U.S. patent application number 15/670963 was filed with the patent office on 2018-02-01 for methods and compositions for detecting target nucleic acids.
The applicant listed for this patent is DXTERITY DIAGNOSTICS INCORPORATED. Invention is credited to Majid R. ABEDI, John Ray CHILDERS, Chang Hee KIM, Yenbou LIU, Robert TERBRUEGGEN.
Application Number | 20180030520 15/670963 |
Document ID | / |
Family ID | 46168641 |
Filed Date | 2018-02-01 |
United States Patent
Application |
20180030520 |
Kind Code |
A1 |
TERBRUEGGEN; Robert ; et
al. |
February 1, 2018 |
METHODS AND COMPOSITIONS FOR DETECTING TARGET NUCLEIC ACIDS
Abstract
The present invention provides compositions, apparatuses and
methods for detecting one or more nucleic acid targets present in a
sample. Methods of the invention include utilizing two or more
ligation probes that reversibly bind a target nucleic acid in close
proximity to each other and possess complementary reactive ligation
moieties. When such probes have bound to the target in the proper
orientation, they are able to undergo a spontaneous chemical
ligation reaction that yields a ligation product that is directly
detected or that is amplified to produce amplicons that are then
detected. The present invention also provides methods to stabilize
sample RNA so that degradation does not significantly affect the
results of the analysis.
Inventors: |
TERBRUEGGEN; Robert;
(Manhattan Beach, CA) ; LIU; Yenbou; (Arcadia,
CA) ; CHILDERS; John Ray; (Long Beach, CA) ;
KIM; Chang Hee; (Rancho Palos Verdes, CA) ; ABEDI;
Majid R.; (Anaheim, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DXTERITY DIAGNOSTICS INCORPORATED |
Rancho Dominguez |
CA |
US |
|
|
Family ID: |
46168641 |
Appl. No.: |
15/670963 |
Filed: |
August 7, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14118524 |
Mar 5, 2014 |
9745616 |
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PCT/US12/38436 |
May 17, 2012 |
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15670963 |
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61486817 |
May 17, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6813 20130101;
C12Q 1/6809 20130101; C12Q 1/6809 20130101; C12Q 1/6855 20130101;
C12Q 1/6862 20130101; C12Q 2563/179 20130101; C12Q 2525/155
20130101; C12Q 2525/161 20130101; C12Q 2565/125 20130101; C12Q
2563/131 20130101; C12Q 2525/155 20130101; C12Q 2525/204 20130101;
C12Q 2533/107 20130101; C12Q 2525/197 20130101; C12Q 2537/143
20130101; C12Q 2525/197 20130101; C12Q 2525/204 20130101; C12Q
2565/125 20130101; C12Q 2523/109 20130101; C12Q 2537/143 20130101;
C12Q 2523/109 20130101; C12Q 2563/179 20130101; C12Q 1/6834
20130101; C12Q 1/6834 20130101; C12Q 1/6855 20130101; C12Q 2523/109
20130101; C12Q 2525/155 20130101; C12Q 2525/15 20130101; C12Q
2525/161 20130101; C12Q 2525/204 20130101; C12Q 2525/155 20130101;
C12Q 2537/143 20130101; C12Q 2565/125 20130101; C12Q 2563/131
20130101; C12Q 2563/131 20130101; C12Q 2525/161 20130101; C12Q
2523/109 20130101; C12Q 2525/204 20130101; C12Q 2533/107 20130101;
C12Q 2533/107 20130101; C12Q 2563/179 20130101; C12Q 2523/109
20130101; C12Q 2525/155 20130101; C12Q 2525/197 20130101; C12Q
2525/204 20130101; C12Q 1/6862 20130101; C12Q 1/6862 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under Grant
No. 1R43HG006656-01 awarded by the Small Business Innovation
Research (SBIR) program. The government has certain rights in the
invention.
Claims
1.-32. (canceled)
33. A method for detecting a plurality of different target nucleic
acids in a blood sample, wherein each target nucleic acid comprises
a first target domain adjacent to a second target domain, said
method comprising: (a) contacting the blood sample comprising the
different target nucleic acids with a lysis buffer to form a
reaction mixture; (b) contacting the reaction mixture directly with
a plurality of different probe sets, each probe set comprising: (i)
a first ligation probe comprising: (1) a first probe domain
complementary to the first target domain of one target nucleic acid
in the plurality of target nucleic acids; (2) a first primer
sequence; and (3) a 5' ligation moiety; and (ii) a second ligation
probe comprising: (1) a second probe domain complementary to the
second target domain of the one target nucleic acid in the
plurality of target nucleic acid; (2) a second primer sequence; and
(3) a 3' ligation moiety; (c) ligating said first and second
ligation probes without the use of a ligase enzyme to form a
plurality of different ligation products; (d) amplifying the
ligation different ligation products; and (e) detecting the
presence of the ligation products
34. A method for detecting a plurality of different target nucleic
acids in a formalin-fixed, paraffin-embedded (FFPE) tissue sample,
wherein each target nucleic acid comprises a first target domain
adjacent to a second target domain, said method comprising: (a)
contacting the FFPE tissue sample comprising the different target
nucleic acids with a lysis buffer to form a reaction mixture; (b)
contacting the reaction mixture directly with a plurality of
different probe sets, each probe set comprising: (i) a first
ligation probe comprising: (1) a first probe domain complementary
to the first target domain of one target nucleic acid in the
plurality of target nucleic acids; (2) a first primer sequence; and
(3) a 5' ligation moiety; and (ii) a second ligation probe
comprising: (1) a second probe domain complementary to the second
target domain of the one target nucleic acid in the plurality of
target nucleic acid; (2) a second primer sequence; and (3) a 3'
ligation moiety; (c) ligating said first and second ligation probes
without the use of a ligase enzyme to form a plurality of different
ligation products; (d) amplifying the different ligation products
to form amplicons; and (e) detecting the amplicons.
35. The method of claim 33, wherein step (a) and (b) are performed
at the same location.
36. The method of claim 33, wherein step (a) and (b) are performed
at different locations.
37. The method of claim 33, wherein step (b) is performed one day
to three months after step (a).
38. The method of claim 33, wherein each of the ligation probes
comprises a primer sequence.
39. The method of claim 33, wherein the 5' ligation moiety
comprises DABSYL and the 3' ligation moiety comprises
phosphorothioate.
40. The method of claim 33, wherein the lysis buffer comprises
gunadinium hydrochloride (GuHCl).
41. The method of claim 40, wherein the lysis buffer comprises 3M
GuHCl.
42. The method of claim 33, wherein the target nucleic acids are
DNA.
43. The method of claim 33, wherein the target nucleic acids are
RNA.
44. A method for assessing the degradation of a target nucleic acid
in a sample, wherein the target nucleic acid comprises a first
target domain adjacent to a second target domain, a third target
domain adjacent to a fourth target domain and a target capture
domain, wherein the third target domain and fourth target domain
are upstream of the first target domain and second target domain,
wherein the first target capture domain is downstream of the first
target domain and second target domain, and wherein the first,
second, third and fourth target domains are at known distances from
the target capture probe domain, the method comprising: (a)
contacting the sample comprising the target nucleic acid with a
lysis buffer to form a reaction mixture; (b) contacting the
reaction mixture with a probe set, the probe set comprising: (i) a
first ligation probe comprising: (1) a first probe domain
complementary to the first target domain of one target nucleic
acid; (2) a first primer sequence; and (3) a 5' ligation moiety;
and (ii) a second ligation probe comprising: (1) a second probe
domain complementary to the second target domain of the target
nucleic acid; (2) a second primer sequence; and (3) a 3' ligation
moiety; (iii) a third ligation probe comprising: (1) a third probe
domain complementary to the third target domain of the target
nucleic acid; (2) a third primer sequence; and (3) a 5' ligation
moiety; (iv) a fourth ligation probe comprising: (1) a fourth probe
domain complementary to the fourth target domain of the target
nucleic acid; (2) a fourth primer sequence; and (3) a 3' ligation
moiety; (c) ligating said first and second ligation probe to form a
first ligation product and said third and fourth ligation probe and
the fifth ligation probe to form a second ligation product without
the use of a ligase enzyme; (d) amplifying the first and second
ligation products; and (e) detecting the presence of the first and
second ligation products, thereby assessing the degradation of the
target nucleic acid.
45. The method of claim 44, wherein each of the 5' ligation
moieties comprises DABSYL and each of the 3' ligation moiety
comprises phosphorothioate.
46. The method of claim 44, wherein the lysis buffer comprises
gunadinium hydrochloride (GuHCl).
47. The method of claim 46, wherein the lysis buffer comprises 3M
GuHCl.
48. The method of claim 44, wherein the target nucleic acid is
DNA.
49. The method of claim 33, wherein the target nucleic acid is
RNA.
50. The method of claim 44, wherein the capture moiety of the
target capture probe comprises a member selected from the group
consisting of a capture nucleic acid, a bead and a bind partner of
a binding partner pair.
51. The method of claim 44, wherein the target nucleic acid further
comprises a fifth target domain adjacent to a sixth target domain,
wherein the fifth and sixth target domains are upstream of the
third and fourth target domains and are at a known distance for the
target capture domain, the probe set further comprising: (v) a
fifth ligation probe comprising: (1) a fifth probe domain
complementary to the fifth target domain of the target nucleic
acid; (2) a fifth primer sequence; and (3) a 5' ligation moiety;
(iv) a sixth ligation probe comprising: (1) a sixth probe domain
complementary to the sixth target domain of the target nucleic
acid; (2) a sixth primer sequence; and (3) a 3' ligation moiety,
wherein (c) further comprises ligating the fifth and sixth ligation
probes to form a third ligation product, wherein (d) amplifying
further comprises amplifying the third ligation product; and
wherein (e) detecting further comprises detecting the third
ligation product.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. patent
application Ser. No. 14/118,524 with a filing date of Mar. 5, 2014
which is a 371 National Phase of International Application No.
PCT/US2012/038436 filed May 17, 2012 which claims priority to U.S.
Provisional Patent Application No. 61/486,817, filed on May 17,
2011, each of which is hereby incorporated by reference in its
entirety.
REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER PROGRAM
LISTING APPENDIX SUBMITTED ON A COMPACT DISK
[0003] The sequence listing contained in the file named
"068433-5003_ST25", created on Aug. 7, 2017 and having a size of
12.2 kilobytes, has been submitted electronically herewith via
EFS-Web, and the contents of the txt file are hereby incorporated
by reference in their entirety.
BACKGROUND OF THE INVENTION
[0004] The detection of specific nucleic acids is an important tool
for diagnostic medicine and molecular biology research. Gene probe
assays currently play a role in a number of spheres of diagnostic
medicine and molecular biology, including for example identifying
infectious organisms such as bacteria and viruses, in probing the
expression of normal and mutant genes and identifying genes
associated with disease or injury, such as oncogenes, in typing
tissue for compatibility preceding tissue transplantation, in
matching tissue or blood samples for forensic medicine, for
responding to emergency response situations like a nuclear incident
or pandemic flu outbreak, in determining disease prognosis or
causation, and in exploring homology among genes from different
species.
[0005] Ideally, a gene probe assay should be sensitive, specific
and easily automatable. The requirement for sensitivity (i.e. low
detection limits) has been greatly alleviated by the development of
the polymerase chain reaction (PCR) and other amplification
technologies which allow researchers to exponentially amplify a
specific nucleic acid sequence before analysis. For example,
multiplex PCR amplification of SNP loci with subsequent
hybridization to oligonucleotide arrays can be used to
simultaneously genotype hundreds of SNPs.
[0006] Specificity is a challenge for gene probe assays. The extent
of molecular complementarity between probe and target defines the
specificity of the interaction. Variations in composition and
concentrations of probes, targets and salts in the hybridization
reaction as well as the reaction temperature, and length of the
probe may all alter the specificity of the probe/target
interaction. It can be possible under some circumstances to
distinguish targets with perfect complementarity from targets with
mismatches, although this is generally difficult using traditional
technology, since small variations in the reaction conditions will
alter the hybridization. Techniques for mismatch detection include
probe digestion assays in which mismatches create sites for probe
cleavage, and DNA ligation assays where single point mismatches
prevent ligation.
[0007] A variety of enzymatic and non-enzymatic methods are
available for detecting sequence variations. Examples of enzyme
based methods include Invader.TM., oligonucleotide ligation assay
(OLA) single base extension methods, allelic PCR, and competitive
probe analysis (e.g. competitive sequencing by hybridization).
Enzymatic DNA ligation reactions are well known in the art and have
been used extensively in SNP detection, enzymatic amplification
reactions and DNA repair. A number of non-enzymatic or template
mediated chemical ligation methods can also be used to detect
sequence variations. These include chemical ligation methods that
utilize coupling reagents, such as N-cyanoimidazole, cyanogen
bromide, and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
hydrochloride.
[0008] A widely recognized problem when analyzing RNA for genetic
studies is the inherent instability of the RNA itself. RNA
naturally has a short lifetime in living organisms because
organisms regulate the RNA concentration which regulates downstream
processes which are dependent on the RNA. There are also many
natural processes which lead to the destruction of an RNA.
