donor/acceptor chromophores that may be used with the
disclosure are not limited to any of the donor/acceptor
chromophores described herein. For example, donor/acceptor
chromophores with improved properties are continually developed,
and these donor/acceptor chromophores could readily be used with
the methods provided in this disclosure. Such improved
donor/acceptor chromophores include quantum dots, which may emit
energy at different wavelengths after being excited at a single
wavelength.
A. Chromophore Combinations
[0215] In some cases, a plurality of chromophores is labeled on a
probe. In some cases, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50 or more chromophores are labeled on a probe. In some
cases, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50
or more chromophores are labeled on a probe. In some cases, less
than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50
chromophores are labeled on a probe. In some cases, one chromophore
is labeled on a probe.
[0216] In some cases, the probe comprises a primer. In some cases,
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,
37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more
chromospheres is labeled on a primer. In some cases, about 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more
chromospheres is labeled on a primer. In some cases, less than 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 chromospheres
is labeled on a primer. In some cases, one chromophore is labeled
on a primer. In some cases, one chromophore is labeled at the 5'
end of a primer. In some cases, a plurality of chromophores is
labeled on a primer with at least one chromophore labeled at its 5'
end.
[0217] In some cases, a plurality of FRET donor chromophores and
FRET acceptor chromophores are labeled on a probe. In some cases,
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,
37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more
donors are labeled on a probe. In some cases, about 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more donors are labeled
on a probe. In some cases, less than 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, or 50 donors are labeled on a probe. In some
cases, one donor is labeled on a probe. In some cases, 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more acceptors are
labeled on a probe. In some cases, about 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,
44, 45, 46, 47, 48, 49, 50 or more acceptors is labeled on a probe.
In some cases, less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49 or 50 acceptors are labeled on a probe. In some cases, one
acceptor is labeled on a probe.
[0218] In some cases, a combination of FRET donor and acceptor
chromophores are labeled on a probe. In some cases, the number of
donors and acceptors on a probe is defined by a ratio. In some
cases, the ratio of donor to acceptor is 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,
44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 200, 300, 400,
500, 600, 700, 800, 900, or 1000. In some cases, the ratio of donor
to acceptor is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800,
900, or 1000. In some cases, the ratio of acceptor to donor is 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90,
100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000. In some
cases, the ratio of acceptor to donor is about 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 200, 300,
400, 500, 600, 700, 800, 900, or 1000.
[0219] In some cases, a plurality of donors and acceptors is
labeled on a primer. In some cases, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, 50 or more donors are labeled on a primer. In
some cases, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, 50 or more donors are labeled on a primer. In some cases, a
primer is labeled with one donor is labeled on a primer. In some
cases, less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,
or 50 donors are labeled on a primer. In some cases, one donor is
labeled at the 5' end of a primer. In some cases, a plurality of
donors re labeled on a primer with at least one donor labeled at
its 5' end. In some cases, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,
47, 48, 49, 50 or more acceptors are labeled on a primer. In some
cases, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50
or more acceptors are labeled on a primer. In some cases, less than
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 acceptors are
labeled on a primer. In some cases, one acceptor is labeled on a
primer. In some cases, one acceptor is labeled at the 5' end of a
primer. In some cases, a plurality of acceptors is labeled on a
primer with at least one acceptor labeled at its 5' end.
[0220] In some cases, a combination of donors and acceptors are
labeled on a primer. In some cases, the number of donors and
acceptors on a primer is defined by a ratio. In some cases, the
ratio of donor to acceptor is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600,
700, 800, 900, or 1000. In some cases, the ratio of acceptor to
donor is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60,
70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or
1000.
[0221] In some cases, multiple donors are paired with one acceptor.
In some cases 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,
50 or more donors are paired with one acceptor. In some cases,
about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or
more donors are paired with one acceptor. In some cases, less than
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 donors are
paired with one acceptor. In some cases, multiple donor and
acceptor pairs are used. In some cases, 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,
44, 45, 46, 47, 48, 49, 50 or more donor and acceptor pairs are
used. In some cases, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,
47, 48, 49, 50 or more donor and acceptor pairs are used. In some
cases, less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,
50 or more donor and acceptor pairs are used. In some cases, 1
donor and acceptor pair is used.
[0222] In some cases, a pair of primers comprises a first primer
and a second primer. In some cases, multiple donors are labeled on
a first primer and one acceptor is labeled on a second primer. In
some cases, at least one donor is labeled on the 5' end of the
first primer. In some cases, the acceptor is labeled on the 5' end
of the second primer. In some cases, multiple donors are labeled on
a second primer and one acceptor is labeled on a first primer. In
some cases, at least one donor is labeled on the 5' end of the
second primer. In some cases, the acceptor is labeled on the 5' end
of the first primer.
[0223] In some cases, the first primer is a forward primer and the
second primer is a reverse primer. The term "FRET-labeled forward
primer" is referred to as a forward primer attached with one
chromophore of an at least one FRET donor-acceptor chromophore
pair. The term "FRET-labeled reverse primer" is referred to as a
reverse primer attached with the other chromophore of the at least
one FRET donor-acceptor chromophore pair.
[0224] In some cases, multiple donors are labeled on the forward
primer and one acceptor is labeled on the reverse primer. In some
cases, at least one donor is labeled on the 5' end of the forward
primer. In some cases, the acceptor is labeled on the 5' end of the
reverse primer. In some cases, multiple donors are labeled on a
reverse primer and one acceptor is labeled on a forward primer. In
some cases, at least one donor is labeled on the 5' end of the
reverse primer. In some cases, the acceptor is labeled on the 5'
end of the forward primer.
[0225] The skilled artisan will realize that the advantages of the
present disclosed probes or primers may be retained while modifying
various aspects of its structure. For example, but not by way of
limitation, the number of donor/acceptor pairs may be modified. The
addition of more donor/acceptor pairs to the probe or primer is
expected to increase the amount of total fluorescence observable
prior to initiation of amplification reaction. There is no upper
limit to the number of donor/acceptor pairs that may be added to
the probe or primer. In one example, the number of donor/acceptor
pairs is at least two. In other examples, the detector contains at
least three or more donor/acceptor pairs. In some examples, the
detector may contain at least 10, 20, 30, or 50 pairs, or it may
contain hundreds of donor/acceptor pairs, as needed to produce, for
example, an optimal signal-to-noise ratio and assay
sensitivity.
[0226] In some cases, the methods provided in this disclosure may
include the use of donor/acceptor pair as a control. The control
donor/acceptor pair may be attached to one or more detector pairs
binding a positive control analyte, and each analyte to be
detected, in a sample. If the same sequence occurs in the positive
control analyte and each analyte to be detected, a single control
primer pair may be used. If the same sequence does not occur in the
positive control analyte and each analyte to be detected, different
primer pairs may be used, but each primer pair may still be
attached to the control donor/acceptor pair.
[0227] For example, building on the methods described above, one
donor/acceptor pair may be used to encode the presence of a control
analyte that is always present in the sample. The control analyte
may be added to the sample, or may be inherently present in the
sample. Additional donor/acceptor pairs may be used to encode the
presence of additional analytes.
B. Signals
[0228] Disclosed herein is a method of utilizing the signal
intensity to detect an analyte. In some cases, the signal is an
increase in intensity. In some cases, the signal is a decay in
intensity. In some cases, a signature profile is generated based on
the changes in intensity at a specific distance. In some cases,
once the length of an analyte is known, additional information can
be extrapolated. In some cases, additional information includes the
molecular weight of an analyte.
[0229] In some cases, the methods presented in this disclosure may
be used with any quantifiable signal. As described herein and else
wherein, a coding scheme may be utilized to indicate a multiplicity
of signal intensity without consideration of color. In some cases,
the coding scheme is equally applicable to any other method
providing a quantifiable signal, including an electrochemical
signal and a chemiluminescent signal.
[0230] The methods presented in this disclosure may also utilize
the measurement of a signal in at least two dimensions, also
referred to as the measurement of at least two components of a
signal. In some cases, the utilization of at least two components
of a signal (e.g., color and intensity) allows the generation of
more unique codes per unit of signal intensity bandwidth.
[0231] In some cases, a quantifiable signal comprises a waveform
that has both a frequency (wavelength) and amplitude (intensity). A
signal may be an electromagnetic signal. An electromagnetic signal
may be a sound, a radio signal, a microwave signal, an infrared
signal, a visible light signal, an ultraviolet light signal, an
x-ray signal, or a gamma-ray signal. In some cases, an
electromagnetic signal may be a fluorescent signal, for example a
fluorescence emission spectrum that may be characterized in terms
of wavelength and intensity.
[0232] In certain portions of this disclosure, the signal is
described and exemplified in terms of a fluorescent signal. This is
not meant to be limiting, and one of ordinary skill in the art will
readily recognize that the principles applicable to the measurement
of a fluorescent signal are also applicable to other signals. For
example, like fluorescent signals, any of the electromagnetic
signals may also be characterized in terms of a wavelength and
intensity. The wavelength of a fluorescent signal may also be
described in terms of color. The color may be determined based on
measuring intensity at a particular wavelength or range of
wavelengths, for example by determining a distribution of
fluorescent intensity at different wavelengths and/or by utilizing
a band pass filter to determine the fluorescence intensity within a
particular range of wavelengths. Such band pass filters are
commonly employed in a variety of laboratory instrumentation,
including quantitative PCR machines. Intensity may be measured with
a photodetector. A range of wavelengths may be referred to as a
"channel."
[0233] In cases when fluorophores are used as FRET-based
chromophore, the FRET signal can be in a range between 480 and 700
nm. The FRET signal intensity generated by FRET fluorophores can
vary largely from a single photon to any level of intensity.
[0234] In some cases, more than two components of a signal may be
measured. For example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 18, 20 or more components of a signal may be measured.
At least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 18, or 20 components of a signal may be measured. At least 2,
but fewer than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 18, or 20 components of a signal may be measured. In some
cases, 2-3, 2-4, 2-5, 2-6, 3-5, 3-6, 3-8, or 5-10 components of a
signal may be measured. These additional components may include
kinetic components, such as a rate of signal decay and rate of
photobleaching.
[0235] Signals can be measured and compared at various points
during a detection method described herein. For example, during an
amplification reaction (e.g., a PCR reaction), pairwise signals can
be measured. In some cases, a signal can be measured: before
annealing and after annealing of the primers to the template (e.g.,
analyte); before and after denaturing the double stranded template;
and before the annealing step and after the denaturing step.
Generally, a fluorescence signal in one or multiple segments of the
spectrum can be detected and correlated with the current
temperature of the sample and phase of the reaction, to generate a
signature. A signal signature as described herein can be generated
using these measurements.