[0009] In recent years researchers have spent considerable effort
developing methods for the analysis of gene expression in cells.
This is generally accomplished by analyzing the cell contents for
the amount of specific mRNA molecules present. Measurements of gene
expression are based on the underlying assumption that the analyzed
RNA sample closely resembles the number of transcripts in vivo.
Hence, maintaining the integrity of the RNA after extraction from
the cell is of paramount importance. Researchers have recognized
that transcripts of different genes (mRNA) possess different
stabilities which implies that that degradation of RNA occurring
during the isolation procedure may be non-uniformly distributed
among different RNA molecules. One comparison of RNA samples of
different degrees of degradation shows that up to 75% of
microarray-based measurements of differential gene expression can
be caused by degradation bias. Auer H, Liyanarachchi S, Newsom D,
Klisovic M I, Marcucci G, Kornacker K (2003), Nature Genetics
35:292-293.
[0010] There remains a need for methods and compositions for
efficient and specific nucleic acid detection and for stabilization
of RNA following extraction from cells.
BRIEF SUMMARY OF THE INVENTION
[0011] Accordingly, the present invention provides methods and
compositions for non-enzymatic chemical ligation reactions which
provides rapid target detection and greatly simplified processes of
detecting and measuring target nucleic acids.
[0012] In one aspect, the present invention provides a method for
detecting a plurality of different target nucleic acids in a
sample, wherein each target nucleic acid comprises a first target
domain adjacent to a second target domain and a third target
capture domain located upstream or downstream from the first and
second target domains. The method comprises the steps of (a)
providing a plurality of ligation substrates each comprising one of
the target nucleic acids; a first set of ligation probes comprising
(i) a first nucleic acid ligation probe comprising a first probe
domain hybridized to a first target domain of the one target
nucleic acid sequence and a 5'-ligation moiety; and (ii) a second
nucleic acid ligation probe comprising a second probe domain
hybridized to a second target domain of the one target nucleic acid
sequence and a 3' ligation moiety; (b) ligating the first and
second ligation probes without the use of a ligase enzyme to form a
first plurality of ligation products; (c) hybridizing target
capture probes comprising a capture moiety to the third target
domain of the target nucleic acids to form target complexes; (d)
capturing the target complexes on a surface using the capture
moiety; amplifying the ligation products to form amplicons; and (0
detecting the amplicons, thereby detecting the target nucleic
acids.
[0013] In a further embodiment and in accordance with the above,
the target nucleic acids further comprise a fourth target domain
adjacent to a fifth target domain and the plurality of ligation
substrates each further comprises a second set of ligation probes
comprising a third nucleic acid ligation probe hybridized to the
fourth target domain and a fourth nucleic acid ligation probe
hybridized to the fifth target domain. In such embodiments, the
method further comprises the steps of ligating the third and fourth
ligation probes in the absence of a ligase enzyme such that the one
target nucleic acid sequence comprises multiple ligation products
and the detecting step (f) comprises detecting amplicons generated
from the multiple ligation products.
[0014] In a further embodiment and in accordance with any of the
above, the target nucleic acids further comprise a sixth target
domain adjacent to a seventh target domain, the plurality of
ligation substrates each further comprises a third set of ligation
probes comprising a fifth nucleic acid ligation probe hybridized to
the sixth target domain and a sixth nucleic acid ligation probe
hybridized to the seventh target domain, and the method further
comprises ligating the third and fourth ligation probes in the
absence of a ligase enzyme such that the one target nucleic acid
sequence comprises multiple ligation products.
[0015] In a still further embodiment and in accordance with any of
the above, each of the ligation probes further comprises a primer
sequence.
[0016] In a still further embodiment and in accordance with any of
the above, at least one of the ligation probes of each set of
ligation probes further comprises a variable spacer sequence such
that the amplicons have a target specific length.
[0017] In a yet further embodiment and in accordance with any of
the above, only one ligation probe of each set of ligation probes
comprises the variable spacer sequence.
[0018] In a yet further embodiment and in accordance with any of
the above, the variable spacer sequence is contained between the
probe domain and the primer of the ligation probe.
[0019] In a further embodiment and in accordance with any of the
above, the capture moiety of the target capture probe comprises a
member selected from: a capture nucleic acid sequence, a bead and a
binding partner of a binding partner pair.
[0020] In a further embodiment and in accordance with any of the
above, the 5' ligation moiety comprises DABSYL and the 3' ligation
moiety comprises phosphorothioate.
[0021] In a further embodiment and in accordance with any of the
above, the 3' ligation moiety comprises DABSYL.
[0022] In a further embodiment and in accordance with any of the
above, the sample was collected into a buffer comprising
guanidinium hydrochloride (GuHCl). In an exemplary embodiment, the
sample is blood.
[0023] In a further embodiment and in accordance with any of the
above, the target nucleic acids comprise DNA or RNA.
[0024] In a still further embodiment and in accordance with any of
the above, the detecting step (f) utilizes a technique selected
from the group consisting of capillary electrophoresis, mass
spectrometry, microarray analysis, sequencing, real-time PCR,
optical detection, fluorescence detection, bioluminescence
detection, chemiluminescence detection, electrochemical detection,
electrochemiluminescence detection and lateral flow detection.
[0025] In a further embodiment and in accordance with any of the
above, the hybridizing step (c) occurs simultaneously with the
ligating step (b).
[0026] In a further aspect and in accordance with any of the above,
the present invention provides a method of detecting a plurality of
different target nucleic acids in a sample, wherein each target
sequence comprises an adjacent first and a second target domain,
the method comprising: (a) providing a plurality of ligation
substrates each comprising one of the different target nucleic
acids, a first nucleic acid ligation probe comprising a first probe
domain complementary to a first target domain of the one target
nucleic acid, a first primer sequence; and a 5'-ligation moiety;
and a second nucleic acid ligation probe comprising: a second probe
domain complementary to a second target domain of the one target
nucleic acid, a second primer sequence, and a 3' ligation moiety.
One of the ligation probe comprises a variable spacer sequence. The
method further comprises the steps of (b) ligating the first and
second ligation probes in the absence of a ligase enzyme to form a
plurality of different ligation products, wherein different
ligation products have different target specific lengths; (c)
amplifying the ligation product; and (d) detecting the presence of
the different ligation products on the basis of the different
target lengths.
[0027] In a further embodiment and in accordance with any of the
above, the target nucleic acid sequences are RNA or DNA.
[0028] In a further embodiment and in accordance with any of the
above, the sample is derived from blood.
[0029] In a still further embodiment and in accordance with any of
the above, the sample is derived from paraffin embedded
samples.
[0030] In a yet further embodiment and in accordance with any of
the above, the detecting is by capillary electrophoresis or by mass
spectrometry.
[0031] In a further embodiment and in accordance with any of the
above, each of the first primers are the same and each of the
second primers are the same.
[0032] In further aspects and in accordance with any of the above,
the present invention provides a kit for detecting a target nucleic
acid sequence, wherein the target sequence comprises an adjacent
first and a second target domain, the kit comprising: (a) 2.times.
lysis buffer comprising 6 M GuHCl; (b) a first ligation probe
comprising: (i) a first probe domain complementary to the first
target domain; (ii) a first primer sequence; and (iii) a
5'-ligation moiety; and (c) a second nucleic acid ligation probe
comprising: (i) a second probe domain complementary the second
target domain; (ii) a second primer sequence; and (iii) a 3'
ligation moiety.
[0033] In a further embodiment and in accordance with any of the
above, one or both of the ligation probes further comprise a
variable spacer sequence.
[0034] In a further aspect and in accordance with any of the above,
the present invention provides a method of detecting a plurality of
different target nucleic acids in a sample, wherein each target
sequence comprises an adjacent first and a second target domain,
the method comprising: (a) providing a reaction mixture comprising:
(i) a target sample comprising blood; and (ii) 1.times. lysis
buffer comprising 3 M GuHCl; (b) contacting the reaction mixture
with a plurality of different probes sets, each probe set
comprising: (i) a first ligation probe comprising: (1) a first
probe domain complementary to a first target domain of the one
target nucleic acid; (2) a first primer sequence; and (3) a
5'-ligation moiety; and (iii) a second nucleic acid ligation probe
comprising: (1) a second probe domain complementary to a second
target domain of the one target nucleic acid; (2) a second primer
sequence; and (3) a 3' ligation moiety; (d) ligating the first and
second ligation probes in the absence of a ligase enzyme to form a
plurality of different ligation products; (e) amplifying the
different ligation products; and (f) detecting the presence of the
ligation products.
[0035] In a further embodiment and in accordance with any of the
above, the ligation probes further comprises a variable spacer
sequence.
[0036] In a further embodiment and in accordance with any of the
above, the target nucleic acids are RNA.
[0037] In a further embodiment and in accordance with any of the
above, one of the first and second ligation probes further
comprises one of a binding partner pair, and prior to amplifying, a
bead comprising the other binding pair is added to capture the
ligated products.
[0038] In a further embodiment and in accordance with any of the
above, the detecting is done using the variable spacer
sequence.
[0039] In a further aspect and in accordance with any of the above,
the present invention provides a method for detecting a plurality
of target RNA sequences in a sample, where each target nucleic acid
sequence comprises an adjacent first and second target domain and a
third target domain. The method includes the steps of collecting a
sample into a buffer to form a stabilized sample; conducting an
assay in accordance with any of the above in the stabilized sample
to detect the plurality of target nucleic acids.
[0040] In a further embodiment and in accordance with any of the
above, the sample is stable for at least 1 week at ambient room
temperature.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIG. 1 is a schematic representation of one embodiment of
the CLPA-CE assay.
[0042] FIG. 2 is a schematic representation of one embodiment of
CLPA-MDM assay.
[0043] FIG. 3 is a schematic representation showing one embodiment
of the 2-probe and the 3-probe CLPA reaction.
[0044] FIG. 4 is a schematic representation of a DNA synthesis
resin that can be used to manufacture DNA with a 3'-DABSYL leaving
group
[0045] FIG. 5 is a schematic representation on the process flow for
one embodiment of the CLPA-CE assay
[0046] FIG. 6 is a schematic representation showing probe design
for a CLPA assay in which the probe contains a size-variant stuffer
sequence.
[0047] FIG. 7 shows an electrophoretic separation profile on a
sample analyzed by CLPA-CE.
[0048] FIG. 8 shows the linear relationship between target
concentration and peak height in CLPA-CE analysis.
[0049] FIG. 9 shows data for an analysis of FFPE tissue samples
comparing a Luminex Signal for various targets with a slice of
prostate FFPE tissue (left hand bar for each target) and the no
target control (pNTC--right hand bar for each target).
[0050] FIG. 10. is a schematic representation of a target capture
method used to separate bound CLPA probe sets from solution
phase/unbound CLPA probe sets.
[0051] FIG. 11 is a schematic illustration of multiple, unique CLPA
probe sets that are bound to the sample target along with a single
target capture probe.
[0052] FIG. 12A-FIG. 12C is a further schematic illustration of
possible orientations of one embodiment of the present invention,
which can find particular use in assessing sample integrity. FIG.
12A depicts a similar orientation to FIG. 11, except with the
capture probe(s) "downstream" of the ligation probe sets. CM is a
capture moiety. As will be appreciated by those in the art, the CM
can be on either the 3' or 5' terminus of the capture probe,
although it usually is depicted on the 3' end. In addition, the
portion of each ligation probe that does not hybridize to a target
domain can contain a number of different functionalities,
including, but not limited to, primer binding domains, size tags,
capture sequences, etc., as is shown in FIG. 11. FIG. 12A shows a
situation where the ligation probe sets are spaced over the length
of the target in roughly 25-30% increments for a sample integrity
assessment. As will be appreciated by those in the art and
described below, the spacing of the different ligation probe sets
can vary as needed. FIG. 12B depicts an alternative orientation.
FIG. 12C depicts an orientation that can be used both for integrity
assessment or redundancy.
[0053] FIG. 13A-FIG. 13C depict several schematic of embodiments of
the invention for use in SNP detection.
DETAILED DESCRIPTION OF THE INVENTION
[0054] The practice of the present invention may employ, unless
otherwise indicated, conventional techniques and descriptions of
organic chemistry, polymer technology, molecular biology (including
recombinant techniques), cell biology, biochemistry, and
immunology, which are within the skill of the art. Such
conventional techniques include polymer array synthesis,
hybridization, ligation, phage display, and detection of
hybridization using a label. Specific illustrations of suitable
techniques can be had by reference to the example herein below.
However, other equivalent conventional procedures can, of course,
also be used. Such conventional techniques and descriptions can be
found in standard laboratory manuals such as Genome Analysis: A
Laboratory Manual Series (Vols. I-IV), Using Antibodies: A
Laboratory Manual, Cells: A Laboratory Manual, PCR Primer: A
Laboratory Manual, and Molecular Cloning: A Laboratory Manual (all
from Cold Spring Harbor Laboratory Press), Stryer, L. (1995)
Biochemistry (4th Ed.) Freeman, New York, Gait, "Oligonucleotide
Synthesis: A Practical Approach" 1984, IRL Press, London, Nelson
and Cox (2000), Lehninger, Principles of Biochemistry 3.sup.rd Ed.,
W. H. Freeman Pub., New York, N.Y. and Berg et al. (2002)
Biochemistry, 5.sup.th Ed., W. H. Freeman Pub., New York, N.Y., all
of which are herein incorporated in their entirety by reference for
all purposes.