C. Signature Profiles
[0236] A signature profile typically comprises a plurality of
signals. In some cases, a signal includes an electrochemical
signal, a chemiluminescence signal and a fluorescence signal. In
some cases, a signature profile contains a plurality of
fluorescence signals. In some cases, a profile curve is generated
from the plurality of florescence signals. In some cases, a
signature profile contains an initial fluorescence signal and an
end-point fluorescence signal. In some cases, a fluorescence signal
is influenced by external factors. In some cases, the external
factors include temperature, pH, organic and inorganic agents (e.g.
salts, urea, DMSO) and addition or removal of chromophores.
[0237] In some cases, signature profiles are generated from
different types of detection experiments. In some cases, a
signature profile generated from a polynucleotide morphology study
is referred to as a morphology curve. In some cases, a signature
profile generated from a denaturation study is referred to as a
melt curve. In some cases, a signature profile generated from a
persistence length study is referred to as a length curve. In some
cases, a signature profile generated from a single-nucleotide
polymorphism (SNP) study is referred to as a SNP curve.
[0238] In some cases, the change in signal can be calculated as a
percentage. In some cases, the percentage of signal change is 0.01,
0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,
50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 300, 400,
500, 600, 700, 800, 900, 1000, 10,000%. In some cases, the
percentage of signal change is about 0.01, 0.1, 0.2, 0.5, 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75,
80, 85, 90, 95, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900,
1000, 10,000%.
VIII. Analytical Techniques and Instrumentation
[0239] The methods described in this disclosure are compatible with
a variety of amplification methods, including polymerase chain
reaction (PCR), ligase chain reaction (LCR), replicase-mediated
amplification, strand-displacement amplification (SDA), "rolling
circle" types of amplification, and various transcription
associated amplification methods. See, e.g., PCR amplification:
U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; LCR
amplification: U.S. Pat. No. 5,516,663 and EP 0320308 B1;
replicase-mediated amplification: U.S. Pat. No. 4,786,600; SDA
amplification: U.S. Pat. Nos. 5,422,252 and 5,547,861; rolling
circle types of amplification: U.S. Pat. Nos. 5,714,320 and
5,834,252; and transcription associated amplification: U.S. Pat.
Nos. 5,399,491, 5,554,516, 5,130,238, 5,437,990, 4,868,105 and
5,124,246, PCT Pub. WO 1988/010315 A1 and US Pub. 2006-0046265 A1,
which are hereby incorporated by reference.
[0240] In some cases, the polymerase chain reaction (PCR) is a
multiplex-PCR, a variable number of tandem repeats (VNTR) PCR, an
asymmetric PCR, long PCR, a nested PCR, a hot-start PCR, a
Touchdown PCR, an assembly PCR, a colony PCR, a quantitative PCR
(qPCR), an end point PCR, a reverse transcriptase PCR, a digital
PCR or a droplet digital PCR. In some cases, the PCR is a
quantitative PCR.
[0241] In some cases, the PCR amplification step of the present
disclosure can be performed by standard techniques well known in
the art (See, e.g., Sambrook, E. F. Fritsch, and T. Maniatis,
Molecular Cloning: A Laboratory Manual, Second. Edition, Cold
Spring Harbor Laboratory Press (1989); U.S. Pat. No. 4,683,202; and
PCR Protocols: A Guide to Methods and Applications, Innis et al.,
eds., Academic Press, Inc., San Diego (1990) which are hereby
incorporated by reference), PCR cycling conditions typically
consist of an initial denaturation step, which can be performed by
heating the PCR reaction mixture to a temperature ranging from
about 80.degree. C. to about 105.degree. C. for times ranging from
about 1 to about 10 min. Heat denaturation is typically followed by
a number of cycles, ranging from about 1 to about 50 cycles, each
cycle usually comprising an initial denaturation step, followed by
a primer annealing step and concluding with a primer extension
step. Enzymatic extension of the primers by a nucleic acid
polymerase, e.g. TAQ polymerase, produces copies of the template
(e.g., an analyte) that can be used as templates in subsequent
cycles. In some cases, the number of cycles ranges from about 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,
65, 70, 75 to about 80 cycles.
[0242] The methods provided herein are suitable for use with a
variety of detection methods. For example, the methods may be
applied using an analytical technique that measures the wavelength
and intensity of a fluorescent signal. This may be accomplished by
measuring the intensity of a signal across a spectrum of
wavelengths, or by using band pass filters that restrict the
passage of certain wavelengths of light, thereby allowing only
light of certain wavelengths to reach a photodetector. Many
real-time PCR and quantitative PCR instruments comprise an
excitation light source and band pass filters that enable the
detection of fluorescent signals. Therefore, the methods of the
present disclosure can be readily applied using instruments widely
used in the art. No separation is necessary. The present disclosure
does not require the use of beads or a solid phase. Of course, one
of ordinary skill in the art would understand that the present
disclosure could be used with separation, beads, or a solid phase,
if desired.
IX. Diseases
[0243] The methods described herein can be used, for example, to
detect one or more analytes associated with a disease or one or
more genetic variations (e.g., a SNP) associated with a disease. A
disease is an abnormal condition of an organism. In some cases, the
organism is a mammal, such as a human, non-human primate, mouse,
rat, rabbit, goat, dog, cat, or cow. In some cases, the mammal is a
human. In some cases, the human is a patient or subject. In some
cases, the disease is a genetic disorder, an autoimmune disease, a
neurological disease, a cardiovascular disease or a cancer.
[0244] A genetic disorder is a disease caused by one or more
abnormalities in the genome. Exemplary genetic disorders include
22q11.2 deletion syndrome, Acrocephaly, Acute cerebral Gaucher's
disease, Adrenal gland disorders, Adrenogenital syndrome,
Alzheimer's disease, Amelogenesis imperfect, androgen insensitivity
syndrome, anemia, Angelman syndrome, Apert syndrome, ataxia
telangiectasia, Canavan disease, Charcot-Marie-Tooth disease, Color
blindness, Cri du chat, Cystic fibrosis, Down syndrome, Duchenne
muscular dystrophy, Haemochromatosis, Haemophilia, Klinefelter
syndrome, Neurofibromatosis, Phenylketonuria, Polycystic kidney
disease, Prader-Willi syndrome, Sickle-cell disease, Tay-Sachs
disease and Turner syndrome.
[0245] An autoimmune disease is a disease caused when the immune
system mistakenly attacks and destroys healthy body tissue.
Exemplary autoimmune diseases include lopecia areata, autoimmune
hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes
(type 1), several forms of juvenile idiopathic arthritis,
glomerulonephritis, Graves' disease, Guillain-Barre syndrome,
idiopathic thrombocytopenic purpura, myasthenia gravis, several
forms of myocarditis, multiple sclerosis, pemphigus/pemphigoid,
pernicious anemia, polyarteritis nodosa, polymyositis, primary
biliary cirrhosis, psoriasis, rheumatoid arthritis,
scleroderma/systemic sclerosis, Sjogren's syndrome, systemic lupus
erythematosus, several forms of thyroiditis, several forms of
uveitis, vitiligo, and granulomatosis with polyangiitis
(Wegener's).
[0246] Exemplary neurological diseases include attention deficit
hyperactivity disorder (ADHD), ALS, Alzheimer's disease, bipolar
disorder, Bell's palsy, birth defects of the brain and spinal cord,
cerebral palsy, chronic fatigue syndrome, dyslexia, epilepsy,
Guillain-Barre syndrome, multiple sclerosis, muscular dystrophy,
neuropathy, neuromuscular and related diseases, Parkinson's
disease, schizophrenia, scoliosis and spinal deformity.
[0247] Exemplary cardiovascular disease include acute myocardial
infarction, angina, arrhythmia, atherosclerosis, cardiomegaly,
cardiomyopathy, carotid artery disease, congenital heart disease,
congestive heart failure, coronary artery disease, endocarditis,
fluid around the heart, hypertension, infective endocarditis,
mitral valve prolapsed, peripheral artery disease, stroke, and
valvular heart disease.
[0248] Cancer is characterized by an abnormal growth of cells.
Exemplary cancer include bladder, brain, breast, bladder, bone,
cervical, colon, esophageal, kidney, liver, lung, ovarian,
pancreatic, proximal or distal bile duct, prostate, skin, stomach,
thyroid, and uterine cancer.
[0249] In some cases, the presence of an analyte or a genetic
variation in an analyte (e.g., a SNP) can serve as a disease
marker. In some cases, the method disclosed herein can be used to
detect a disease marker. In some cases, the method disclosed herein
can be applicable in determining the presence or absence or the
type of diseases affecting a patient. For example, FIG. 5
illustrates an overview of a method of providing a treatment in
conjunction with a detection method described herein. 601
illustrates a clinician preparing to take a sample from a patient.
In some cases, the sample can be a blood sample. In some cases, the
sample can be a tissue sample. 602 illustrates the sample in an
Eppendorf tube. 603 indicates the amplification step. 604
illustrates products from the PCR assay that have been performed to
amplify the hybridized biomarkers. 605 depicts a clinician
returning the results of an analysis to a subject.
X. Compositions and Kits
[0250] This disclosure also provides compositions and kits for use
with the methods described herein. The compositions may comprise
any component, reaction mixture and/or intermediate described
herein, as well as any combination thereof. For example, the
disclosure provides detection reagents for use with the methods
provided herein. Any suitable detection reagents may be provided,
including a primer pair labeled with two different chromophores
(e.g., a donor chromophore and an acceptor chromophore), as
described elsewhere in the specification.
[0251] In some cases, compositions comprise a first and a second
primer or probe for the detection of at least one analyte wherein
the primers are labeled with either a FRET donor or a FRET acceptor
at the 5' end. In some cases, compositions comprise primers labeled
at the 5' end with either a FRET donor or a FRET acceptor for the
detection of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 100, 500, 1000, 5000, or 10000 analytes. In some cases,
compositions comprise primers labeled with multiple different FRET
donors or acceptors wherein at least one donor or acceptor is at
the 5' end for the detection of at least 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,
44, 45, 46, 47, 48, 49, 50, 100, 500, 1000, 5000, or 10000
analytes. In some cases the compositions comprise multiple pairs of
first and second primers, wherein each pair of first and second
primers comprise either a FRET donor or a FRET acceptor at the 5'
end. In some cases each pair of first and second primers comprise a
different FRET donor and FRET acceptor from the remaining set of
primers. In some cases the compositions comprise at least 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 500, 1000,
5000, or 10000 pairs of first and second primers.
[0252] The present disclosure also provides kits for carrying out
the methods of the invention. Accordingly, a variety of kits are
provided in suitable packaging. The kits may be used for any one or
more of the uses described herein, and, accordingly, may contain
instructions for detecting the presence or absence of each analyte
or a plurality of analytes. A kit may be a diagnostic kit, for
example, a diagnostic kit suitable for the detection of one or more
analytes, including the analytes recited herein. A kit may contain
any of the compositions provided in this disclosure, including
those recited above.
XI. Services
[0253] The methods provided herein may also be performed as a
service. For example, a service provider may obtain the identity of
a plurality of analytes that a customer wishes to analyze. The
service provider may then encode each analyte to be detected by any
of the methods described herein and provide appropriate reagents to
the customer for the assay. The customer may perform the assay and
provide the results to the service provider for decoding. The
service provider may then provide the decoded results to the
customer. The customer may also encode analytes, generate probes,
and/or decode results by interacting with software installed
locally (at the customer's location) or remotely (e.g., on a server
reachable through a network). Exemplary customers include clinical
laboratories, physicians, manufacturers of food and consumer
products, industrial manufacturers (e.g., petroleum companies) and
the like. A customer or party may be any suitable customer or party
with a need or desire to use the methods, systems, compositions,
and kits of the invention.
A. Server
[0254] The methods provided herein may be processed on a server or
a computer server (FIG. 6). The server 1101 includes a central
processing unit (CPU, also "processor") 1105 which can be a single
core processor, a multi core processor, or plurality of processors
for parallel processing. A processor used as part of a control
assembly may be a microprocessor. The server 1101 also includes
memory 1110 (e.g. random access memory, read-only memory, flash
memory); electronic storage unit 1115 (e.g. hard disk);
communications interface 1120 (e.g. network adaptor) for
communicating with one or more other systems; and peripheral
devices 1125 which may include cache, other memory, data storage,
and/or electronic display adaptors. The memory 1110, storage unit
1115, interface 1120, and peripheral devices 1125 are in
communication with the processor 1105 through a communications bus
(solid lines), such as a motherboard. The storage unit 1115 can be
a data storage unit for storing data. The server 1101 is
operatively coupled to a computer network ("network") 1130 with the
aid of the communications interface 1120. A processor with the aid
of additional hardware described herein, may also be operatively
coupled to a network. The network 1130 can be the Internet, an
intranet and/or an extranet, an intranet and/or extranet that is in
communication with the Internet, a telecommunication or data
network. The network 1130 in some cases, with the aid of the server
1101, can implement a peer-to-peer network, which may enable
devices coupled to the server 1101 to behave as a client or a
server. In general, the server may be capable of transmitting and
receiving computer-readable instructions (e.g., device/system
operation protocols or parameters) or data (e.g., sensor
measurements, raw data obtained from detecting nucleic acids,
analysis of raw data obtained from detecting nucleic acids,
interpretation of raw data obtained from detecting nucleic acids,
etc.) via electronic signals transported through the network 1130.
Moreover, a network may be used, for example, to transmit or
receive data across an international border.
[0255] The server 1101 may be in communication with one or more
output devices 1135 such as a display or printer, and/or with one
or more input devices 1140 such as, for example, a keyboard, mouse,
or joystick. The display may be a touch screen display, in which
case it may function as both a display device and an input device.
Different and/or additional input devices may be present such an
enunciator, a speaker, or a microphone. The server may use any one
of a variety of operating systems, such as for example, any one of
several versions of Windows, or of MacOS, or of Unix, or of
Linux.
[0256] The storage unit 1115 can store files or data associated
with the operation of a device or method described herein.
[0257] The server can communicate with one or more remote computer
systems through the network 1130. The one or more remote computer
systems may be, for example, personal computers, laptops, tablets,
telephones, Smart phones, or personal digital assistants.
[0258] In some situations a control assembly includes a single
server 1101. In other situations, the system includes multiple
servers in communication with one another through an intranet,
extranet and/or the Internet.
[0259] The server 1101 can be adapted to store device operation
parameters, protocols, methods described herein, and other
information of potential relevance. Such information can be stored
on the storage unit 1115 or the server 1101 and such data can be
transmitted through a network.
[0260] The details of one or more embodiments of the disclosure are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages will be apparent from the
description and drawings, and from the claims.
EXAMPLES
[0261] The FRET labeled primers methods and system herein described
are further illustrated in the following examples, which are
provided by way of illustration and are not intended to be
limiting. In particular, in the following examples a further a
description of the FRET-labeled primers and related methods and
systems of the present disclosure is provided with reference to
detection of KRAS mutation performed by FRETplexing in combination
with qPCR. A person skilled in the art will appreciate the
applicability of the features described in detail for
FRET-chromophore-labeled primers designed to detect different SNPs
or other genetic variations or other variations including deletion,
mutations and insertions involving more than one residue in the
polynucleotide analytes with different FRET labels in accordance
with the present disclosure. In particular, a skilled person
reading the present disclosure will appreciate that the
FRET-chromophore-labeled primers illustrated in the Examples, are
only one exemplary of FRET-chromophore-labeled primers and that
FRET-chromohore-labeled primers can include also other fluorophores
identifiable to a skilled person.
Example 1: Exemplary Experimental Approach for FRET Detection of
SNPs
[0262] An approach involving use of FRET-chromophore-labeled
primers in standard q-PCR has been designed to achieve SNPs
detection. In particular, in the following approach, primers are
designed and labeled in view of the specific SNPs to be detected
and then used in q-PCR experiments for the related detection.
[0263] In order to design the primers first, at least one target
sequence is selected which is associated with the SNP (or other
mutation) to be detected. In particular, at least one of the target
sequences corresponds to the primers, preferably both target
sequences includes the sequence where the SNP to be detected might
be located. In particular, in cases in which both primers carry
corresponding FRET donor-acceptor, the target sequences are
identified so that upon specific binding of the primers the FRET
donor and FRET acceptor attached to the primers are located at a
distance up to four times their related Forster distance,
preferably within three times the related Forster distance one with
respect to the other, more preferably within two times the Forster
distance one with respect to the other, even more preferably within
or at the Forster distance one with respect to the other.
[0264] For example for the FRET donor-acceptor chromophore pair Cy3
and Cy5 the target sequence are selected on the polynucleotide
analyte so that upon binding on the respective primer Cy3 and Cy5
are placed at the related Forster distance of 5.6 nm one with
respect to the other. Therefore the target sequences will be
selected on the polynucleotide to be investigated so that upon
specific hybridization a pair of suitable primers will locate the
Cy3 and Cy5 label within 5.6 nm. Based on the base pair to length
conversion factor identifiable to a skilled person in the art, the
optimal FRET distance between one FRET chromophore and the other is
preferably in a range between 15-250 bp, more preferably in a range
between 15-80 bp, possibly up to 1,000 bp. Such ranges can be
different depending on particular chemical environments,
instrumentation used in the qPCR experiments and many other factors
that may contribute to the FRET signal detection.
[0265] A pair of forward and reverse primers is then constructed to
be specific to a corresponding target sequence at the reaction
conditions of the q-PCR. Identification and design of specific
primer for a target sequence can be performed by applying, the
general concepts for PCR primer design which are identifiable by a
person skilled in the art. In general, the primers can be selected
to ensure that the forward and reverse primers are specific to the
corresponding target sequence and have similar melting temperatures
(preferably within 1 or 2.degree. C. one from the other and no more
than 5.degree. C.) so that specific hybridization and extension of
both forward and reverse primers takes place under same reaction
conditions in reaction mixture. Under reaction conditions of
commercially available q-PCR kits, primers can be typically between
20 to 35 bp long to allow for enough specificity at the related
melting temperatures, typically within a range between
55-65.degree. C., possibly between 50-75.degree. C. The limit for
the melting temperatures is below the boiling point of water and
the enzyme hybridization temperature. When the target sequence
includes a SNP or point mutation, the primers typically have the 3'
end base overlapping the point mutation to maximize sensitivity. In
particular, for detection of a SNP a base complementary to the
expected SNP in the target sequence is included in the primer at
the 3' end of the primer within a range of 1 to 3 bp from such 3'
end towards the 5' end direction, preferably at the 3' terminus.
The mutations cannot be located beyond the 3'end in the 5'->3'
direction of the primer as such location renders the primer
non-specific for the SNP to be detected.
[0266] A similar approach can be used for a different variation of
a reference polynucleotide analyte, e.g. a mutation, deletion or
insertion, wherein the primers can be designed so that they
specifically bind to a target sequence where the expected variation
might be located one or more consecutive base pairs that are
indicative of the variation. In particular, the primer are designed
to specifically bind the sequence indicative of the variation
preferably with their 3' end.
[0267] Each forward and reverse primer is then labeled with a FRET
chromophore (in the following examples Cy3-Cy5). The FRET
chromophore labels can be located anywhere along the primer
sequence except for the 3' end as long as upon binding the related
location one with respect to the other is within the related
forester distance.
[0268] In general, a donor chromophore can be attached to the 5'
end of one primer and an acceptor chromophore can be attached to
the 5' end of the second primer to minimize interference of the
label with primer hybridization. The attachment can be done through
NHS chemistry, peptide bonding or other approaches identifiable to
a person skilled in the art. When the donor is attached to the 5'
end of one primer and the acceptor is attached to the 5' end of the
other primer, the distance from the 5' end of one primer to the 5'
end of the other primer along the template sequence should be short
enough to allow efficient FRET between the donor and acceptor,
preferably in a range between 15-80 bp, more preferably between
15-45 bp.
[0269] As described previously, the donor and acceptor fluorophores
are selected to form a FRET donor-acceptor chromophore pair so that
when placed within a Forster distance the receptor can emit a
fluorescence signal generated upon excitation by the energy
released from the donor with at least 50% energy transfer
efficiency.
[0270] The primers are then mixed with the sample and PCR reagents
(e.g. a PCR mastermix) to perform PCR experiments. Signal detection
can be performed after each annealing step and each denaturing
step. Each such detection is done by exciting the donor at a
certain wavelength and detecting the donor emission and/or the
acceptor emission at the same or different wavelengths. In
particular, typically detection can be performed after one or more
annealing cycle with respect to the acceptor and possibly also the
donor. In cases where multiple detection is performed with
different FRET donor-acceptor chromophore pair, detection can be
performed of the different acceptors possibly in combination with
related donors after one or more of the annealing cycle in
accordance with the experimental design.
[0271] In the FRET detection herein described, the donor is
expected to generate a fluorescence emission signal both after the
denaturing and annealing steps, while the acceptor typically
produces little to no detectable emission signal as donor
excitation is not efficient in exciting the acceptor directly as
this point when FRET has not occurred. Thus, no change in donor
and/or acceptor emission indicates that the target sequence is
absent from the sample. When the target sequence is present, primer
extension and amplicon hybridization will take place, resulting in
a decrease of donor emission and a concomitant increased in
acceptor emission.
[0272] Following detection of FRET labels signals, the related
result can be plotted in function of the time and/or annealing
cycle to derive a signature profile with respect to one or both of
the FRET labels and/or with respect to all the FRET labels used.
For example a measured FRET signal after annealing in each cycle
can be used to form a real-time curve that can be used for
quantitative measurement of the target analyte. The speed of the
reaction, e.g. as measured by Ct, can be compared to a positive
control to determine the relative amount of the target present. The
total present of the target and the wild type can be estimated
using different techniques in order to produce an abundance
measurement.