[0055] Note that as used herein and in the appended claims, the
singular forms "a," "an," and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to "a polymerase" refers to one agent or mixtures of such
agents, and reference to "the method" includes reference to
equivalent steps and methods known to those skilled in the art, and
so forth.
[0056] 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 this invention belongs. All
publications mentioned herein are incorporated herein by reference
for the purpose of describing and disclosing devices, compositions,
formulations and methodologies which are described in the
publication and which might be used in connection with the
presently described invention.
[0057] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range is encompassed within the invention. The
upper and lower limits of these smaller ranges may independently be
included in the smaller ranges is also encompassed within the
invention, subject to any specifically excluded limit in the stated
range. Where the stated range includes one or both of the limits,
ranges excluding either both of those included limits are also
included in the invention.
[0058] In the following description, numerous specific details are
set forth to provide a more thorough understanding of the present
invention. However, it will be apparent to one of skill in the art
that the present invention may be practiced without one or more of
these specific details. In other instances, well-known features and
procedures well known to those skilled in the art have not been
described in order to avoid obscuring the invention.
[0059] All numerical designations, e.g., pH, temperature, time,
concentration, and molecular weight, including ranges, are
approximations which are varied (+) or (-) by increments of 0.1. It
is to be understood, although not always explicitly stated that all
numerical designations are preceded by the term "about". The term
"about" also includes the exact value "X" in addition to minor
increments of "X" such as "X+0.1" or "X-0.1." It also is to be
understood, although not always explicitly stated, that the
reagents described herein are merely exemplary and that equivalents
of such are known in the art.
1. Overview
[0060] The present invention includes methods and compositions for
detecting target nucleic acids in a sample. In general, target
nucleic acids are detected through methods that include a step of
chemical ligation in which two ligation probes hybridized to
adjacent domains of a target nucleic acid are ligated without the
use of an exogenous ligase enzyme.
[0061] The invention provides methods utilizing two or more
ligation probes (also referred to herein as "oligonucleotide
probes") that reversibly bind a target nucleic acid in close
proximity to each other and possess complementary reactive ligation
moieties. In the ligation reaction, when the probes have bound to
the target in the proper orientation, they are able to undergo a
spontaneous chemical ligation reaction that yields a ligated
oligonucleotide product that does not rely on the use of a ligase
enzyme. The presence of the target(s) of interest can then be
determined by measuring the presence or amount of ligated
oligonucleotide product (also referred to herein as a "ligation
product") in a number of different ways. As is described below, the
ligation probes can contain a variety of additional
functionalities, including, but not limited to, detectable labels
to aid in the identification, quantification or detection of the
ligated oligonucleotide product, including, for example, direct
labels such as optical, and electrochemical labels, etc. as more
fully described below, variable spacer sequences or "size tags"
comprising nucleic acid sequences that are sized to be specific for
a particular target, such that detecting ligation products (or
amplicons generated from such ligation products) of a particular
size identifies the presence and/or amount of a particular target
nucleic acid sequence. Another optional functionality for inclusion
in one or more of the ligation probes are capture moieties designed
for subsequent capture on a solid support (e.g. microarrays,
microbeads, nanoparticles, etc.), which include, but are not
limited to, binding partners such as biotin, anchoring
oligonucleotide sequences (also referred to herein as "anchor
sequences" or "capture sequences") molecular handles that promote
the concentration or manipulation of the ligated product (magnetic
particles, oligonucleotide coding sequences), and promoter and
primer sequences to facilitate subsequent secondary amplification
of the ligated product via an enzyme like a DNA or RNA
polymerase.
[0062] Preferably, the ligation reactions of the invention do not
require the presence of exogeneously added ligases, nor additional
enzymes, although some secondary reactions may rely on the use of
enzymes such as polymerases, as described below. Amplification of
the target may also include turnover of the ligation product, in
which the ligation product has a lower or comparable affinity for
the template or target nucleic acid than do the separate ligation
probes. Thus, upon ligation of the hybridized probes, the ligation
product is released from the target, freeing the target to serve as
a template for a new ligation reaction. Alternatively, thermal
cycling can be done to remove a ligation product from the target
sequence and allow new ligation probes to hybridize for another
cycle of ligation.
[0063] The invention provides compositions, apparatus and methods
for the detection of one or more nucleic acid targets in a sample
including, but not limited to, DNA and RNA targets. Advantages of
using non-enzymatic approaches for nucleic acid target detection
include lower sensitivity to non-natural DNA analog structures,
ability to use RNA target sequences and lower cost and greater
robustness under varied conditions. In particular, the methods
described herein do not require significant sample preparation;
that is, the ligation reactions can be performed in the presence of
contaminants and buffers that would inhibit or inactivate enzymatic
processes for detection. For example, blood samples can be
collected into highly denaturing stabilization buffers, the probes
added and the reactions occur, under conditions that would denature
an enzymatic process. This ability to analyze target nucleic acids,
particularly RNA, in impure samples is of particular use in
applications such as medical diagnostics (including gene expression
profiling and SNP detection), forensic applications, and testing
for damage due to environmental toxins and/or radiation. In
addition, methods and compositions of the present invention are
useful in detection of nucleic acids from samples that are
degraded, including paraffin-embedded samples in which the process
of fixing and embedding in paraffin resulted in degradation of the
samples' nucleic acids.
[0064] In addition, one embodiment of the invention provides for
assays relating to target nucleic acid "integrity". That is, as is
known in the art with mRNA, for example, or nucleic acids in fixed
samples, the nucleic acids are degraded over time. As is shown in
FIG. 11 and FIG. 12 and more fully described below, the present
invention allows for the use of multiple ligation complexes to
allow for an assessment of the integrity of the sample. Similarly,
the use of these multiple ligation complexes per target sequence
can also be used for data and assay integrity through redundancy,
similar to running samples in duplicate or triplicate, for
example.
[0065] In further aspects, the present invention provides buffers
that serve to stabilize nucleic acids in a sample. Such buffers in
general include a denaturant comprising a chaotropic cation,
including in a non-limiting embodiment, guanidinium hydrochloride.
In specific embodiments of the invention, a sample is collected
directly into a buffer of the invention, and then subsequent
hybridization and ligation of ligation probes is conducted in that
buffer without need of purification of the nucleic acids from the
sample. In certain embodiments, the sample collected into the
buffer is first diluted and then subsequently methods described
herein of hybridizing and ligating two or more ligation probes are
conducted within that diluted sample without need of purification
of the target nucleic acids in the sample.
[0066] As discussed above, ligation probes of the invention are
hybridized to a target nucleic acids and then ligated without the
use of a ligase enzyme. Following ligation, the new product
generated (the "ligation product") can optionally be amplified by
an enzymatic or chemical reaction. In the preferred embodiment, the
chemical ligation reaction joins two probes that have PCR primer
sites on them, e.g. universal PCR primers. Additionally, in one
embodiment of the invention, one or both ligation probes contain a
stuffer sequence, or variable spacer sequence, which is designed to
have differing lengths for each probe set (i.e. each target
sequence) thereby resulting in a ligation product having a
target-specific length. Following ligation a defined length
oligonucleotide can now be exponentially amplified by PCR. In
accordance with one aspect of the invention, the probes can possess
detectable labels (e.g. fluorescent labels, electrochemical labels,
magnetic beads, nanoparticles, biotin, etc.) to aid in the
identification, purification, quantification or detection of the
ligated oligonucleotide product. The probes may also optionally
include in their structure: anchoring oligonucleotide sequences
designed for subsequent capture on a solid support (microarrays,
microbeads, nanoparticles), molecule handles that promote the
concentration or manipulation of the ligated product (magnetic
particles, oligonucleotide coding sequences), and promoter
sequences to facilitate subsequent secondary amplification of the
ligated product via an enzyme like a DNA or RNA polymerase.
[0067] The ligation reactions of the invention proceed rapidly, are
specific for the target(s) of interest, and can produce multiple
copies of the ligated product for each target(s), resulting in an
amplification (sometimes referred to herein as "product turnover")
of the detectable signal. The ligation reactions of the invention
do not require the presence of exogeneously added ligases, nor
additional enzymes, although some secondary reactions may rely on
the use of enzymes such as polymerases, as described below.
Ligation chemistries can be chosen from many of the previously
described chemical moieties. Preferred chemistries are ones that
can be easily incorporated into routine manufacture techniques, are
stable during storage, and demonstrate a large preference for
target specific ligation when incorporated into a properly designed
ligation probe set. Additionally, for embodiments which involve
subsequent amplification by an enzyme, ligation chemistries and
probe designs (including unnatural nucleotide analogs) that result
in a ligation product that can be efficiently processed by an
enzyme are preferred. Amplification of the target may also include
turnover of the ligation product, either by destabilization, e.g.
in which the ligation product has a lower or comparable affinity
for the template or target nucleic acid than do the separate
ligation probes, or by standard thermocycling in the presence of
excess probes. Thus, upon ligation of the hybridized probes, the
ligation product is released from the target, freeing the target to
serve as a template for a new ligation reaction.
[0068] In further aspects of the invention and as is discussed in
further detail below, specificity of the assays of the invention
are optionally improved through the use of target capture probes.
Target capture probes of the invention include a domain
complementary to a domain on the target nucleic acid and a capture
moiety. The target capture probes do not participate in the
ligation reaction with the ligation probes, but are instead
designed to hybridize to the target nucleic acid upstream or
downstream from the ligation probes. Hybridization of the target
capture probe to the target nucleic acid produces a target complex
that includes the target nucleic acid, the target capture probe,
and any ligation products formed on the target nucleic acid. The
target complex can then be bound to a surface or substrate (such as
a bead), and any unbound reactants can be separated from the target
complexes bound to the surface or substrate. Thus, since any
subsequent amplification and/or detection steps are performed on
the subset of the original sample of target nucleic acids that were
successfully hybridized with ligation probes, the specificity of
the subsequent assays is improved.
[0069] The above and further aspects and embodiments of the
invention are described in further detail in the following
sections.
II. Samples
[0070] In one aspect, the present invention provides compositions
and methods for detecting the presence or absence of target nucleic
acids (also referred to herein as "target sequences") in samples.
As will be appreciated by those in the art, the samples may
comprise any number of things, including, but not limited to,
bodily fluids (including, but not limited to, blood, urine, serum,
lymph, saliva, anal and vaginal secretions, perspiration and semen,
of virtually any organism, with mammalian samples being preferred
and human samples being particularly preferred); environmental
samples (including, but not limited to, air, agricultural, water
and soil samples); plant materials; biological warfare agent
samples; research samples (for example, the sample may be the
product of an amplification reaction, for example general
amplification of genomic DNA); purified samples, such as purified
genomic DNA, RNA, proteins, etc.; raw samples (bacteria, virus,
genomic DNA, etc.); as will be appreciated by those in the art,
virtually any experimental manipulation may have been done on the
sample. Some embodiments utilize siRNA and microRNA as target
sequences (Zhang et al., J Cell Physiol. (2007) 210(2):279-89;
Osada et al., Carcinogenesis. (2007) 28(1):2-12; and Mattes et al.,
Am J Respir Cell Mol Biol. (2007) 36(1):8-12, each of which is
incorporated herein by reference in its entirety for all purposes,
and in particular all teachings related to target sequences).
[0071] Some embodiments of the invention utilize samples from
stored (e.g. frozen and/or archived) or fresh tissues.
Paraffin-embedded samples are of particular use in many
embodiments, as these samples can be very useful for diagnosis and
prognosis, due to the presence of additional data associated with
the samples. Fixed and paraffin-embedded tissue samples as
described herein refers to storable or archival tissue samples.
Most patient-derived pathological samples are routinely fixed and
paraffin-embedded to allow for histological analysis and subsequent
archival storage. Such samples are often not useful for traditional
methods of nucleic acid detection, because such studies require a
high integrity of the nucleic acid sample so that an accurate
measure of nucleic acid expression can be made. Often, gene
expression studies in paraffin-embedded samples are limited to
qualitative monitoring by using immunohistochemical staining to
monitor protein expression levels.
[0072] A number of techniques exist for the purification of nucleic
acids from fixed paraffin-embedded samples as described in WO
2007/133703 the entire contents of which is herein incorporated by
reference for all purposes and in particular for all teachings
related to the purification of nucleic acids from paraffin-embedded
samples. Methods described by Foss, et al Diagnostic Molecular
Pathology, (1994) 3:148-155 and Paska, C., et al Diagnostic
Molecular Pathology, (2004) 13:234-240 as well as commercially
available kits like Ambion's Recoverall Total Nucleic acid
Isolation kit are included by reference in their entirety. Common
methods start with a step that removes the paraffin from the tissue
via extraction with Xylene or other organic solvent, followed by
treatment with heat and a protease like proteinase K which cleaves
the tissue and proteins and helps to release the genomic material
from the tissue. The released nucleic acids are then captured on a
membrane or precipitated from solution, washed to removed
impurities and for the case of mRNA isolation, a DNase treatment
step is sometimes added to degrade unwanted DNA.