Example 2: Exemplary Mutations Detected and Characterized by
FRET-Based Methods and Systems
[0273] FRET-based methods and systems herein described have been
used in connection with detection and characterization of KRAS
gene. KRAS mutations have been selected as they are a major source
of drug resistance in colorectal cancer (CRC).
[0274] CRC is responsible for 140,000 new cases and 49,000 deaths
per year in the US alone. One of the most effective and widely used
drugs against CRC is Cetuximab. The total cost of Cetuximab therapy
is estimated to be $1.75 billion per year world-wide. However, KRAS
mutations lead to resistance to Cetuximab in at least 35% of the
patients. As a result, the American Society of Clinical Oncology
(ASCO) issued a recommendation in 2009 that patients should be
tested for KRAS mutations before being prescribed the drug.
Nevertheless, half of the patients that are tested negative by
traditional allele-specific assays still fail the 8-week regimen
with high cost.
[0275] FRET-labeled primers have been designed to detect KRAS
mutation carriers. FRET-based characterization and detection
methods are expected to provide carrier detection with sensitivity
and accuracy.
[0276] Experiments were conducted on six of the top eight KRAS
mutations: G216A, G216C, G219A, G215A, G215T and G216T. These six
mutations are responsible for over 90% of all observed KRAS
mutations, and are present in 18% of all CRC tumors as well as 90%
of all pancreatic cancers.
[0277] While these mutations are not exhaustive, such mutations
have a strong clinical representation and significance, as many of
these same mutations are also found in other cancers, affording
general cancer screening test that covers several of the most
common cancers.
Example 3: Selection of KRAS Related Target DNA Sequences
[0278] Synthetic nucleic acid targets within KRAS gene sequences,
including wild type and mutant nucleic acid were chosen for
exemplary detection studies using a PCR amplification
technique.
[0279] In particular, the selected wild type and six mutant
sequences can be found in Table 2, in which the positions of the
SNPs are underlined. Each of the mutant sequences represents one of
the six above mentioned SNP mutations found in KRAS. For example,
in the 216T mutant, base-pair G at locus 216 is mutated into T,
while in 216C mutant, base-pair G is mutated into C. "G" is omitted
from the notation for the simplicity purpose.
TABLE-US-00002 TABLE 2 Sequences of the wild type and mutant
templates WT/Mutants Sequence SEQ ID NO WT 5'- ATG ACT GAA TAT AAA
CTT GTG GTA GTT SEQ ID NO: 1 GGA GCT GGT GGC GTA GGC AAG AGT GCC
TTG ACG ATA CAG CTA ATT CAG AAT CAT TTT GTG GAC -3' 216A 5'- ATG
ACT GAA TAT AAA CTT GTG GTA GTT SEQ ID NO: 2 GGA GCT GAT GGC GTA
GGC AAG AGT GCC TTG ACG ATA CAG CTA ATT CAG AAT CAT TTT GTG GAC -3'
216T 5'- ATG ACT GAA TAT AAA CTT GTG GTA GTT SEQ ID NO: 3 GGA GCT
GTT GGC GTA GGC AAG AGT GCC TTG ACG ATA CAG CTA ATT CAG AAT CAT TTT
GTG GAC -3' 216C 5'- ATG ACT GAA TAT AAA CTT GTG GTA GTT SEQ ID NO:
4 GGA GCT GCT GGC GTA GGC AAG AGT GCC TTG ACG ATA CAG CTA ATT CAG
AAT CAT TTT GTG GAC -3' 219A 5'- ATG ACT GAA TAT AAA CTT GTG GTA
GTT SEQ ID NO: 5 GGA GCT GCT GAC GTA GGC AAG AGT GCC TTG ACG ATA
CAG CTA ATT CAG AAT CAT TTT GTG GAC -3' 215A 5'- ATG ACT GAA TAT
AAA CTT GTG GTA GTT SEQ ID NO: 6 GGA GCT ACT GGC GTA GGC AAG AGT
GCC TTG ACG ATA CAG CTA ATT CAG AAT CAT TTT GTG GAC -3' 215T 5'-
ATG ACT GAA TAT AAA CTT GTG GTA GTT SEQ ID NO: 7 GGA GCT TCT GGC
GTA GGC AAG AGT GCC TTG ACG ATA CAG CTA ATT CAG AAT CAT TTT GTG GAC
-3'
Example 4: Design of Primers for Detection of KRAS Target DNA
Sequence Using FRETplexing
[0280] Specific primers were designed according to each sequence of
the mutants listed in Table 2. For each of the six mutants listed
in Table 2, a forward primer and a reverse primer are specifically
designed so that the 5'-end of a forward primer is tagged with a
donor (Cy3), the 5'-end of a reverse primer is tagged with a
receptor (Cy5) and both their 3'-end basepairs match the
corresponding SNP. Thus, both primers are mutation-specific. For
example, the forward primer for 216A mutation has an "A" at the 3'
end position and the reverse primer has a complementary "T" at the
3' end position. Table 3 shows a list of forward and reverse
primers used for each of the six mutants in Table 2.
TABLE-US-00003 TABLE 3 Exemplary forward and reverse primers
designed for detecting KRAS mutations KRAS SEQ ID SEQ ID mutation
Forward Primer NO Reverse Primers NO 216A /5Cy3/GAATATAAACTTGT SEQ
ID /5Cy5/GCACTCTTGCCTA SEQ ID GGTAGTTGGAGCTGA NO: 8 CGCCAT NO: 9
216T /5Cy3/GAATATAAACTTGT SEQ ID /5Cy5/GCACTCTTGCCTA SEQ ID
GGTAGTTGGAGCTGT NO: 10 CGCCAA NO: 11 216C /5Cy3/GAATATAAACTTGT SEQ
ID /5Cy5/GCACTCTTGCCTA SEQ ID GGTAGTTGGAGCTGC NO: 12 CGCCAG NO: 13
219A /5Cy3/GAATATAAACTTGT SEQ ID /5Cy5/TCAAGGCACTCT SEQ ID
GGTAGTTGGAGCTGGTG NO: 14 TGCCTACGT NO: 15 A 215A
/5Cy3/GAATATAAACTTGT SEQ ID /5Cy5/CACTCTTGCCTAC SEQ ID
GGTAGTTGGAGCTA NO: 16 GCCACT NO: 17 215T /5Cy3/GAATATAAACTTGT SEQ
ID /5Cy5/CACTCTTGCCTAC SEQ ID GGTAGTTGGAGCTT NO: 18 GCCACA NO:
19
Example 5: FRET Labels and Related Attachment to the KRAS Specific
Primers
[0281] Cy3-Cy5 is commonly used as a FRET pair. Green excitation
(Cy3) together with FRET can be used for any tissue assay as it is
known from experience that the tissue tends to scatter most
strongly from blue excitation.
[0282] The KRAS specific primers of Example 4 were labeled with
Cy3-Cy5 FRET labels in which the forward primer is attached to Cy3
FRET donor chromophore and the reverse primer is attached to Cy5
FRET acceptor chromophore. The Cy3-Cy5 labels are attached by the
manufacturer of DNA oligonucleotides as part of the fabrication
process.
[0283] For FRET and primer design, for the oligos of the examples
the fluorophore was attached at the 5' terminus. The linkage
between the label and the base is very short (shorter than 1 by
width).
Example 6: Polymerase Chain Reactions on the KRAS DNA Target
Sequences with KRAS Specific FRET-Chromophore-Labeled Primers
[0284] The KRAS DNA target sequences obtained in Example 3 were
amplified using the KRAS specific FRET-labeled primers of Example
5.
[0285] In particular, PCR reactions were performed on a Roche 480
lightcycler I and Roche 480 lightcycler II instrument. The PCR
cycling conditions were set up with an initial heating step of 5
minutes at 95.degree., followed by 40 PCR cycles each cycle
comprising a 10-second denature step at 95.degree., a 50-second
primer annealing step at 50.degree. and a 5-second primer extension
step at 72.degree.. Fluorescence measurements in 523 nm-568 m (Cy3)
and 615 nm-670 nm (Cy5) were taken at the end of annealing for
every cycle. Melt curve analysis can be performed for the
amplification products to ensure that desired amplicons have been
detected. Different double-stranded DNA molecules melt at different
temperature, dependent upon a number of factors including GC
content, amplicon length, secondary and tertiary structure etc. To
produce melt curve, the PCR product is exposed to a temperature
gradient from about 50.degree. C. to 95.degree. C. while
fluorescence readouts are continually collected. The increase in
temperature causes the denaturation of the dsDNA. The point at
which the dsDNA melts into ssDNA is observed as a drop in
fluorescence as the chromophore/dye labels dissociate. The melt
temperature of the amplicon can be determined from the melt
curve.
[0286] The results of the PCR experiments on each KRAS target
sequence with KRAS specific FRET-chromophore-labeled primers are
discussed in the Examples 7-12 with reference to FIGS. 7-16.
Example 7: Primer Titration Experiments for Calibration of
Fluorescence Output in FRETplexing
[0287] Primer concentration usually has an effect on the quality
and intensity of the fluorescence output. A number of PCR titration
experiments were carried out for each of the six mutant sequences
synthesized in Example 3 to determine optimum primer concentration
to be used in subsequent detection experiments. For each mutation,
a pair of forward and reverse primers specifically designed for
that mutation in Example 4 was used. Each primer of the primer pair
is labeled with a single color by attaching a FRET donor
chromophore (Cy3) and a FRET acceptor chromophore (Cy5),
respectively. Each of the PCR titration experiments includes
reagents, templates and primers.
TABLE-US-00004 TABLE 4 primer titration experiment cocktail for
each of the six mutations Components Concentration Volume Reagents
100 uM 20 uL Templates 10 uM 10 uL Primers 10 uM 1-10 uL in 1 uL
integers DI Water 60-69 uL
[0288] A calibration curve was produced for each mutation
documenting the end-point fluorescence output as a function of the
primer concentration.
Example 8: Proof Testing FRETplexing with Synthetic DNA
[0289] The calibration curves obtained from Example 7 were then
used to design appropriate ratio of the primer concentrations when
combined together in a multiplexed assay. Primers at different
concentration obtained from the calibration curves were combined
and tested. Tables 5-6 show different combinations of primer
concentration used in multiplexed assays, each containing a mixture
of at least one of the six mutant templates listed in Table 2 and
at least one of the six pairs of primer pairs listed in Table 3 at
various concentration ratios. The output for each of the
combinations was reported in chromatograms.
TABLE-US-00005 TABLE 5 Combinations of primer concentration ratios
used in multiplexed assays of mutations 216A, 216C and 216T Number
Combination of Primer Concentration 1 216A: 8 ul of 10 uM; 216T: 4
ul of 10 uM 2 216A: 8 ul of 10 uM; 216C: 2 ul of 10 uM 3 216T: 4 ul
of 10 uM; 216C: 2 ul of 10 uM 4 216A: 8 ul of 10 uM; 216T: 4 ul of
10 uM; 216C: 2 ul of 10 uM 5 216A: 8 ul of 10 uM; 216T: 8 ul of 10
uM; 216C: 8 ul of 10 uM 6 216T: 8 ul of 10 uM; 216C: 8 ul of 10 uM
7 216A: 8 ul of 10 uM; 216C: 8 ul of 10 uM 8 216A: 8 ul of 10 uM;
216T: 8 ul of 10 uM
TABLE-US-00006 TABLE 6 Combinations of primer concentration ratios
used in multiplexed assays of mutations 215A, 215T and 219C Number
Combination of Primer Concentration 1 215A: 8 ul of 10 uM; 215T: 4
ul of 10 uM 2 215A: 8 ul of 10 uM; 219C: 2 ul of 10 uM 3 215T: 4 ul
of 10 uM; 219C: 2 ul of 10 uM 4 215A: 8 ul of 10 uM; 215T: 4 ul of
10 uM; 219C: 2 ul of 10 uM 5 215A: 8 ul of 10 uM; 215T: 8 ul of 10
uM; 219C: 8 ul of 10 uM 6 215T: 8 ul of 10 uM; 219C: 8 ul of 10 uM
7 215A: 8 ul of 10 uM; 219C: 8 ul of 10 uM 8 215A: 8 ul of 10 uM;
215T: 8 ul of 10 uM
Example 9: Wild Type Calibration for Mutant Primers
[0290] WT concentration can affect the specificity and sensitivity
of mutant primers. A set of PCR titration experiments were carried
out using a mastermix to compare the real-time PCR data with the WT
sequence at various concentrations. The mastermix is a premixed
solution containing a combination of DNA polymerase, dNTPs,
reaction buffers and other reagents needed for a PCR reaction.
Optimum WT concentrations were then determined from the calibration
experiments.
[0291] The qPCR experiments were carried out with the WT sequence
at five different concentrations: 10 uM, 100 uM, 1000 uM, 10,000 uM
and 100,000 uM. The components for two of the qPCR WT calibration
experiments including reagents, WT, and mutant primers are
tabulated in Tables 7-8 with two different WT concentrations. Note
that no mutant sequence is present in the WT calibration
experiments. The mutant primers contained in the assay represent a
mixture of 216A, 216T, 216C primer pairs each at an 8 uL of 10 uM
volume/concentration.
TABLE-US-00007 TABLE 7 WT concentration titration experiment
cocktail Components Concentration Volume Reagents 100 uM 20 uL WT
10-100,000 uM 10 uL Each primer 10 uM for each forward and reverse
8 uL pair primer
TABLE-US-00008 TABLE 8 WT concentration titration experiment
cocktail Components Concentration Volume Reagents 100 uM 20 uL WT
10-100,000 uM 10 uL Each primer 10 uM for each forward and reverse
8 uL pair primer
[0292] FIG. 7 shows a chart reporting the wild type concentration
calibration experiments using FRET-chromophore-labeled primers with
qPCR. Each multiplexed assay contains 20 uL of Taq 5.times.
matermix, 8 uL of 10 uM for each primer 216C, 216A and 216T and the
WT sequence at different concentrations (10 uM, 100 uM, 1000 uM,
10,000 uM and 100,000 uM). The y-axis shows averaged relative FRET
emission intensities obtained from 22 qPCR replica experiments. For
each qPCR experiment, the relative FRET emission intensity (delta
value) is calculated by subtracting the maximum FRET emission
intensity with the minimum FRET emission intensity.
[0293] The results indicate that as the concentration of wild type
sequence increases, the delta value remains relatively leveled
until the WT concentration reaches to 100,000 uM at which an
increase in delta value is noted. The false positive signal
observed at the high concentration suggests that mispriming has
occurred due to non-specific annealing and subsequent extension
upon addition of nucleotide by DNA polymerase.
Example 10: Detection of Single Mutation 216T, 216A, and 216C Using
FRET-Chromophore-Labeled Primers with OCR
[0294] Example described in this section is a proof of concept
experiment indicating that FRET detection herein described can be
used in conjunction with a qPCR without affecting the amplification
reaction.
[0295] qPCR experiments were conducted to detect single mutation
216T, 216A, and 216C from KRAS gene. Tables 9-11 lists the
components contained in 216T, 216A, and 216C detection experiments.
Initial experiment results show a progressive increase of the
emission intensity as the number of PCR cycles increase.
TABLE-US-00009 TABLE 9 Experiment cocktail for detection of a
single 216T mutation Components Volume (uL)/Concentration(uM)
Reagents 20 uL of 100 uM Taq 5.times. matermix WT 10 uL of 100 uM
216T 10 uL of 10 uM 216T primer 1.times. 2 uL of 10 uM forward and
reverse primer
TABLE-US-00010 TABLE 10 Experiment cocktail for detection of a
single 216A mutation Components Volume (uL)/Concentration(uM)
Reagents 20 uL of 100 uM Taq 5.times. matermix WT 10 uL of 100 uM
216A 10 uL of 10 uM 216A primer 4.times. 48 uL of 10 uM forward and
reverse primer
TABLE-US-00011 TABLE 11 Experiment cocktail for detection of a
single 216C mutation Components Volume (uL)/Concentration(uM)
Reagents 20 uL of 100 uM Taq 5x matermix WT 10 uL of 100 uM 216C 10
uL of 10 uM 216C primer 2.times. 4 uL of 10 uM forward and reverse
primer
[0296] FIG. 8 shows a chart reporting detection of 216T mutant
sequence from KRAS gene using FRET-chromophore-labeled primers with
quantitative PCR (qPCR). The assay contains 2 uL of 10 uM forward
and reverse primer concentration, 20 uL of Taq 5.times. mastermix,
10 uL of 10 uM 216T mutant sequence and 10 uL of 100 uM wild type
sequence. The forward primer is labeled with Cy3 as a FRET donor
chromophore and the reverse primer is labeled with Cy5 as a FRET
acceptor chromophore. The chart shows an averaged FRET emission
signal intensity of 10 qPCR replica experiments (y-axis) plotted as
a function of PCR cycles (x-axis) with a standard deviation of
1.sigma.. The FRET emission signal intensity is generated by Cy5
(y-axis) and detected after each annealing step.
[0297] Generally, in a PCR curve, a baseline region in the early
stage of the PCR amplification is followed by an exponential phase
where an exponential increase in signal intensity is observed. The
exponential phase is then followed by a plateau phase where the
growth is slowed down and eventually levels off at the saturation
stage where changes in signal intensity can no longer be
recognized. In FIG. 8 a typical PCR curve is observed in which a
baseline region in the early stage of the PCR amplification (cycles
1-6) is followed by an exponential phase (cycles 7-17) and finished
with a plateau phase at cycle 15-17. The final saturation stage is
reached at cycles 19.
[0298] FIG. 9 shows a chart reporting detection of 216A mutant
sequence from KRAS gene using FRET-chromophore-labeled primers with
quantitative PCR (qPCR). The assay contains 8 uL of 10 uM forward
and reverse primer concentration, 20 uL of Taq 5.times. mastermix,
10 uL of 10 uM 216A mutant sequence and 10 uL of 100 uM wild type
sequence. The forward primer is labeled with Cy3 as a FRET donor
chromophore and the reverse primer is labeled with Cy5 as a FRET
acceptor chromophore. The chart shows an averaged FRET emission
signal intensity of 10 qPCR replica experiments (y-axis) plotted as
a function of PCR cycles (x-axis) with a standard deviation of
1.sigma.. The FRET emission signal intensity is generated by Cy5
(y-axis) and detected after each annealing step. The delta value
calculated by subtracting the maximum FRET signal recorded during
the PCR saturation period and the minimum FRET signal recorded at
the beginning of the PCR cycles is 0.35 A.U. The system appears to
already be in the amplification cycle at the start of the
experiment and reaches saturation at cycle 25.
[0299] FIG. 10 shows a chart reporting detection of 216C mutant
sequence from KRAS gene using FRET-chromophore-labeled primers with
quantitative PCR (qPCR). The assay contains 4 uL of 10 uM forward
and reverse primer concentration, 20 uL of Taq 5.times. mastermix,
10 uL of 10 uM 216C mutant sequence and 10 uL of 100 uM wild type
sequence. The forward primer is labeled with Cy3 as a FRET donor
chromophore and the reverse primer is labeled with Cy5 as a FRET
acceptor chromophore. The chart shows an averaged FRET emission
signal intensity of 10 qPCR replica experiments (y-axis) plotted as
a function of PCR cycles (x-axis) with a standard deviation of
1.sigma.. The FRET emission signal intensity is generated by Cy5
(y-axis) and detected after each annealing step. The delta value
calculated by subtracting the maximum FRET signal recorded during
the PCR saturation period and the minimum FRET signal recorded at
the beginning of the PCR cycles is 0.6 A.U. The PCR reaction system
shown in FIG. 10 reaches saturation at cycle 20 and the exponential
growth begins at cycle 5.
[0300] FIGS. 8-10 in particular show the successful detection of
216T, 216A and 216C mutants using the templates, primer pair
designed for the specific mutant and methods described in the above
examples.
[0301] The observed effect suggest an increase in the emission
signal generated by the attached Cy5 upon electron transfer from
Cy3 donor chromophore to Cy5 receptor chromophore following the
formation of a labeled amplicon (see FIGS. 4, S4 and S5).
[0302] Using the graph shown in FIGS. 8-10, a delta value can be
calculated by subtracting the maximum FRET signal recorded during
the PCR saturation period or at the end of the PCR cycles when the
saturation period has not been in certain qPCR experiments and the
minimum FRET signal recorded at the beginning of the PCR cycles
(cycle 0). For example, the delta value for 216T mutant at 1.times.
primer concentration ratio is estimated at about 0.27 A.U. Delta
values from other PCR amplifications having different mutant
sequence and primer concentration ratios can be obtained similarly.
The delta values can then be used to chart the relatively output
intensity changes that correspond to that specific mutation or a
mixture of various mutations in a multiplexed assay, as shown in
FIG. 15
[0303] Accordingly the experiments described herein in connection
with the detection of the mutant 216T, 216A and 216C can be
performed as part of FRETplex detection of multiple SNPs within the
KRAS gene.
[0304] Reference is made in this connection to the illustration of
FIGS. 8-10 where the notation #.times. used for detecting 216T,
216C and 216A mutants specifies a ratio between the concentration
of one primer pair specific to a mutation and the concentration of
another primer pair specific to a different mutation in a
multiplexed assay.
[0305] 1.times. denotes that the primer pair is at the lowest
concentration. The lowest concentration used in the mutation
detection experiments using FRET-chromophore-labeled primers with
qPCR herein described is 2 uL of 10 uM for both the forward and
reverse primers. 2.times. denotes that the primer concentration is
twice the lowest concentration, which is 4 uL of 10 uM for both the
forward and reverse primers. 4.times. denotes that the primer
concentration is four times the lowest concentration, which is 8 uL
of 10 uM for both the forward and reverse primers. The primer
concentration level is directly related to the FRET emission
intensity level, that is, the higher the primer concentration, the
more intense is the FRET emission signal.