[0073] As will be discussed in further detail herein, both the
target analytes and the ligation probes used to detect the target
analytes may in accordance with the invention comprise nucleic
acids. By "nucleic acid" or "oligonucleotide" or grammatical
equivalents herein means at least two nucleotides covalently linked
together. The target nucleic acids may comprise DNA or RNA. A
nucleic acid of the present invention will generally contain
phosphodiester bonds (for example in the case of the target
sequences), although in some cases, as outlined below, nucleic acid
analogs are included that may have alternate backbones
(particularly for use with the ligation, label or capture probes),
comprising, for example, phosphoramide (Beaucage et al.,
Tetrahedron (1993) 49(10):1925 and references therein; Letsinger,
J. Org. Chem. (1970) 35:3800; Sprinzl et al., Eur. J. Biochem.
(1977) 81:579; Letsinger et al., Nucl. Acids Res. (1986) 14:3487;
Sawai et al, Chem. Lett. (1984) 805; Letsinger et al., J. Am. Chem.
Soc. (1988) 110:4470; and Pauwels et al., Chemica Scripta (1986)
26:141), phosphorothioate (Mag et al., Nucleic Acids Res. (1991)
19:1437; and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et
al., J. Am. Chem. Soc. (1989) 111:2321, O-methylphophoroamidite
linkages (see Eckstein, Oligonucleotides and Analogues: A Practical
Approach, Oxford University Press), and peptide nucleic acid
backbones and linkages (see Egholm, J. Am. Chem. Soc.
(1992)114:1895; Meier et al., Chem. Int. Ed. Engl. (1992) 31:1008;
Nielsen, Nature, (1993) 365:566; Carlsson et al., Nature (1996)
380:207, all of which are incorporated herein by reference in their
entirety). Other analog nucleic acids include those with bicyclic
structures including locked nucleic acids, Koshkin et al., J. Am.
Chem. Soc. (1998) 120:13252 3); positive backbones (Denpcy et al.,
Proc. Natl. Acad. Sci. USA (1995) 92:6097; non-ionic backbones
(U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and
4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English (1991)
30:423; Letsinger et al., J. Am. Chem. Soc. (1988) 110:4470;
Letsinger et al., Nucleoside & Nucleotide (1994) 13:1597;
Chapters 2 and 3, ASC Symposium Series 580, Ed. Y. S. Sanghui and
P. Dan Cook; Mesmaeker et al., Bioorganic & Medicinal Chem.
Lett. (1994) 4:395; Jeffs et al., J. Biomolecular NMR (1994) 34:17;
Xu et al., Tetrahedron Lett. (1996) 37:743) and non-ribose
backbones, including those described in U.S. Pat. Nos. 5,235,033
and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, Ed.
Y. S. Sanghui and P. Dan Cook. Nucleic acids containing one or more
carbocyclic sugars are also included within the definition of
nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995) pp
169-176). Several nucleic acid analogs are described in Rawls, C
& E News Jun. 2, 1997 page 35. All of these references are
herein expressly incorporated by reference in their entirety for
all purposes, and in particular for all teachings related to
nucleic acids. These modifications of the ribose-phosphate backbone
may be done to facilitate the addition of labels or other moieties,
to increase or decrease the stability and half-life of such
molecules in physiological environments, etc.
[0074] For example, the use of the ligation moieties of the
invention can, in some cases depending on the chemistry utilized,
result in nucleic acid analogs as the ligation product. For
example, as shown in Scheme 1, below, the use of certain ligation
moieties result in a phosphothioester bonds.
[0075] As will be appreciated by those in the art, all of these
nucleic acid analogs may find use in the present invention. In
addition, mixtures of naturally occurring nucleic acids and analogs
can be made; for example, at the site of a ligation moiety, an
analog structure may be used. Alternatively, mixtures of different
nucleic acid analogs, and mixtures of naturally occurring nucleic
acids and analogs may be made.
[0076] Nucleic acid analogue may include, for example, peptide
nucleic acid (PNA, WO 92/20702, incorporated herein by reference in
its entirety) and Locked Nucleic Acid (LNA, Koshkin A A et al.
Tetrahedron (1998) 54:3607-3630, Koshkin A A et al. J. Am. Chem.
Soc. (1998) 120:13252-13253, Wahlestedt C et al. PNAS (2000)
97:5633-5638, each of which is incorporated herein by reference in
its entirety). In some applications analogue backbones of this type
may exhibit improved hybridization kinetics, improved thermal
stability and improved sensitivity to mismatch sequences.
[0077] The nucleic acids may be single stranded or double stranded,
as specified, or contain portions of both double stranded or single
stranded sequences. The nucleic acid may be DNA, both genomic and
cDNA, RNA or a hybrid, where the nucleic acid contains any
combination of deoxyribo- and ribo-nucleotides, and any combination
of bases, including naturally occurring nucleobases (uracil,
adenine, thymine, cytosine, guanine) and non-naturally occurring
nucleobases (inosine, xathanine hypoxathanine, isocytosine,
isoguanine, 5-methylcytosine, pseudoisocytosine, 2-thiouracil and
2-thiothymine, 2-aminopurine, N9-(2-amino-6-chloropurine),
N9-(2,6-diaminopurine), hypoxanthine, N9-(7-deaza-guanine),
N9-(7-deaza-8-aza-guanine) and N8-(7-deaza-8-aza-adenine).
5-propynyl-uracil, 2-thio-5-propynyl-uracil) etc. As used herein,
the term "nucleobase" includes both "nucleosides" and
"nucleotides", and monomers of nucleic acid analogs. Thus, for
example, the individual units of a peptide nucleic acid, each
containing a base, are referred to herein as a nucleobase.
[0078] Nucleic acid samples (e.g. target sequences) that do not
exist in a single-stranded state in the region of the target
sequence(s) are generally rendered single-stranded in such
region(s) prior to detection or hybridization. Generally, nucleic
acid samples can be rendered single-stranded in the region of the
target sequence using heat or chemical denaturation. For
polynucleotides obtained via amplification, methods suitable for
generating single-stranded amplification products are preferred.
Non-limiting examples of amplification processes suitable for
generating single-stranded amplification product polynucleotides
include, but are not limited to, T7 RNA polymerase run-off
transcription, RCA, Asymmetric PCR (Bachmann et al., Nucleic Acid
Res. (1990) 18:1309), and Asynchronous PCR (WO 01/94638). Commonly
known methods for rendering regions of double-stranded
polynucleotides single stranded, such as the use of PNA openers
(U.S. Pat. No. 6,265,166), may also be used to generate
single-stranded target sequences on a polynucleotide.
[0079] Target Nucleic Acids
[0080] As discussed further herein, the invention provides methods
and compositions for detecting target sequences. By "target
sequence" or "target nucleic acid" or grammatical equivalents
herein means a nucleic acid sequence on a single strand of nucleic
acid. The target sequence may be a portion of a gene, a regulatory
sequence, genomic DNA, cDNA, RNA including mRNA, MicroRNA and rRNA,
or others. As is outlined herein, the target sequence may be a
target sequence from a sample, or a secondary target such as a
product of an amplification reaction, etc. It may be any length,
with the understanding that longer sequences are more specific. As
will be appreciated by those in the art, the complementary target
sequence may take many forms. For example, it may be contained
within a larger nucleic acid sequence, i.e. all or part of a gene
or mRNA, a restriction fragment of a plasmid or genomic DNA, among
others. Any and all combinations of these may serve as target
nucleic acids in a particular assay. In many cases, multiplex
assays are done, where a plurality of target sequences are
simultaneously detected, such as for gene expression profiling as
is more fully described below.
[0081] In general, each target sequence is comprised of a plurality
of different target domains. Each target sequence has at least a
pair of ligation domains for hybridization to a set of ligation
probes, or more, as described below. For example, a first target
domain of a sample target sequence may hybridize to a first
ligation probe, and a second target domain in the target sequence
may hybridize to a second ligation probe, such as to bring the
chemical ligation moieties into spatial proximity sufficient to
allow spontaneous chemical ligation.
[0082] In general, each pair of target ligation domains is adjacent
to each other, that is, there are no nucleotides separating the two
domains. This finds use in both general detection of target
sequences (e.g. gene expression profiling using mRNA as the target
sequences), transfer reactions as discussed below, as well as for
single nucleotide polymorphism (SNP) detection. For SNP detection,
the target sequence comprises a position for which sequence
information is desired, generally referred to herein as the
"detection position". In some embodiments, the detection position
is a single nucleotide, although in some embodiments, it may
comprise a plurality of nucleotides, either contiguous with each
other or separated by one or more nucleotides. By "plurality" as
used herein is meant at least two. As used herein, the base of a
ligation probe which basepairs with the detection position base in
a hybrid is termed the "interrogation position".
[0083] Each sample target nucleic acid can additionally have
multiple pairs of ligation domains. That is, 1, 2, 3 or more sets
of ligation probes can hybridize to the same target sequence at
multiple locations, as is generally depicted in FIG. 11 or 12. As
is more fully outlined below, the use of multiple ligation domains
per target nucleic acid can serve as the basis to assess the
integrity of the target nucleic acids (and/or the original sample)
in the sample.
[0084] The sample target nucleic acids may contain other domains,
in addition to ligation domains. In certain embodiments, the target
nucleic acids of the invention include a target capture domain to
which a target capture domain is able to hybridize. In general, as
depicted in FIG. 11 and depending on the purpose of the assay, a
target capture domain can be "upstream", "downstream" or
"in-between" one or more of the ligation domains of the target
nucleic acid.
[0085] Unless specified, the terms "first" and "second" are not
meant to confer an orientation of the sequences with respect to the
5'-3' orientation of the target sequence. For example, assuming a
5'-3' orientation of the complementary target sequence, the first
target domain may be located either 5' to the second domain, or 3'
to the second domain. For ease of reference and not to be limiting,
these domains are sometimes referred to as "upstream" and
"downstream", with the normal convention being the target sequence
being displayed in a 5' to 3' orientation. However, it should be
noted that ligation domains have an orientation such that the 3'
and 5' ligation moieties of the ligation probe sets hybridize
either completely adjacently (e.g. no intervening nucleobases) or
within a distance that the linkers attaching the ligation moieties
allow for ligation.
[0086] In some embodiments, the pair of target ligation domains may
be separated. For example, in some cases, when ligation
amplification is desired, the ligation probes may utilize linkers
and be separated when hybridized by one or more nucleobases of the
target sequence to confer hybridization instability on the ligated
product. In other applications, As will be discussed in further
detail below, buffers of the invention are of use in stabilizing
nucleic acids, particularly RNA, in a sample. In some aspects of
the invention, samples are collected into buffers of the invention.
In further embodiments, and as is discussed in further detail
below, such buffers include one or more of a denaturant, a reducing
agent, a surfactant, a pH buffer, EDTA, and any combination
thereof.
III. Buffers
[0087] In one aspect, the invention provides methods and
compositions which stabilize nucleic acids (also referred to herein
as "sample nucleic acid" or "target nucleic acids"). By "stabilize"
as used herein is meant that the nucleic acids in a sample are
resistant to degradation even when stored at ambient room
temperature or above for a period of time. In some embodiments,
nucleic acids contained in buffers of the invention are stable at
room temperature or above for about one day to about three months.
Stability can be measured using any means known in the art,
including assays for nucleic acid integrity as further discussed
below. In further embodiments, a sample comprising nucleic acids
contained in a buffer of the invention is assessed as having
increased stability as compared to a sample that was not stored in
the buffer if at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
95% of the nucleic acids in the sample stored in the buffer show
less degradation than those in the sample that was not stored in
the buffer. In yet further embodiments, a sample is identified as
being stabilized by the buffers of the present invention if at
least a majority of the nucleic acids in the sample show reduced
degradation as compared to a sample that was not stored in the
buffer. Stability of RNA samples are often assessed by Capillary
Electrophoresis methodologies that look to measure the average size
of the nucleic acid sample. Stabilized samples will have a longer
average size than non-stabilized samples. Another aspect of this
invention is the use of multiple ligation probe sets combined with
one or more target capture probes that can be used to assess the
average size of a target nucleic acid, and by correlation, the
level of degradation of the target nucleic acid.
[0088] Buffers of the invention can optionally and in any
combination include one or more of a denaturant, a reducing agent,
a surfactant, a pH buffer, a chelator such as EDTA, and any
combination thereof. As will be appreciated, buffers of the
invention may include multiple types of components within the same
class--e.g., buffers of the invention may include one or more
different kinds of denaturants in combination with one or more
types of surfactants, and so on.