[0306] The related FRETplexing experiments are reported in Example
11.
Example 11: Detection of Multiple Mutations Based on Emission
Intensity Using FRETplexing
[0307] To detect multiple mutations in a single sample, multiplex
assays described in Examples 3-9 were used. The goal with
multiplexing is to have multiple mutations coded in a single color
with each mutation assigned with different emission intensities,
which can be achieved by adjusting the relative concentrations of
primers when comparing two constituent assay that form a
multiplexed assay. For example, a first mutation can be assigned
with a single intensity (1.times.) with the lowest primer
concentration (2 uL of 10 uM), the second mutation can be assigned
with a double intensity (2.times.) with a primer concentration at 4
uL of 10 uM, the third mutation can be assigned with a quadruple
intensity (4.times.) with a primer concentration at 8 uL of 10 uM,
and so on. Thus, a combination of two mutations will accumulate a
3.times. intensity response and a combination of 3 mutations will
accumulate a 7.times. intensity response. The level of intensity,
such as 1.times., 2.times., 4.times., and 7.times. can be
controlled by adjusting the concentration of the primer pair for
that specific mutation. Table 12 lists combinations of mutant
primers at different concentrations for seven multiplexed assays.
Note that the lowest prime volume is 2 uL of 10 uM for 216T primer,
but any primer volume can be used as long as the FRET signal
intensity is strong enough to be detected over background noise
signal generated during a qPCR reaction. The concentrative primer
volume for each of other primer pairs can be incremented by a
multiplicative factor of 2, such as 2.times., 4.times., 8.times.,
16.times., 32.times. and so on.
TABLE-US-00012 TABLE 12 Combinations of mutant primers at different
concentration levels for multiplexed assays Multiplex
number/accumulated Combination of Primer concentration level
Concentration Volume 1x 216T: 2 ul of 10 uM 10 uL 2x 216C: 4 ul of
10 uM 10 uL 4x 216A: 8 ul of 10 uM; 10 uL 3x 216C: 4 ul of 10 uM;
10 uL 216T: 2 ul of 10 uM 10 uL 5x 216A: 8 ul of 10 uM; 10 uL 216T:
2 ul of 10 uM 10 uL 6x 216A: 8 ul of 10 uM; 10 uL 216C: 4 ul of 10
uM 10 ul 7x 216A: 8 ul of 10 uM; 10 uL 216C: 4 ul of 10 uM; 10 uL
216T: 2 ul of 10 uM 10 uL
[0308] Tables 13-16 list the components contained in the
multiplexed detection experiments. For each PCR experiment, the
concentration of the WT is 10 times the concentration of the
mutant.
TABLE-US-00013 TABLE 13 Experiment cocktail for detection of a
double mutation 216C at 2.times. and 216T at 1.times. primer
concentrations Components Concentration Volume Reagents 100 uM 20
uL WT 100 uM 10 uL 216C 10 uM 10 uL 216T 10 uM 10 uL 216C Primer 10
uM 4 uL 216T Primer 10 uM 2 uL
TABLE-US-00014 TABLE 14 Experiment cocktail for detection of a
double mutation 216C at 2.times. and 216A at 4.times. primer
concentrations. Components Concentration Volume Reagents 100 uM 20
uL WT 100 uM 10 uL 216C 10 uM 10 uL 216A 10 uM 10 uL 216C Primer 10
uM 4 uL 216A Primer 10 uM 8 uL
TABLE-US-00015 TABLE 15 Experiment cocktail for detection of a
double mutation 216T at 1.times. and 216A at 4.times. primer
concentrations. Components Concentration Volume Reagents 100 uM 40
uL WT 100 uM 10 uL 216T 10 uM 10 uL 216A 10 uM 10 uL 216T Primer 10
uM 2 uL 216A Primer 10 uM 8 uL
TABLE-US-00016 TABLE 16 Experiment cocktail for detection of a
triple mutation 216C at 2.times. and 216T at 1.times. and 216A at
4.times. primer concentrations. Components Concentration Volume
Reagents 100 uM 40 uL WT 100 uM 10 uL 216C 10 uM 10 uL 216T 10 uM
10 uL 216A 10 uM 10 uL 216C Primer 10 uM 4 uL 216T Primer 10 uM 2
uL 216A Primer 10 uM 8 uL
[0309] FIG. 11 shows a chart reporting detection of double mutation
216T and 216A from KRAS gene using FRET-chromophore-labeled primers
with quantitative PCR (qPCR). The assay contains 8 uL of 10 uM
forward and reverse primer concentration for 216A, 2 uL of 10 uM
forward and reverse primer concentration for 216T, 20 uL of Taq
5.times. mastermix, 10 uL of 10 uM for each 216A and 216T mutant
and 10 uL of 100 uM wild type sequence. The forward primer is
labeled with Cy3 as a FRET donor chromophore and the reverse primer
is labeled with Cy5 as a FRET acceptor chromophore. The chart shows
an averaged FRET emission signal intensity of 10 qPCR replica
experiments (y-axis) plotted as a function of PCR cycles (x-axis)
with a standard deviation of 1.sigma.. The FRET emission signal
intensity is generated by Cy5 (y-axis) and detected after each
annealing step. The delta value calculated by subtracting the
maximum FRET signal recorded during the PCR saturation period and
the minimum FRET signal recorded at the beginning of the PCR cycles
is 2.0 A.U. The PCR reaction system shown in FIG. 11 reaches
saturation at cycle 20 and the exponential growth begins at cycle
5
[0310] FIG. 12 shows a chart reporting detection of double mutation
216T and 216C from KRAS gene using FRET-chromophore-labeled primers
with quantitative PCR (qPCR). The assay contains 4 uL of 10 uM
forward and reverse primer concentration for 216C, 2 uL of 10 uM
forward and reverse primer concentration for 216T, 20 uL of Taq
5.times. mastermix, 10 uL of 10 uM for each 216C and 216T mutant
and 10 uL of 100 uM wild type sequence. The forward primer is
labeled with Cy3 as a FRET donor chromophore and the reverse primer
is labeled with Cy5 as a FRET acceptor chromophore. The chart shows
an averaged FRET emission signal intensity of 10 qPCR replica
experiments (y-axis) plotted as a function of PCR cycles (x-axis)
with a standard deviation of 1.sigma.. The FRET emission signal
intensity is generated by Cy5 (y-axis) and detected after each
annealing step. The delta value calculated by subtracting the
maximum FRET signal recorded during the PCR saturation period and
the minimum FRET signal recorded at the beginning of the PCR cycles
is 1.6 A.U. The PCR reaction system shown in FIG. 12 reaches
saturation at cycle 25 and the exponential growth begins at cycle
6.
[0311] FIG. 13 shows a chart reporting detection of double mutation
216A and 216C from KRAS gene using FRET-chromophore-labeled primers
with quantitative PCR (qPCR). The assay contains 4 uL of 10 uM
forward and reverse primer concentration for 216C, 8 uL of 10 uM
forward and reverse primer concentration for 216A, 20 uL of Taq
5.times. mastermix, 10 uL of 10 uM for each 216A and 216C mutant
and 10 uL of 100 uM wild type sequence. The forward primer is
labeled with Cy3 as a FRET donor chromophore and the reverse primer
is labeled with Cy5 as a FRET acceptor chromophore. The chart shows
an averaged FRET emission signal intensity of 10 qPCR replica
experiments (y-axis) plotted as a function of PCR cycles (x-axis)
with a standard deviation of 1.sigma.. The FRET emission signal
intensity is generated by Cy5 (y-axis) and detected after each
annealing step. The delta value calculated by subtracting the
maximum FRET signal recorded during the PCR saturation period and
the minimum FRET signal recorded at the beginning of the PCR cycles
is 1.9 A.U. The PCR reaction system shown in FIG. 13 reaches
saturation at cycle 30 and the exponential growth begins at cycle
5.
[0312] FIG. 14 shows a chart reporting detection of triple mutation
216A, 216T and 216C from KRAS gene using FRET-chromophore-labeled
primers with quantitative PCR (qPCR). The assay contains 4 uL of 10
uM forward and reverse primer concentration for 216C, 8 uL of 10 uM
forward and reverse primer concentration for 216A, 2 uL of 10 uM
forward and reverse primer concentration for 216T, 20 uL of Taq
5.times. mastermix, 10 uL of 10 uM for each 216A, 216T and 216C
mutant and 10 uL of 100 uM wild type sequence. The forward primer
is labeled with Cy3 as a FRET donor chromophore and the reverse
primer is labeled with Cy5 as a FRET acceptor chromophore. The
chart shows an averaged FRET emission signal intensity of 10 qPCR
replica experiments (y-axis) plotted as a function of PCR cycles
(x-axis) with a standard deviation of 1.sigma.. The FRET emission
signal intensity is generated by Cy5 (y-axis) and detected after
each annealing step. The delta value calculated by subtracting the
maximum FRET signal recorded during the PCR saturation period and
the minimum FRET signal recorded at the beginning of the PCR cycles
is 2.2 A.U. The PCR reaction system shown in FIG. 14 reaches
saturation at cycle 25 and the exponential growth begins at cycle
5.
[0313] In FIG. 15, the relative FRET emission intensities (delta
values at y-axis) obtained from FIGS. 8-14 are plotted as a
function of the accumulated primer concentration ratio (x-axis)
with a standard deviation of 1.sigma..
[0314] The x-axis of FIG. 15 is the accumulated intensity level
calculated from summing up the primer concentration ratios in
various mutations mixtures. For example, a multiplexed assay
containing 216T at 1.times. primer concentration and 216C at
2.times. primer concentration results in an accumulated 3.times.
primer concentration on the x-axis. A multiplexed assay containing
216T with 1.times. primer concentration, 216C at 2.times. primer
concentration and 216A at 4.times. primer concentration results in
an accumulated 7.times. primer concentration on the x-axis. The
y-axis of FIG. 15 shows the relative FRET emission intensity (delta
values) calculated from the qPCR curves shown in FIGS. 8-14.
[0315] FIG. 15 indicates that the intensity in A.U. shown on y-axis
initially increases linearly with the level of accumulated primer
concentration shown on the x-axis. As the level of accumulated
primer concentration increases, the degree of multiplexing
increases, the linearity is decreasing. Such effect is expected to
be associated to a high level of sequence similarity between the
variation to be detected (here are SNPs) In particular, it is
expected that as the concentration of polynucleotide analyte in the
solution increases with increased PCR reaction time, the
probability of a primer finding a corresponding target sequence
decreases. This issue is expected to have a lower impact in FRET
detection were described where different variations to be detected
have a higher lower level of sequence similarity as will be
understood by a skilled person. Accuracy of the detection can be
improved by detecting a FRET signals in signature profiles curve
where detection of intensity is performed for a certain number of
cycles to identify the deviation from the expected result due to
the impact of sequence similarities or other experimental
conditions and the deviation due to a lack of target sequence. The
accuracy of the results can also be improved by adjusting the PCR
reaction time, concentrations of the WT, mutants or primers or
other PCR reaction factors.
Example 12: Detection of Multiple Mutations Based on Presence or
Absence of Signal Using FRETplexing
[0316] Alternatively, instead of detecting of multiple mutations
based on emission intensity, the detection can also be carried out
by determining the presence or absence of any mutation with a
simple yes/no system, in which a given intensity indicates that a
mutation is present. When no given intensity is detected, the
mutation is absent.
Instead of attempting to detect a specific mutation from
quantifying the signal intensity, the experiments were re-designed
to determine whether a particular mutation is present or not. Table
17 list the components contained in such mutation detection
experiments with the WT sequence at 10 uL of 100 uM
volume/concentration. The primer pair contained in Table 17 is
specific for the given mutation to be detected. Table 18 lists the
components in control experiments in which mutant sequences are
absent and the WT sequence has a 10 uL of 100 uM
volume/concentration in one control experiment and 10 uL of 1000 uM
volume/concentration in the other control experiment.
TABLE-US-00017 TABLE 17 Experiment cocktail for detection of any
given mutation in a multiplexed assay Components Concentration
Reagents 20 uL of Taq 5x matermix WT 10 uL of 100 uM 216T 10 uL of
10 uM 216A 10 uL of 10 uM 216C 10 uL of 10 uM 216T or 216A 8 uL of
10 uM or 216C primer
TABLE-US-00018 TABLE 18 Experiment cocktail for control experiments
Components Concentration Reagents 20 uL of Taq 5x matermix WT 10 uL
of 100 uM or 10 uL of 1000 uM 216T primer 8 uL of 10 uM 216A primer
8 uL of 10 uM 216C primer 8 uL of 10 uM
[0317] FIG. 16 shows a chart reporting the detection of the
presence or absence of a KRAS mutation 216A, 216T and 216C using
FRETplexing with qPCR. Each multiplexed assay contains 20 uL of Taq
5.times. matermix, 10 uL of 10 uM for a given mutant sequence, 8 uL
of 10 uM primer concentration for each of three primer pairs. The
forward primer is labeled with Cy3 as a FRET donor chromophore and
the reverse primer is labeled with Cy5 as a FRET acceptor
chromophore.
[0318] The chart shows averaged relative FRET emission intensities
(delta values at y-axis) of 12 qPCR replica experiments plotted for
each mutation also in comparison with wild type assay absence of
mutants at two different concentrations: 10 uL of 100 uM wild type
in one experiment and 10 uL of 1000 uM wild type in the other
experiment. Note that the false intensity signal was observed in
the wild-type samples, which may be caused by the mispriming of the
amplification enzyme. An intensity difference when a mutant
sequence is present or absent is also noted. The experiment reveals
that even though there is a high concentration of wild type primers
in the tested samples, the signal intensity is still greater for
the mutant sequence at a much lower concentration compared to that
of the wild type. In addition, the data also shows that the mutant
primers are inhibited when the WT sequence concentration is
increased to 100.times. and 1000.times. greater than the
concentrations of the mutant sequences. Therefore, the data
suggests that the FRETplex system can specifically amplify mutant
sequences and can be used to determine the presence or absence of a
mutant sequence when a response threshold is reached.
Example 13: PCR Amplification of Mutations 216C, 216T and 216A
Using Quenchiplexing
[0319] Described below are a number of PCR experiments of mutations
216C, 216T and 216A in different combinations according to the
FRETplexing experiments described in Examples 10-11. For each PCR
experiment, the concentration of the WT sequence is 10 times the
concentration mutant WT sequence. The data are shown to provide a
comparison with the above results obtained with FRETplex
approach.
[0320] Similar to the FRETplexing described in the above Examples,
the notation #.times. specifies a relative concentration of one
primer pair with respect to another primer pair that forms a
multiplexed assay. For each primer pair, the forward primer is
designed to be specific to a given mutation and the reverse primer
is a common primer. The two primers, forward and reverse primers,
are labeled with a fluorophore and a quencher, respectively. Table
19 shows the forward and reverse primers used for the
quenchiplexing experiments. The forward primer is labeled with Cy3
and the reverse primer is labeled with iABkFQ (Iowa black FQ). The
components for each of the PCR experiments including reagents, WT,
mutant and primers are tabulated in Tables 20-26. The fluorescence
intensity was recorded at the end of each PCR cycle and the data
are plotted into graphs shown in FIGS. 17-23.
TABLE-US-00019 TABLE 19 Exemplary forward and reverse primers
designed for detecting KRAS mutations using quenchiplexing KRAS SEQ
mutation Forward Primer ID NO Reverse Primers SEQ ID NO 216T /5Cy3
SEQ ID /5iABkFQ/ SEQ ID NO: 20 /GAATATAAACTTGT NO 10 GTCCACAAAATGA
GGTAGTTGGAGCTGT ATCTGAAT 216C /5Cy3 SEQ ID /5iABkFQ SEQ ID NO; 20
/GAATATAAACTTGT NO: 12 /GTCCACAAAATG GGTAGTTGGAGCTG AATCTGAAT C
219A /5Cy3 SEQ ID /5iABkFQ SEQ ID NO: 20 /GAATATAAACTTGT NO 14
/GTCCACAAAATG GGTAGTTGGAGCTG AATCTGAAT GTGA
TABLE-US-00020 TABLE 20 Experiment cocktail for detection of a
single 216C mutation at a 1.times. primer concentration Components
Volume/Concentration Reagents 20 uL of 100 uM Taq 5x matermix WT 10
uL of 100 uM 216C 10 uL of 10 uM 216C Primer 0.5 uL of 10 uM
TABLE-US-00021 TABLE 21 Experiment cocktail for detection of a
single 216T mutation at a 2.times. primer concentration Components
Concentration Reagents 20 uL of 100 uM Taq 5x matermix WT 10 uL of
100 uM 216T 10 uL of 10 uM 216T Primer 1 uL of 10 uM
TABLE-US-00022 TABLE 22 Experiment cocktail for detection of a
single 216A mutation at a 4.times. primer concentration Components
Concentration Reagents 20 uL of 100 uM Taq 5x matermix WT 10 uL of
100 uM 216A 10 uL of 10 uM 216A Primer 2 uL of 10 uM
TABLE-US-00023 TABLE 23 Experiment cocktail for detection of a
double mutation 216C at 1.times. and 216T at 2.times. primer
concentrations. Components Concentration Reagents 20 uL of 100 uM
Taq 5x matermix WT 10 uL of 100 uM 216C 10 uL of 10 uM 216T 10 uL
of 10 uM 216C Primer 0.5 uL of 10 uM 216T primer 1 uL of 10 uM
TABLE-US-00024 TABLE 24 Experiment cocktail for detection of a
double mutation 216C at 1.times. and 216A at 4.times. primer
concentrations. Components Concentration Reagents 20 uL of 100 uM
Taq 5x matermix WT 10 uL of 100 uM 216C 10 uL of 10 uM 216A 10 uL
of 10 uM 216C primer 0.5 uL of 10 uM 216A Primer 2 uL of 10 uM
TABLE-US-00025 TABLE 25 Experiment cocktail for detection of a
double mutation 216T at 2.times. and 216A at 4.times. primer
concentrations. Components Concentration Reagents 20 uL of 100 uM
Taq 5x matermix WT 10 uL of 100 uM 216T 10 uL of 10 uM 216A 10 uL
of 10 uM 216T primer 1 uL of 10 uM 216A Primer 2 uL of 10 uM
TABLE-US-00026 TABLE 26 Experiment cocktail for detection of a
triple mutation 216C at 1.times. and 216T at 2.times. and 216A at
4.times. primer concentrations. Components Concentration Reagents
20 uL of 100 uM Taq 5x matermix WT 10 uL of 100 uM 216T 10 uL of 10
uM 216A 10 uL of 10 uM 216C 10 uL of 10 uM 216T primer 1 uL of 10
uM 216C primer 0.5 uL of 10 uM 216A Primer 2 uL of 10 uM
[0321] FIGS. 17-23 show the results from the above described qPCR
amplification experiments using quenchiplexing.
[0322] FIG. 17 shows a chart reporting detection of 216C mutant
sequence from KRAS gene using quenchiplexing with qPCR. The
multiplexed assay contains 0.5 uL of 10 uM forward and reverse
primer concentration, 20 uL of Taq 5.times. matermix and 10 uL of
10 uM mutant 216C. The forward primer is labeled with Cy3 as a
fluorophore and the reverse primer is labeled with iABkFQ as a
quencher. The chart shows averaged fluorescence emission signal of
12 qPCR replica experiments (at y-axis) plotted as a function of
PCR cycles with a standard deviation of 1.sigma..
[0323] FIG. 18 shows a chart reporting detection of 216T mutant
sequence from KRAS gene using quenchiplexing with qPCR. The
multiplexed assay contains 1 uL of 10 uM forward and reverse primer
concentration, 20 uL of Taq 5.times. matermix and 10 uL of 10 uM
mutant 216T. The forward primer is labeled with Cy3 as a
fluorophore and the reverse primer is labeled with iABkFQ as a
quencher. The chart shows averaged fluorescence emission signal of
12 qPCR replica experiments (at y-axis) plotted as a function of
PCR cycles with a standard deviation of 1.sigma.. The amplification
phase starts at cycle 8 and the saturation phase at cycle 20.
[0324] FIG. 19 shows a chart reporting detection of 216A mutant
sequence from KRAS gene using quenchiplexing with qPCR. The
multiplexed assay contains 2 uL of 10 uM forward and reverse primer
concentration, 20 uL of Taq 5.times. matermix and 10 uL of 10 uM
mutant 216A. The forward primer is labeled with Cy3 as a
fluorophore and the reverse primer is labeled with iABkFQ as a
quencher. The chart shows averaged fluorescence emission signal of
12 qPCR replica experiments (at y-axis) plotted as a function of
PCR cycles with a standard deviation of 1.sigma.. The amplification
phase starts at cycle 8 and the saturation phase at cycle 32.
[0325] FIG. 20 shows a chart reporting detection of 216C and 216T
mutant sequence from K-RAS gene using quenchiplexing with qPCR. The
multiplexed assay contains 0.5 uL of 10 uM forward and reverse
primer concentration for 216C, 1 uL of 10 uM forward and reverse
primer concentration for 216T, 20 uL of Taq 5.times. matermix, 10
uL of 10 uM mutant 216C and 10 uL of 10 uM 216T. The forward primer
is labeled with Cy3 as a fluorophore and the reverse primer is
labeled with iABkFQ as a quencher. The chart shows averaged
fluorescence emission signal of 12 qPCR replica experiments (at
y-axis) plotted as a function of PCR cycles with a standard
deviation of 1.sigma.. The amplification phase starts at cycle 9
and the saturation phase at cycle 35.