[0089] An advantage of the buffers of the present invention is that
they can be used to stabilize nucleic acids such as RNA in a sample
and then the sample can be directly analyzed from the buffer
solutions in accordance with the methods described herein. In other
words, samples contained in buffer solutions of the invention can
be subjected to the chemical ligation and detection methods
described herein without isolation or purification of the RNA.
Another advantage of the buffers of the invention is that cell
lysis occurs upon the collection of the sample in the buffer, thus
not requiring an additional lysis step to release the target
nucleic acids from the sample.
[0090] In an exemplary embodiment, a sample comprising RNA can be
combined in a buffer solution comprising guanidinium hydrochloride,
ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT),
Triton X-100, and Tris-HCL at a pH of 7.5. In another embodiment,
the sample comprising RNA can be combined in a buffer solution
comprising guanidinium isothiocyanate, EDTA, DTT, Triton X-100, and
Tris-HCl at a pH of 7.5. The RNA is stable in such buffer solutions
and it is not necessary to isolate the RNA from other sample
constituents which may enhance degradation of the RNA.
[0091] In further embodiments, the buffers of the invention
preferably include a denaturant, particularly a chaotropic cation,
that has the effect of increasing reaction and binding efficiency
in the methods and assays described herein by helping to unfold the
secondary structure of the RNA. Common Chaotropic molecules are
guanidinium hydrochloride, guanidinium isothiocyanate, betaine or
glycline betaine, urea, thiourea, and lithium perchlorate. Without
being bound by theory, chaotropic agents that are effective in
breaking of tertiary structure in nucleic acids are preferred and
chaotropic agents that also maintain the solubility of the nucleic
acid target in solution are particularly beneficial. An advantage
of buffers of the invention, particularly buffers comprising a
chaotropic cation, is that the buffer keeps the nucleic acids of
the sample in solution. This is in contrast to other traditional
buffers used in transport systems for blood-based tests, which tend
to precipitate/form a cationic shell around the nucleic acids of
the sample (particularly RNA). Since the buffers of the invention
keep the nucleic acids in solution, and since the chemical ligation
methods of the assays of the invention do not require enzymes, a
sample can be collected into a buffer and the ligation probes (and
in many embodiments, target capture probes) can be added to the
sample and ligation products formed. To change hybridization
conditions to then release the ligation products or target
complexes for further analysis, the sample plus buffer can simply
be diluted to dilute the denaturant and thereby change the
hybridization conditions, thus allowing analysis of the nucleic
acids using any of the methods described herein and known in the
art.
[0092] In further embodiments, the buffers of the invention have a
pH of about 5 to about 8.5. More preferably the buffer solution has
a pH of about 6 to 8 and even more preferably, a pH of
approximately 7.3 or 7.5.
[0093] The following sections discuss exemplary buffer components
in further detail. Although each of these components is discussed
separately, the present invention encompasses any combination of
the following buffer components as well as any other components
known in the art.
[0094] Denaturants
[0095] In preferred embodiments, buffers of the present invention
include one or more denaturants. By denaturant as used herein is
meant any substance that serves to unfold the double helix of
nucleic acids with loss of secondary and tertiary structure. In
further embodiments, the denaturants comprise a chaotropic cation,
including without limitation guanidinium hydrochloride (GuHCl) and
guanidinium isothiocyanate.
[0096] In further embodiments, the denaturant is guanidinium
hydrochloride, which is present in a concentration from about 1
molar to about 8 molar and more preferably, a concentration of
about 2 molar to about 4 molar, and even more preferably, a
concentration of approximately 3 molar. In further embodiments,
concentration of GuHCl in buffers of the invention range from about
0.2-10, 0.5-9, 1-8, 1.5-7, 2-6, 2.5-5, and 3.0-4.0 molar. In still
further embodiments, concentrations of GuHCl in buffers of the
invention are about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14 or 15 molar.
[0097] In other embodiments, the denaturant is guanidinium
isothiocyanate, which is present in a concentration from about 1
molar to about 8 molar and more preferably, a concentration of
about 2 molar to about 4 molar, and even more preferably, a
concentration of approximately 3 molar. In further embodiments,
concentration of guanidinium isothiocyanate in buffers of the
invention range from about 0.2-10, 0.5-9, 1-8, 1.5-7, 2-6, 2.5-5,
and 3.0-4.0 molar. In still further embodiments, concentrations of
guanidinium isothiocyanate in buffers of the invention are about
0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 molar.
[0098] As will be appreciated, other denaturants known in the art
can be used in buffers of the invention at similar concentrations
as those listed above for guanidinium hydrochloride and guanidinium
isothiocyanate.
[0099] In some embodiments, such as with the use of high
concentrations of salts such as guanidinium salts, these reagents
also serve as lysis agents. As will be appreciated by those in the
art, in general, the use of a denaturant that also serves as a cell
lysis agent is of particular use, although the present invention
also contemplates the use of a first separate lysis step followed
by the addition of the denaturant.
[0100] Surfactants
[0101] In some embodiments, buffers of the present invention
include one or more surfactants. In further embodiments, the
surfactant includes without limitation Triton X-100 and sodium
N-lauroylsarcosine.
[0102] In further embodiments, the surfactant is present in buffers
of the invention at a concentration from about 0.1% to about 5% by
weight. In still further embodiments, the surfactant is present in
a concentration of about 0.1%-10%, 0.5%-9.5%, 1%-9%, 1.5%-8.5%,
2%-8%, 2.5%-7.5%, 3%-7%, 3.5%-6.5%, 4%-6%, and 4.5%-5.5% by weight.
In preferred embodiments, the surfactant has a concentration of
about 0.5% to about 3%. In a further embodiment, the surfactant has
a concentration of approximately 1.5% by weight.
[0103] ph Buffer
[0104] In some embodiments, buffers of the present invention
include one or more pH buffers. Such pH buffers include without
limitation Tris. In other embodiments the pH buffer can be one of
many known by those skilled in the art. Generally the pH buffer
used in the present invention includes an agent that has a pKa
within one pH unit of the operating pH.
[0105] In some embodiments, the pH buffer is present in buffers of
the invention at a concentration from about 10 mM to about 100 mM.
In preferred embodiments, the pH buffer has a concentration of
about 20 mM to about 50 mM and more preferably, a concentration of
approximately 30 mM. In further embodiments, the pH buffer has a
concentration of about 5-150, 10-140, 15-130, 20-120, 25-110,
30-100, 35-90, 40-80, 45-70, and 50-60 mM.
[0106] Reducing Agents
[0107] In some embodiments, buffers of the present invention
include one or more reducing agents. Such reducing agents can
include without limitation Dithiothreitol (DTT) and
mercaptoethanol.
[0108] In further embodiments, the reducing agents have a
concentration from about 1 mM to about 100 mM. In preferred
embodiments, the reducing agent has a concentration of about 4 mM
to about 7 mM and even more preferably, a concentration of
approximately 5 mM. In still further embodiments, the reducing
agents have a concentration of about 0.5-10, 1-9.5, 1.5-9, 2-8.5,
2.5-8, 3-7.5, 3.5-7, 4-6.5 mM. In yet further embodiments, the
reducing agents have a concentration of about 1-150, 10-140,
15-130, 20-120, 25-110, 30-100, 35-90, 40-80, 45-70, and 50-60
mM.
[0109] EDTA
[0110] In further embodiments, buffers of the invention include
EDTA at a concentration of from about 1 mM to about 100 mM. More
preferably the EDTA has a concentration of about 10 mM to about 50
mM and even more preferably, a concentration of approximately 20
mM. In further embodiments, the EDTA is present at a concentration
of about 1-150, 10-140, 15-130, 20-120, 25-110, 30-100, 35-90,
40-80, 45-70, and 50-60 mM. In still further embodiments, the EDTA
has a concentration of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,
55, 60 mM.
[0111] Additional Buffer Components
[0112] The buffers of the invention may further include any
additional components known in the art, particularly components
known in the art to be of use in reactions involving nucleic acids.
Additional components may include without limitation: adjuvants,
diluents, binders, stabilizers, salts (including NaCl and
MgCl.sub.2), lipophilic solvents, preservatives, or the like.
Buffer components may also include pharmaceutical excipients and
additives, proteins, peptides, amino acids, lipids, and
carbohydrates (e.g., sugars, including monosaccharides, di-, tri-,
tetra-, and oligosaccharides; derivatized sugars such as alditols,
aldonic acids, esterified sugars and the like; and polysaccharides
or sugar polymers), which can be present singly or in combination,
comprising alone or in combination 1-99.99% by weight or volume.
Exemplary protein excipients include serum albumin such as human
serum albumin (HSA), recombinant human albumin (rHA), gelatin,
casein, and the like. Representative amino acid/antibody
components, which can also function in a buffering capacity,
include alanine, glycine, arginine, betaine, histidine, glutamic
acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine,
methionine, phenylalanine, aspartame, and the like. Carbohydrate
excipients are also intended within the scope of this invention,
examples of which include but are not limited to monosaccharides
such as fructose, maltose, galactose, glucose, D-mannose, sorbose,
and the like; disaccharides, such as lactose, sucrose, trehalose,
cellobiose, and the like; polysaccharides, such as raffinose,
melezitose, maltodextrins, dextrans, starches, and the like; and
alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol
sorbitol (glucitol) and myoinositol.
[0113] Uses of Buffers of the Invention
[0114] Buffers of the invention can be used with any of the sample
collection, chemical ligation, or assays discussed herein.
[0115] In some embodiments, samples containing target nucleic acids
are collected into buffers of the invention. The advantage of
buffers of the invention is that they tend to stabilize nucleic
acids contained in the samples. Thus, degradation that often begins
to occur, particularly with RNA, immediately upon collection is
prevented to at least some degree by the buffers of the invention,
providing the advantage of a robust set of target nucleic acids for
further analysis using methods of the invention.
[0116] In further embodiments, upon collection of samples into
buffers of the invention, ligation probes and optionally target
capture probes (which are further discussed in detail below) can be
added directly to the sample in the buffer without purification of
the target nucleic acids. In some embodiments, the sample in the
buffer is first diluted prior to the addition of the probes
(including ligation and target capture probes). Because the
ligation methods of the present invention are not reliant on
enzymes, hybridization and ligation of ligation probes can occur
without purifying the nucleic acids from the sample and without
relying on the use of a ligase enzyme. This further serves to limit
the degradation that occurs in the nucleic acids of the sample.
IV. Ligation Probes and Chemical Ligation Methods
[0117] In one aspect, ligation probes of the invention comprise any
polymeric species that is capable of interacting with a nucleic
acid target(s) in a sequence specific manner and possess chemical
moieties allowing the probes to undergo a spontaneous chemical
ligation reaction with another polymeric species possessing
complementary chemical moieties. In one embodiment, the ligation
probes can be DNA, RNA, PNA, LNA, modified versions of the
aforementioned and/or any hybrids of the same (e.g. DNA/RNA
hybrids, DNA/LNA hybrids, DNA/PNA hybrids). In a preferred
embodiment, the ligation probes comprise DNA or RNA
oligonucleotides.
[0118] Ligation probes of the invention are designed such that when
the probes bind to a part of the target polynucleotide in close
spatial proximity, a chemical ligation reaction occurs between the
probes. In general, the probes comprise chemically reactive
moieties (herein generally referred to as "ligation moieties") and
bind to the target polynucleotide in a particular orientation, such
that the chemically reactive moieties come into close spatial
proximity, thus resulting in a spontaneous ligation reaction that
can take place without the use of a ligase enzyme.
[0119] In one embodiment, the invention provides sets of ligation
probes, usually a first and a second ligation probe, although as is
described herein some embodiments utilize more than two. In
addition, as noted herein, in some cases a transfer reaction is
done rather than ligation; "ligation probes" includes "transfer
probes". Each ligation probe comprises a nucleic acid portion,
sometimes referred to herein as a "ligation domain" or "probe
domain" that is substantially complementary to one of the target
domains. Probes of the present invention are designed to be
complementary to a target sequence such that hybridization of the
target sequence and the probes of the present invention occurs. As
outlined herein, this complementarity need not be perfect; there
may be any number of base pair mismatches which will interfere with
hybridization between the target sequence and the probes of the
present invention. However, if the number of mutations is so great
that no hybridization can occur under even the least stringent of
hybridization conditions, the sequence is not a complementary
sequence. Thus, by "substantially complementary" herein is meant
that the probes are sufficiently complementary to the target
sequences to hybridize under normal reaction conditions.
"Identical" sequences are those that over the length of the shorter
sequence of nucleobases, perfect complementarity exists. A variety
of hybridization conditions may be used in the present invention,
including high, moderate and low stringency conditions; see for
example Maniatis et al., Molecular Cloning: A Laboratory Manual, 2d
Edition, 1989, and Ausubel, et al, Short Protocols in Molecular
Biology, herein incorporated by reference. The hybridization
conditions may also vary when a non-ionic backbone, e.g. PNA is
used, as is known in the art.
[0120] In one aspect of the invention, the length of the probe is
designed to vary with the length of the target sequence, the
specificity required, the reaction (e.g. ligation or transfer) and
the hybridization and wash conditions. Generally, in this aspect
ligation probes range from about 5 to about 150 nucleobases, with
from about 15 to about 100 being preferred and from about 25 to
about 75 being especially preferred. In general, these lengths
apply equally to ligation and transfer probes.