[0326] FIG. 21 shows a chart reporting detection of 216C and 216A
mutant sequence from K-RAS gene using quenchiplexing with qPCR. The
multiplexed assay contains 0.5 uL of 10 uM forward and reverse
primer concentration for 216A, 2 uL of 10 uM forward and reverse
primer concentration for 216T, 20 uL of Taq 5.times. matermix, 10
uL of 10 uM mutant 216C and 10 uL of 10 uM 216A. The forward primer
is labeled with Cy3 as a fluorophore and the reverse primer is
labeled with iABkFQ as a quencher. The chart shows averaged
fluorescence emission signal of 12 qPCR replica experiments (at
y-axis) plotted as a function of PCR cycles with a standard
deviation of 1.sigma.. The amplification phase starts at cycle 11
and the saturation phase at cycle 36.
[0327] FIG. 22 shows a chart reporting detection of 216T and 216A
mutant sequence from K-RAS gene using quenchiplexing with qPCR. The
multiplexed assay contains 1 uL of 10 uM forward and reverse primer
concentration for 216T, 2 uL of 10 uM forward and reverse primer
concentration for 216A, 20 uL of Taq 5.times. matermix and 10 uL of
10 uM mutant 216T and 10 uL of 10 uM 216A. The forward primer is
labeled with Cy3 as a fluorophore and the reverse primer is labeled
with iABkFQ as a quencher. The chart shows averaged fluorescence
emission signal of 12 qPCR replica experiments (at y-axis) plotted
as a function of PCR cycles with a standard deviation of 1.sigma..
The amplification phase starts at cycle 11 and the saturation phase
at cycle 38.
[0328] FIG. 23 shows a chart reporting detection of 216C, 216T and
216A mutant sequence from KRAS gene using quenchiplexing with qPCR.
The multiplexed assay contains 0.5 uL of 10 uM forward and reverse
primer concentration for 216C, 1 uL of 10 uM forward and reverse
primer concentration for 216T, 2 uL of 10 uM forward and reverse
primer concentration for 216A, 20 uL of Taq 5.times. matermix, and
10 uL of 10 uM for each mutant 216C, 216T and 216A. The forward
primer is labeled with Cy3 as a fluorophore and the reverse primer
is labeled with iABkFQ as a quencher. The chart shows averaged
fluorescence emission signal of 12 qPCR replica experiments (at
y-axis) plotted as a function of PCR cycles with a standard
deviation of 1.sigma.. The amplification phase starts at cycle 11
and the saturation phase at cycle 36.
[0329] In FIGS. 17-18, the amplification phase starts at cycle 8
and the saturation phase at cycle 18. The slight increase in
fluorescence intensity observed after cycle 25 can be caused by the
reduction of the quencher efficiency. In FIGS. 19-23, the
amplification phase starts at cycle 8. However, the saturation
phase is not observed in the reactions, which can be due to the
fact that the PCR amplification appears to be linear instead of
undergoing a traditional exponential amplification.
[0330] The fluorescence intensity in A.U. decreases as the PCR
amplification progresses. Since in quenchiplexing the primers are
labeled with fluorophore and quencher, respectively, the
incorporation of fluorophore and quencher in the newly generated
amplicon can lead to quenching of fluorescence intensity from the
fluorophore. Therefore, with each iterative amplification or
polymerization reaction, the fluorescence intensity is reduced
gradually.
[0331] As the labeled forward and reverse primer concentrations are
increased, the initial fluorescence intensity (i.e. the
fluorescence intensity recorded at cycle 0) is increased and so is
the delta fluorescence intensity (i.e. the fluorescence intensity
difference between cycle 0 and the last cycle 40 shown in FIGS.
17-23). However, the change in delta values calculated from each of
the above PCR experiments with respect to particular primer
concentrations do not correspond well with the increase in the
primer concentrations. Similar to the FRETplexing experimental
results obtained in Example 11, the loss of linearity observed in
the quenchiplexing experiments also indicates that as the
concentration of mutant sequences in the solution increases with
the progress of PCR reaction, the probability of a primer finding
its correct target sequence becomes lower, which promotes
mispriming and accumulation of nonspecific amplification products.
Consequently, primer concentrations may be exhausted before the
reaction is completed, resulting in lower yields of desired
products, which is directly related to the lower than expected
emission intensity.
[0332] In addition, the change in the slope of the graphs shown in
FIGS. 17-23 is observed as the primer concentrations change. The
slope of the graphs in FIGS. 17-23 becomes less steep as the total
primer concentration increases. Such results also suggest that PCR
reactions may be inhibited as higher primer concentrations may
promote mispriming and accumulation of nonspecific products as the
PCR reaction progresses.
[0333] In comparison with FRETplexing shown in Examples 1-12, the
main difference between FRETplex and quenchiplex results from the
fact that FRETplex produces positive signal for a positive outcome
while quenchiplex produces a negative signal for a positive
outcome. From perspective of metrology and statistics, the former
is typically preferable. FRETplex also generally has a better
signal to noise ratio compared with quenchiplex, since contrary to
quenchplex FRET minimizes noise from scattering and
autofluorescence. Due to increased signal sensitivity, FRETplex can
preferably be applied to measurement of analytes where primers
result in longer double-stranded amplicons.
Example 14. Quantification and Abundance Measurements
[0334] Methods and kits herein described can be used to perform
quantification and abundance measurement of genetic variations such
as SNPs. Accordingly methods and kits herein described can be used
to investigate the effect of a mutation in quantitative terms. For
example, in the field of oncology, if a mutation confers drug
resistance, an issue to investigate is whether there a critical
cutoff in its abundance in the population above which enough cancer
cells survive therapy to repopulate and kill the patient. Such
issue and similar require mutation abundance measurements on tumor
samples of different outcomes or stages of development. To measure
the abundance accurately, one must measure the total DNA present
(mutant and WT) and the amount of mutant DNA. FRETplex can be used
to quantify the mutant by using the FRETplex signal in a manner
similar to real-time PCR, e.g. measuring threshold cycles and
comparing them to a quantified control. This is an example of use
of signatures of FRETplex signal, as the shape of the curve of
signal vs cycle number reveals important properties of the
sample.
Example 15. Multiplexed Detection
[0335] Multiplexed FRETplex assays are expected to allow several
mutations to be tracked at the same time. The cumulative FRETplex
signal in a same color can be considered essentially as a sum of
the FRETplex real-time curves of each constituent assay. Then
peculiarities of that curve, e.g. inflection points, step-wise
saturations, threshold cycles, etc., can be used to decode the
presence and characteristics of particular analytes. For example,
if the curve experiences a Ct, then saturates, then shows another
Ct then saturates at an even higher level, the decoding analysis
will identify that a mutation that was amplitude-coded by the
magnitude of the first plateau was present at a starting
concentration consistent with the first Ct, while a mutation that
was amplitude-coded by a magnitude that is the difference between
the heights of the two plateau was present at a starting
concentration consistent with the second Ct.
Example 16. Coding by FRET Distance
[0336] An additional application of methods and kits herein
described is to perform coding by FRET distance. One can build a
Multiplexed FRETplex assay e.g. for two analytes using the FRET
distance as a means to code signal amplitude. For example, the
first assay can be coded by a primer pair that will result in
amplicons with FRET distance of say 10 nm (.about.30 bp), while the
second analyte is coded by primers using a FRET distance of say 15
nm (.about.45 bp). If both assays are included in the multiplexed
assay at the same primer concentration, and the resulting
multiplexed assay is run to completion, there will be four possible
levels of output signal: signal close to background means neither
analyte was present; low signal above background means the second
analyte was present alone; medium signal above background.
[0337] The examples set forth above are provided to give those of
ordinary skill in the art a complete disclosure and description of
how to make and use the embodiments of the materials, compositions,
systems and methods of the disclosure, and are not intended to
limit the scope of what the inventors regard as their
disclosure.
[0338] All patents and publications mentioned in the specification
are indicative of the levels of skill of those skilled in the art
to which the disclosure pertains.
[0339] The entire disclosure of each document cited (including
patents, patent applications, journal articles, abstracts,
laboratory manuals, books, or other disclosures) in the Background,
Summary, Detailed Description, and Examples is hereby incorporated
herein by reference. All references cited in this disclosure are
incorporated by reference to the same extent as if each reference
had been incorporated by reference in its entirety individually.
However, if any inconsistency arises between a cited reference and
the present disclosure, the present disclosure takes
precedence.
[0340] The terms and expressions which have been employed herein
are used as terms of description and not of limitation, and there
is no intention in the use of such terms and expressions of
excluding any equivalents of the features shown and described or
portions thereof, but it is recognized that various modifications
are possible within the scope of the disclosure claimed. Thus, it
should be understood that although the disclosure has been
specifically disclosed by embodiments, exemplary embodiments and
optional features, modification and variation of the concepts
herein disclosed can be resorted to by those skilled in the art,
and that such modifications and variations are considered to be
within the scope of this disclosure as defined by the appended
claims.
[0341] It is also to be understood that the terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to be limiting. As used in this specification and
the appended claims, the singular forms "a," "an," and "the"
include plural referents unless the content clearly dictates
otherwise. The term "plurality" includes two or more referents
unless the content clearly dictates otherwise. Unless defined
otherwise, all technical and scientific terms used herein have the
same meaning as commonly understood by one of ordinary skill in the
art to which the disclosure pertains.
[0342] When a Markush group or other grouping is used herein, all
individual members of the group and all combinations and possible
subcombinations of the group are intended to be individually
included in the disclosure. Every combination of components or
materials described or exemplified herein can be used to practice
the disclosure, unless otherwise stated. One of ordinary skill in
the art will appreciate that methods, device elements, and
materials other than those specifically exemplified may be employed
in the practice of the disclosure without resort to undue
experimentation. All art-known functional equivalents, of any such
methods, device elements, and materials are intended to be included
in this disclosure. Whenever a range is given in the specification,
for example, a temperature range, a frequency range, a time range,
or a composition range, all intermediate ranges and all subranges,
as well as, all individual values included in the ranges given are
intended to be included in the disclosure. Any one or more
individual members of a range or group disclosed herein may be
excluded from a claim of this disclosure. The disclosure
illustratively described herein suitably may be practiced in the
absence of any element or elements, limitation or limitations which
is not specifically disclosed herein.
[0343] A number of embodiments of the disclosure have been
described. The specific embodiments provided herein are examples of
useful embodiments of the invention and it will be apparent to one
skilled in the art that the disclosure can be carried out using a
large number of variations of the devices, device components,
methods steps set forth in the present description. As will be
obvious to one of skill in the art, methods and devices useful for
the present methods may include a large number of optional
composition and processing elements and steps.
[0344] In particular, it will be understood that various
modifications may be made without departing from the spirit and
scope of the present disclosure. Accordingly, other embodiments are
within the scope of the following claims.
* * * * *