[0121] In another embodiment of the invention, referred to herein
as "CLPA-CE" which is described more fully below, probe length is
designed to vary for each target of interest thereby generating
ligation products that can be identified and analyzed based on
length variance.
[0122] Ligation probes of the invention are designed to be specific
for the polynucleotide target. These probes bind to the target in
close spatial proximity to each other and are oriented in such a
manner that the chemically reactive ligation moieties are in close
spatial proximity. In one aspect, two or more probes are designed
to bind near adjacent sites on a target polynucleotide. In a
preferred embodiment, two probes bind to the target such that the
ligation moiety at the 5' end of one oligonucleotide probe is able
to interact with the ligation moiety at the 3' end of the other
probe. In preferred embodiments, the ligation reaction between the
ligation moieties occurs without the use of an exogenous ligase
enzyme, also referred to herein as "chemical ligation." It should
be noted that the sets of ligation probes do not ligate using
ligases due to the presence of the ligation moieties.
[0123] In the case of SNP detection, a set of ligation probes may
in some cases actually comprise be three or four or five different
ligation probes, some of which are allele specific. By "allele
specific" probe or primer is meant a probe or primer that
hybridizes to a target sequence and discriminates between alleles.
In this case, for example, when a SNP is biallelic (such as
depicted in FIG. 13), a set of three ligation probes are used, two
of which contain a different nucleotide at the detection position.
That is, as is known in the art, mismatches at the junction of a
ligation complex will either result in no ligation or a decreased
amount of ligation. So, depending on the orientation of the probes,
the detection position can either be on the terminal position of
the "upstream" probe or the downstream probe as is depicted in
FIGS. 13A and 13C). The mismatch can also be positioned internally
to a probe and the mismatch discrimination is based on differences
in the binding strength or Tm of the mismatch discriminating probes
(as depicted in FIG. 13B. In the case of a biallelic SNP, two of
the probes have the same probe domain except that the nucleotide at
the detection position, corresponding to the interrogation position
on the target sequence, is different. Thus, depending on whether
the patient is homozygotic (T/T or C/C) or heterozygotic (T/C or
C/T), the ligation between probes occurs. Furthermore, in this
invention, there is generally an additional difference between the
mismatch detection ligation probes that enables the easy
identification of which probe was preferentially ligated. For
example, the "A" allelic probe may have a variable spacer sequence
of 15 nucleotides (15 mer) and the "G" allelic probe may have a
variable spacer sequence of 20 nucleotides (20 mer), the probes may
have different capture domains or label sequences, etc.
[0124] Similarly, as will be appreciated by those in the art,
triallelic or quadallelic SNPs use 4 (3 probes with the same target
domain and one with the other target domain) or 5 (4 probes with
the same target domain and one with the other target domain)
ligation probes in the set.
[0125] The size of the primer and probe nucleic acid may vary, as
will be appreciated by those in the art with each portion of the
probe and the total length of the probe in general varying from 5
to 500 nucleotides in length. Each portion is preferably between 10
and 100 being preferred, between 15 and 50 being particularly
preferred, and from 10 to 35 being especially preferred, depending
on the use and amplification technique. Thus, for example, the
universal priming site(s) of the probes are each preferably about
15-20 nucleotides in length, with 18 being especially preferred.
The adapter sequences of the probes are preferably from 15-25
nucleotides in length, with 20 being especially preferred. The
target specific portion of the probe is preferably from 15-50
nucleotides in length. In addition, the primer may include an
additional amplification priming site. In a preferred embodiment
the additional amplification priming site is a T7 RNA polymerase
priming site.
[0126] A number of non-enzymatic or template mediated chemical
ligation methods can be used in accordance with the present
invention. These include chemical ligation methods that utilize
coupling reagents, such as N-cyanoimidazole, cyanogen bromide, and
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride. See
Metelev, V. G., et al., Nucleosides & Nucleotides (1999)
18:2711; Luebke, K. J., and Dervan, P. B. J. Am. Chem. Soc. (1989)
111:8733; and Shabarova, Z. A., et al., Nucleic Acids Research
(1991)19:4247, each of which is incorporated herein by reference in
its entirety for all purposes and in particular for all teachings
related to chemical ligation. Kool (U.S. Pat. No. 7,033,753), which
is incorporated herein by reference in its entirety, describes the
use of chemical ligation and fluorescence resonance energy transfer
(FRET) to detect genetic polymorphisms. The readout in this process
is based on the solution phase change in fluorescent intensity.
Terbrueggen (US Patent Publication No. 12008/0124810) which is
incorporated herein by reference in its entirety describes the use
of chemical ligation methods, compositions and reagents for the
detection of nucleic acids via microarray detection. Other chemical
ligation methods react a 5'-tosylate or 5'-iodo group with a
3'-phosphorothioate group, resulting in a DNA structure with a
sulfur atom replacing one of the bridging phosphodiester oxygen
atoms. See Gryanov, S. M., and Letsinger, R. L., Nucleic Acids
Research (1993) 21:1403; Xu, Y. and Kool, E. T. Tetrahedron Letters
(1997) 38:5595; and Xu, Y. and Kool, E. T., Nucleic Acids Research
(1999) 27:875, each of which is herein incorporated by reference in
its entirety. Letsinger et al (U.S. Pat. No. 5,780,613, herein
incorporated by reference in its entirety) have previously
described an irreversible, nonenzymatic, covalent autoligation of
adjacent, template-bound oligonucleotides wherein one
oligonucleotide has a 5' displaceable group and the other
oligonucleotide has a 3' thiophosphoryl group. Each of these
references describing chemical ligation methods is incorporated
herein by reference in its entirety for all purposes and in
particular for all teachings related to chemical ligation.
[0127] In one aspect, the ligation reactions of the invention
include transfer reactions. In this embodiment, the probes
hybridize to the target sequence, but rather than oligonucleotide
probes being ligated together to form a ligation product, a nucleic
acid-directed transfer of a molecular entity (including reporter
molecules such as fluorophores, quenchers, etc) from one
oligonucleotide probe to other occurs. This transfer reaction is
analogous to a ligation reaction, however instead of joining of two
or more probes, one of the probes is ligated to the transfer
molecule and the other probe is the "leaving" of the chemical
reaction. Importantly, similar to the ligation reaction, the
transfer reaction is facilitated by the proximal binding of the
transfer probes onto a nucleic acid target, such that significant
signal is detected only if the probes have hybridized to the target
nucleic acid in close enough proximity to one another (e.g., at
adjacent sites) for the transfer reaction to take place.
[0128] In one aspect, the invention relates to methods of chemical
ligation that include the binding of at least a first and a second
ligation probe to the target nucleic acid to form a "ligation
substrate" under conditions such that the ligation moieties of the
first and second ligation probes are able to spontaneously react,
ligating the probes together, in the absence of exogenous ligase;
that is, no exogenous ligase is added to the reaction and instead
the reaction proceeds without the use of a ligase. In the case of
the transfer reaction, this may be referred to as either a
"ligation substrate" or a "transfer substrate". By "ligation
substrate" herein is meant a substrate for chemical ligation
comprising at least one target nucleic acid sequence and two or
more ligation probes. Similarly, included within the definition of
"ligation substrate" is a "transfer substrate", comprising at least
one target nucleic acid sequence and two or more transfer probes.
Once the chemical ligation step has occurred, the product of
reaction is sometimes referred to as a "ligation complex",
comprising the ligated probes and the target to which they are
still hybridized.
[0129] In some embodiments of the invention, for example when
additional specificity is desired, more than two ligation probes
can be used, as is generally depicted in FIG. 3. In this
embodiment, the "middle" ligation probe(s) can also be adjacent or
separated by one or more nucleobases of the target sequence. In a
preferred embodiment, the ligation reaction does not require the
presence of a ligase enzyme and occurs spontaneously between the
bound probes in the absence of any addition (e.g. exogeneous)
ligase.
[0130] Chemical ligation can, under appropriate conditions, occur
spontaneously without the addition of any additional activating
reagents or stimuli. Alternatively, "activating" agents or external
stimuli can be used to promote the chemical ligation reaction.
Examples of activating agents include, without limitation,
carbodiimide, cyanogen bromide (BrCN), imidazole,
1-methylimidazole/carbodiimide/cystamine, N-cyanoimidazole,
dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP) and
other reducing agents as well as external stimuli like ultraviolet
light, heat and/or pressure changes.
[0131] As is outlined herein, the ligation moieties of the
invention may take a variety of configurations, depending on a
number of factors. Most of the chemistries depicted herein are used
in phosphoramidite reactions that generally progress in a 3' to 5'
direction. That is, the resin contains chemistry allowing
attachment of phosphoramidites at the 5' end of the molecule.
However, as is known in the art, phosphoramidites can be used to
progress in the 5' to 3' direction; thus, the invention includes
moieties with opposite orientation to those outlined herein.
[0132] Each set of ligation probes (or transfer probes) contains a
set of a first ligation moiety and a second ligation moiety. The
identification of these ligation moiety pairs depends on the
chemistry of the ligation to be used. In addition, as described
herein, linkers (including but not limited to destabilization
linkers) may be present between the probe domain and the ligation
moiety of one or both ligation probes. In general, for ease of
discussion, the description herein may use the terms "upstream" and
"downstream" ligation probes, although this is not meant to be
limiting.
[0133] Different sets of ligation probes can be designed for use in
any of the assays and methods described herein. The sets of
ligation probes can be designed to include ligation probes directed
to one or more target domains of a target nucleic acid, or
different probe sets can be designed to detect different target
nucleic acids. For example, for detection of a particular target
nucleic acid in embodiments in which two ligation probes are used,
a set of ligation probes is designed such that the first probe
(e.g., the "upstream probe") comprises a probe domain directed to a
first target domain. The set also includes a second ligation probe
(e.g., the "downstream probe") that is directed to a second target
domain that is adjacent to and downstream from the first target
domain. The first and second target domains may in some embodiments
be separated by a gap of one, two or three nucleotides. This set of
ligation probes thus produces ligation products that can be
detected to identify the presence of the target nucleic acid to
which they are directed. In further embodiments, different sets of
ligation probes can be designed to detect different target nucleic
acids. In still further embodiments, different sets of ligation
probes are designed to detect the same target nucleic acid--in such
embodiments, the different sets of ligation probes are directed to
different domains of the same target nucleic acid, such that the
same target nucleic acid may have multiple ligation products,
depending on the number of different probe sets used. As will be
appreciated, similar designs can be used when designing sets of
probes for use in embodiments in which more than two ligation
probes are used to form a single ligation product.
[0134] Halo Leaving Group Chemistry
[0135] In one embodiment of the invention, the chemistry is based
on 5' halogen leaving group technology such as is generally
described in Gryanov, S. M., and Letsinger, R. L., (1993) Nucleic
Acids Research, 21:1403; Xu, Y. and Kool, E. T. (1997) Tetrahedron
Letters, 38:5595; Xu, Y. and Kool, E. T., (1999) Nucleic Acids
Research, 27:875; Arar et al., (1995), BioConj. Chem., 6:573; Kool,
E. T. et. al, (2001) Nature Biotechnol 19:148; Kool, E. T. et. al.,
(1995) Nucleic Acids Res, 23 (17):3547; Letsinger et al., U.S. Pat.
No. 5,476,930; Shouten et al., U.S. Pat. No. 6,955,901; Andersen et
al., U.S. Pat. No. 7,153,658, all of which are expressly
incorporated by reference herein. In this embodiment, the first
ligation probe includes at its 5' end a nucleoside having a 5'
leaving group, and the second ligation probe includes at its 3' end
a nucleoside having 3' nucleophilic group such as a 3'
thiophosphoryl. The 5' leaving group can include many common
leaving groups know to those skilled in the art including, for
example the halo-species (I, Br, Cl) and groups such as those
described by Abe and Kool, J. Am. Chem. Soc. (2004)
126:13980-13986, which is incorporated herein by reference in its
entirety. In a more preferred embodiment of this aspect of the
invention, the first ligation probe has a 5' leaving group attached
through a flexible linker and a downstream oligonucleotide which
has a 3' thiophosphoryl group. This configuration leads to a
significant increase in the rate of reaction and results in
multiple copies of ligated product being produced for every
target.
[0136] The "upstream" oligonucleotide, defined in relation to the
5' to 3' direction of the polynucleotide template as the
oligonucleotide that binds on the "upstream" side (i.e., the left,
or 5' side) of the template includes, as its 5' end, a 5'-leaving
group. Any leaving group capable of participating in an S.sub.N2
reaction involving sulfur, selenium, or tellurium as the
nucleophile can be utilized. The leaving group is an atom or group
attached to carbon such that on nucleophilic attack of the carbon
atom by the nucleophile (sulfur, selenium or tellurium) of the
modified phosphoryl group, the leaving group leaves as an anion.
Suitable leaving groups include, but are not limited to a halide,
such as iodide, bromide or chloride, a tosylate, benzenesulfonate
or p-nitrophenylester, as well as RSO.sub.3 where R is phenyl or
phenyl substituted with one to five atoms or groups comprising F,
Cl, Br, I, alkyl (C1 to C6), nitro, cyano, sulfonyl and carbonyl,
or R is alkyl with one to six carbons. The leaving group is
preferably an iodide, and the nucleoside at the 5' end of the
upstream oligonucleotide is, in the case of DNA, a
5'-deoxy-5'-iodo-2'-deoxynucleoside. Examples of suitable
5'-deoxy-5'-iodo-2'-deoxynucleosides include, but are not limited
to, 5'-deoxy-5'-iodothymidine (5'-1-T),
5'-deoxy-5'-iodo-2'-deoxycytidine (5'-I-dC),
5'-deoxy-5'-iodo-2'-deoxyadenosine (5'-I-dA),
5'-deoxy-5'-iodo-3-deaza-2'-deoxyadenosine (5'-I-3-deaza-dA),
5'-deoxy-5'-iodo-2'-deoxyguanosine (5'-I-dG) and
5'-deoxy-5'-iodo-3-deaza-2'-deoxyguanosine (5'-I-3-deaza-dG), and
the phosphoroamidite derivatives thereof (see FIG. 2). In the case
of RNA oligonucleotides, analogous examples of suitable
5'-deoxy-5'-iodonucleosides include, but are not limited to,
5'-deoxy-5'-iodouracil (5'-I-U), 5'-deoxy-5'-iodocytidine (5'-I-C),
5'-deoxy-5'-iodoadenosine (5'-I-A),
5'-deoxy-5'-iodo-3-deazaadenosine (5'-I-3-deaza-A),
5'-deoxy-5'-iodoguanosine (5'-I-G) and
5'-deoxy-5'-iodo-3-deazaguanosine (5'-I-3-deaza-G), and the
phosphoroamidite derivatives thereof. In a preferred embodiment, an
upstream ligation probe contains 2'-deoxyribonucleotides except
that the modified nucleotide on the 5' end, which comprises the 5'
leaving group, is a ribonucleotide. This embodiment of the upstream
nucleotide is advantageous because the bond between the penultimate
2'-deoxyribonucleotide and the terminal 5' ribonucleotide is
susceptible to cleavage using base. This allows for potential reuse
of an oligonucleotide probe that is, for example, bound to a solid
support, as described in more detail below. In reference to the
CLPA assay, which is described more fully below, the 5' leaving
group of the "upstream" probe is most preferably DABSYL.
[0137] The "downstream" oligonucleotide, which binds to the
polynucleotide template "downstream" of, i.e., 3' to, the upstream
oligonucleotide, includes, as its 3' end, a nucleoside having
linked to its 3' hydroxyl a phosphorothioate group (i.e., a
"3'-phosphorothioate group"), a phosphoroselenoate group (i.e., a
"3'-phosphoroselenoate group), or a phosphorotelluroate group
(i.e., a "3'-phosphorotelluroate group"). The chemistries used for
autoligation are thus sulfur-mediated, selenium-mediated, or
tellurium mediated. Self-ligation yields a ligation product
containing a 5' bridging phosphorothioester
(--O--P(O)(O.sup.-)--S--), phosphoroselenoester
(--O--P(O)(O.sup.-)--Se--) or phosphorotelluroester
(--O--P(O)(O.sup.-)--Te--), as dictated by the group comprising the
3' end of the downstream oligonucleotide. This non-natural, achiral
bridging diester is positioned between two adjacent nucleotides and
takes the place of a naturally occurring 5' bridging
phosphodiester. Surprisingly, the selenium-mediated ligation is 3
to 4 times faster than the sulfur-mediated ligation, and the
selenium-containing ligation product was very stable, despite the
lower bond strength of the Se--P bond. Further, the bridging
phosphoroselenoester, as well as the bridging
phosphorotelluroester, are expected to be cleavable selectively by
silver or mercuric ions under very mild conditions (see Mag et al.,
Nucleic Acids Res. (1991) 19:1437 1441).
[0138] In one embodiment, a downstream oligonucleotide contains
2'-deoxyribonucleotides except that the modified nucleotide on the
3' end, which comprises the 3' phosphorothioate,
phosphoroselenoate, or phosphorotelluroate, is a ribonucleotide.
This embodiment of the upstream nucleotide is advantageous because
the bond between the penultimate 2'-deoxyribonucleotide and the
terminal ribonucleotide is susceptible to cleavage using base,
allowing for potential reuse of an oligonucleotide probe that is,
for example, bound to a solid support. In reference to the CLPA
assay, as described more fully below, the "downstream" probe most
preferably includes at its 3' end 3'-phosphorothioate.
[0139] It should be noted that the "upstream" and "downstream"
oligonucleotides can, optionally, constitute the two ends of a
single oligonucleotide, in which event ligation yields a circular
ligation product. The binding regions on the 5' and 3' ends of the
linear precursor oligonucleotide must be linked by a number of
intervening nucleotides sufficient to allow binding of the 5' and
3' binding regions to the polynucleotide target.
[0140] Compositions provided by the invention include a
5'-deoxy-5-'iodo-2'-deoxynucleoside, for example a
5'-deoxy-5'-iodothymidine (5'-I-T),
5'-deoxy-5'-iodo-2'-deoxycytidine (5'-I-dC),
5'-deoxy-5'-iodo-2'-deoxyadenosine (5'-I-dA),
5'-deoxy-5'-iodo-3-deaza-2'-deoxyadenosine (5'-I-3-deaza-dA),
5'-deoxy-5'-iodo-2'-deoxyguanosine (5'-I-dG) and
5'-deoxy-5'-iodo-3-deaza-2'-deoxyguanosine (5'-I-3-deaza-dG), and
the phosphoroamidite derivatives thereof, as well as an
oligonucleotide comprising, as its 5' end, a
5'-deoxy-5'-iodo-2'-deoxynucleoside of the invention. Compositions
provided by the invention further include a
5'-deoxy-5'-iodonucleoside such as 5'-deoxy-5'-iodouracil (5'-I-U),
5'-deoxy-5'-iodocytidine (5'-I-C), 5'-deoxy-5'-iodoadenosine
(5'-1-A), 5'-deoxy-5'-iodo-3-deazaadenosine (5'-I-3-deaza-A),
5'-deoxy-5'-iodoguanosine (5'-I-G) and
5'-deoxy-5'-iodo-3-deazaguanosine (5'-I-3-deaza-G), and the
phosphoroamidite derivatives thereof, as well as an oligonucleotide
comprising, as its 5' end, a 5'-deoxy-5'-iodonucleoside of the
invention. Also included in the invention is a nucleoside
comprising a 3'-phosphoroselenoate group or a
3'-phosphorotelluroate group, and an oligonucleotide comprising as
its 3' end a nucleoside comprising a 3'-phosphoroselenoate group or
a 3'-phosphorotelluroate group. Oligonucleotides containing either
or both of these classes of modified nucleosides are also included
in the invention, as are methods of making the various nucleosides
and oligonucleotides. Oligonucleotides that are modified at either
or both of the 5' or 3' ends in accordance with the invention
optionally, but need not, include a detectable label, preferably a
radiolabel, a fluorescence energy donor or acceptor group, an
excimer label, or any combination thereof.
[0141] In addition, in some cases, substituent groups may also be
protecting groups (sometimes referred to herein as "PG"). Suitable
protecting groups will depend on the atom to be protected and the
conditions to which the moiety will be exposed. A wide variety of
protecting groups are known; for example, DMT is frequently used as
a protecting group in phosphoramidite chemistry (as depicted in the
figures; however, DMT may be replaced by other protecting groups in
these embodiments. A wide variety of protecting groups are
suitable; see for example, Greene's Protective Groups in Organic
Synthesis, herein incorporated by reference for protecting groups
and associated chemistry.
[0142] By "alkyl group" or grammatical equivalents herein is meant
a straight or branched chain alkyl group, with straight chain alkyl
groups being preferred. If branched, it may be branched at one or
more positions, and unless specified, at any position. The alkyl
group may range from about 1 to about 30 carbon atoms (C1-C30),
with a preferred embodiment utilizing from about 1 to about 20
carbon atoms (C1-C20), with about C1 through about C12 to about C15
being preferred, and C1 to C5 being particularly preferred,
although in some embodiments the alkyl group may be much larger.
Also included within the definition of an alkyl group are
cycloalkyl groups such as C5 and C6 rings, and heterocyclic rings
with nitrogen, oxygen, sulfur or phosphorus. Alkyl also includes
heteroalkyl, with heteroatoms of sulfur, oxygen, nitrogen, and
silicone being preferred. Alkyl includes substituted alkyl groups.
By "substituted alkyl group" herein is meant an alkyl group further
comprising one or more substitution moieties "R", as defined
above.
[0143] By "amino groups" or grammatical equivalents herein is meant
NH.sub.2, --NHR and --NR.sub.2 groups, with R being as defined
herein. In some embodiments, for example in the case of the peptide
ligation reactions, primary and secondary amines find particular
use, with primary amines generally showing faster reaction
rates.
[0144] By "nitro group" herein is meant an --NO.sub.2 group.
[0145] By "sulfur containing moieties" herein is meant compounds
containing sulfur atoms, including but not limited to, thia-, thio-
and sulfo-compounds, thiols (--SH and --SR), and sulfides
(--RSR--). A particular type of sulfur containing moiety is a
thioester (--(CO)--S--), usually found as a substituted thioester
(--(CO)--SR). By "phosphorus containing moieties" herein is meant
compounds containing phosphorus, including, but not limited to,
phosphines and phosphates. By "silicon containing moieties" herein
is meant compounds containing silicon.
[0146] By "ether" herein is meant an --O--R group. Preferred ethers
include alkoxy groups, with --O--(CH.sub.2).sub.2CH.sub.3 and
--O--(CH.sub.2).sub.4CH.sub.3 being preferred.
[0147] By "ester" herein is meant a --COOR group.
[0148] By "halogen" herein is meant bromine, iodine, chlorine, or
fluorine. Preferred substituted alkyls are partially or fully
halogenated alkyls such as CF.sub.3, etc.
[0149] By "aldehyde" herein is meant --RCOH groups.
[0150] By "alcohol" herein is meant --OH groups, and alkyl alcohols
--ROH.
[0151] By "amido" herein is meant --RCONH-- or RCONR-- groups.
[0152] By "ethylene glycol" herein is meant a
--(O--CH.sub.2--CH.sub.2).sub.n-- group, although each carbon atom
of the ethylene group may also be singly or doubly substituted,
i.e. --(O--CR.sub.2--CR.sub.2).sub.n--, with R as described above.
Ethylene glycol derivatives with other heteroatoms in place of
oxygen (i.e. --(N--CH.sub.2--CH.sub.2).sub.n-- or
--(S--CH.sub.2--CH.sub.2).sub.n--, or with substitution groups) are
also preferred.
[0153] Additionally, in some embodiments, the R group may be a
functional group, including quenchers, destabilization moieties and
fluorophores (as defined below). Fluorophores of particular use in
this embodiment include, but are not limited to Fluorescein and its
derivatizes, TAMRA (Tetramethyl-6-carboxyrhodamine), Alexa dyes,
and Cyanine dyes (e.g. Cy3 and Cy5).
[0154] Quencher moieties or molecules are known in the art, and are
generally aromatic, multiring compounds that can deactivate the
excited state of another molecule. Fluorophore-quencher pairs are
well known in the art. Suitable quencher moieties include, but are
not limited to Dabsyl (Dimethylamini(azobenzene) sulfonyl) Dabcyl
(Dimethylamino(azobenzene)carbonyl), Eclipse Quenchers (Glen
Research Catalog) and blackhole Quenchers (BHQ-1, BHQ-2 and BHQ-3)
from Biosearch Technologies.
[0155] Methods of making probes having halo leaving groups is known
in the art; see for example Abe et al., Proc Natl Acad Sci USA
(2006)103(2):263-8; Silverman et al., Nucleic Acids Res. (2005)
33(15):4978-86; Cuppolletti et al., Bioconjug Chem. (2005)
16(3):528-34; Sando et al., J Am Chem Soc. (2004) 4; 126(4):1081-7;
Sando et al., Nucleic Acids Res Suppl. (2002) 2:121-2; Sando et
al., J Am Chem Soc. (2002) 124(10):2096-7; Xu et al., Nat
Biotechnol. (2001) 19(2):148-52; Xu et al., Nucleic Acids Res.
(1998) 26(13):3159-64; Moran et al., Proc Natl Acad Sci USA (1997)
94(20):10506-11; Kool, U.S. Pat. No. 7,033,753; Kool, U.S. Pat. No.
6,670,193; Kool, U.S. Pat. No. 6,479,650; Kool, U.S. Pat. No.
6,218,108; Kool, U.S. Pat. No. 6,140,480; Kool, U.S. Pat. No.
6,077,668; Kool, U.S. Pat. No. 5,808,036; Kool, U.S. Pat. No.
5,714,320; Kool, U.S. Pat. No. 5,683,874; Kool, U.S. Pat. No.
5,674,683; and Kool, U.S. Pat. No. 5,514,546, each of which is
incorporated herein by reference in its entirety.
[0156] Nucleophile Ligation Moieties
[0157] In some embodiments, ligation probes of the invention
comprise a ligation moiety comprising a nucleophile such as an
amine. Ligation moieties comprising both a thiol and an amine find
particular use in certain reactions. In general, the nucleophile
ligation moieties can include a wide variety of potential amino,
thiol compounds as long as the nucleophile ligation moiety contains
a thiol group that is proximal to a primary or secondary amino and
the relative positioning is such that at least a 5 or 6 member ring
transition state can be achieve during the S to N acyl shift.
[0158] Accordingly, nucleophile ligation molecules that comprise 1,
2 or 1, 3 amine thiol groups find particular use. Primary amines
find use in some embodiments when reaction time is important, as
the reaction time is generally faster for primary than secondary
amines, although secondary amines find use in acyl transferase
reactions that contribute to destabilization as discussed below.
The carbons between the amino and thiol groups can be substituted
with non-hydrogen R groups, although generally only one
non-hydrogen R group per carbon is utilized. Additionally, adjacent
R groups may be joined together to form cyclic structures,
including substituted and unsubstituted cycloalkyl and aryl groups,
including heterocycloalkyl and heteroaryl and the substituted and
unsubstituted derivatives thereof. In the case where a 1,2 amino
thiol group is used and adjacent R groups are attached, it is
generally preferred that the adjacent R groups form cycloalkyl
groups (including heterocycloalkyl and substituted derivatives
thereof) rather than aryl groups.
[0159] In this embodiment, for the generation of the 4 sigma bond
contraction of the chain for destabilization, the replacement
ligation moiety relies on an acyl transferase reaction.
[0160] Linkers
[0161] In many embodiments, linkers (sometimes shown herein as "L"
or "-(linker).sub.n-), (where n is zero or one) may optionally be
included at a variety of positions within the ligation probe(s).
Suitable linkers include alkyl and aryl groups, including
heteroalkyl and heteroaryl, and substituted derivatives of these.
In some instances, for example when Native Peptide Ligation
reactions are done, the linkers may be amino acid based and/or
contain amide linkages. As described herein, some linkers allow the
ligation probes to be separated by one or more nucleobases, forming
abasic sites within the ligation product, which serve as
destabilization moieties, as described below.
[0162] Destabilization Moieties
[0163] In accordance with one aspect of the invention, it is
desirable to produce multiple copies of ligated product for each
target molecule without the aid of an enzyme. One way to achieve
this goal involves the ligated product disassociating from the
target following the chemical ligation reaction to allow a new
probe set to bind to the target. To increase ligation product
turnover, probe designs, instrumentation, and chemical ligation
reaction chemistries that increase product disassociation from the
target molecule are desirable. Suitable destabilization moieties
are discussed below and include, but are not limited to molecule
entities that result in a decrease in the overall binding energy of
an oligonucleotide to its target site. Potential examples include,
but are not limited to alkyl chains, charged complexes, and ring
structures.
[0164] Previous work has shown one way to achieve product
disassociation and increase product turnover is to "heat cycle" the
reaction mixture. Heat cycling is the process of varying the
temperature of a reaction so as to facilitate a desired outcome.
Most often heat cycling takes the form of briefly raising the
temperature of the reaction mixture so that the reaction
temperature is above the melting temperature of the ligated product
for a brief period of time causing the product to disassociate from
the target. Upon cooling, a new set of probes is able to bind the
target, and undergo another ligation reaction. This heat cycling
procedure has been practiced extensively for enzymatic reactions
like PCR.
[0165] While heat cycling is one way to achieve product turnover,
it is possible to design probes such that there is significant
product turnover without heat cycling. Probe designs and ligation
chemistries that help to lower the melting temperature of the
ligated product increase product turnover by decreasing product
inhibition of the reaction cycle.
[0166] Accordingly, in one aspect, the probes are designed to
include elements (e.g. destabilization moieties), which, upon
ligation of the probes, serve to destabilize the hybridization of
the ligation product to the target sequence. As a result, the
ligated substrate disassociates after ligation, resulting in a
turnover of the ligation product, e.g. the ligation product
comprising the two ligation probes dehybridizes from the target
sequence, freeing the target sequence for hybridization to another
probe set.
[0167] In addition, increasing the concentration of the free (e.g.
unhybridized) ligation probes can also help drive the equilibrium
towards release of the ligation product (or transfer product) from
the target sequence. Accordingly, some embodiments of the invention
use concentrations of probes that are 1,000,000 fold higher than
that of the target while in other embodiments the probes are 10,000
to 100 fold higher than that of the target. As will be appreciated
by those skilled in the art, increasing the concentration of free
probes can be used by itself or with any embodiment outlined herein
to achieve product turnover (e.g. amplification). While increasing
the probe concentration can result in increased product turnover,
it can also lead to significant off target reactions such as probe
hydrolysis and non-target mediated ligation.
[0168] In one aspect, probe elements include structures which lower
the melting temperature of the ligated product. In some
embodiments, probe elements are designed to hybridize to
non-adjacent target nucleobases, e.g. there is a "gap" between the
two hybridized but unligated probes. In general, this is done by
using one or two linkers between the probe domain and the ligation
moiety. That is, there may be a linker between the first probe
domain and the first ligation moiety, one between the second probe
domain and the second ligation moiety, or both. In some
embodiments, the gap comprises a single nucleobase, although more
can also be utilized as desired. As will be appreciated by those
skilled in the art, there may be a tradeoff between reaction
kinetics and length of the linkers; if the length of the linker(s)
are so long that contact resulting in ligation is kinetically
disfavored, shorter linkers may be desired. However, in some cases,
when kinetics are not important, the length of the gap and the
resulting linkers may be longer, to allow spanning gaps of 1 to 10
nucleobases. Generally, in this embodiment, what is important is
that the length of the linker(s) roughly corresponds to the number
of nucleobases in the gap.
[0169] In another aspect of this embodiment of the invention, the
formation of abasic sites in a ligation product as compared to the
target sequence serves to destabilize the duplex. For example, Abe
and Kool (J. Am. Chem. Soc. (2004) 126:13980-13986) compared the
turnover when two different 8-mer oligonucleotide probes (Bu42 and
DT40) were ligated with the same 7-mer probe (Thio 4). When Thio4
is ligated with DT40, a continuous 15-mer oligonucleotide probe
with a nearly native DNA structure is formed that should be
perfectly matched with the DNA target. However, when Thio4 is
ligated with Bu42, a 15-mer oligonucleotide probe is formed, but
when the probe is bound to the target, it has an abasic site in the
middle that is spanned by an alkane linker. Comparison of the
melting temperature (Tm) of these two probes when bound to the
target shows approximately a 12.degree. C. difference in melting
temperature (58.5 for Bu42 versus 70.7.degree. C. for DT40). This
12.degree. C. difference in melting temperature led to roughly a
10-fold increase in product turnover (91.6-Bu42 versus 8.2 DT40) at
25.degree. C. when the probe sets (10,000-fold excess, 10 .mu.M
conc) were present in large excess compared to the target (1 nM).
Similarly, Dose et al (Dose 2006) showed how a 4.degree. C.
decrease in Tm for two identical sequences, chemically ligated PNA
probes (53.degree. C. versus 57.degree. C.) results in
approximately a 4-fold increase in product turnover.
[0170] Recent work has demonstrated the use of chemical ligation
based Quenched Auto-Ligation (QUAL) probes to monitor RNA
expression and detect single base mismatches inside bacterial and
human cells (WO 2004/0101011 herein incorporated by reference).
[0171] In one embodiment, destabilization moieties are based on the
removal of stabilization moieties. That is, if a ligation probe
contains a moiety that stabilizes its hybridization to the target,
upon ligation and release of the stabilization moiety, there is a
drop in the stability of the ligation product. Accordingly, one
general scheme for reducing product inhibition is to develop probes
that release a molecular entity like a minor groove binding
molecule during the course of the initial chemical ligation
reaction or following a secondary reaction post ligation. Depending
on the oligonucleotide sequence, minor groove binders like the
dihydropyrroloindole tripeptide (DPI.sub.3) described by Kutyavin
(Kutyavin 1997 and Kutyavin 2000) can increase the Tm of a duplex
nucleic acid by up to 40.degree. C. when conjugated to the end of
an oligonucleotide probe. In contrast, the unattached version of
the DPI3 only increases the Tm of the same duplex by 2.degree. C.
or so. Thus, minor groove binders can be used to produce probe sets
with enhanced binding strengths, however if the minor groove binder
is released during the course of the reaction, the binding
enhancement is loss and the ligated product will display a
decreased Tm relative to probes in which the minor groove binder is
still attached.
[0172] Suitable minor groove binding molecules include, but are not
limited to, dihydropyrroloindole tripeptide (DPI.sub.3), distamycin
A, and pyrrole-imidazole polyamides (Gottesfeld, J. M., et al., J.
Mol. Biol. (2001) 309:615-629.
[0173] In addition to minor groove binding molecules tethered
intercalators and related molecules can also significantly increase
the melting temperature of oligonucleotide duplexes, and this
stabilization is significantly less in the untethered state.
(Dogan, et al., J. Am. Chem Soc. (2004) 126:4762-4763 and
Narayanan, et al., Nucleic Acids Research, (2004)
32:2901-2911).
[0174] Similarly, as will be appreciated by those in the art,
probes with attached oligonucleotide fragments (DNA, PNA, LNA, etc)
capable of triple helix formation, can serve as stabilization
moieties that upon release, results in a decrease of stabilization
of the ligation product to the target sequence (Pooga, M, et al.,
Biomolecular Engineering (2001) 17:183-192.
[0175] Another general scheme for decreasing product inhibition by
lowering the binding strength of the ligated product is to
incorporate abasic sites at the point of ligation. This approach
has been previously demonstrated by Abe (J. Am. Chem. Soc. (2004)
126:13980-13986), however it is also possible to design secondary
probe rearrangements to further amplify the decrease in Tm via
straining the alignment between the ligated probes and the target.
For example, Dose et al. (Org. Letters (2005) 7:20 4365-4368)
showed how a rearrangement post-ligation that changed the spacing
between PNA bases from the ideal 12 sigma bonds to 13 resulted in a
lowering of the Tm by 4.degree. C. Larger rearrangements and
secondary reactions that interfere with the binding of the product
to the target or result in the loss of oligonucleotide bases can
further decrease the Tm.
[0176] The present invention provides methods and compositions for
a ligation reaction that results in a chain contraction of up to 4
sigma bonds during the rearrangement, which should have a
significant effect on the Tm post-rearrangement compared to the 1
base expansion using the chemistry described by Dose. This
chemistry is based on the acyl transfer auxiliary that has been
described previously (Offer et al., J Am Chem Soc. (2002)
124(17):4642-6). Following completion of the chain contraction, a
free-thiol is generated that is capable of undergoing another
reaction either with a separate molecule or with itself. For
example, this thiol could react with an internal thioester to
severely kink the oligonucleotide and thus further decrease the
ligation product's ability to bind to the target.
[0177] Thus, in this embodiment, ligation reactions that release
functional groups that will undergo a second reaction with the
ligation product can reduce stabilization of the hybrid of the
ligation product and the target sequence.
[0178] Variable Spacer Sequences
[0179] In addition to the target domains, ligation moieties, and
optional linkers, one or more of the ligation probes of the
invention can have variable spacer sequences (also referred to as
"stuffer sequences"). These variable spacer sequences are of
particular use in the CLPA-CE assays described in further detail
herein. That is, the variable spacer sequence can serve as a type
of "label" or "barcode" to identify the target sequence when the
products are detected on the basis of length, for example using
capillary electrophoresis (CE).
[0180] Variable spacer sequences are domains of ligation probes
that can have varying lengths. These varying lengths will in some
embodiments be specific to a particular target sequence to which
other domains of the ligation probe (e.g., the probe domain) are
able to hybridize, such that the length of the ligation product
resulting from ligation of ligation probes containing stuffer
sequences is a target-specific length. As a result, any amplicons
generated from such ligation products will also have a
target-specific length. In other embodiments, the variable spacer
sequence is designed to render all ligation products of similar
length so as to facilitate the efficiency of subsequent
amplification reactions.
[0181] In some embodiments, only one of the ligation probes of a
ligation probe pair comprises a variable spacer sequence. In other
embodiments, both ligation probes comprise the variable spacer
sequence. In embodiments in which three or more ligation probes are
used, one or more of the ligation probes forming a particular
ligation product can contain the variable spacer sequence, although
in this embodiment is it generally one or both of the terminal
probes that contains the variable spacer sequence.
[0182] In further embodiments and in accordance with any of the
above, the variable spacer sequence is contained within a region of
the ligation probe that is substantially non-complementary to both
any other domains of the target nucleic acid as well as to any of
the target nucleic acids in a sample.
[0183] In still further embodiments, the ligation probes (as will
be discussed in further detail below) contain a primer sequence. In
accordance with any of the above, the variable spacer sequence is
in some embodiments contained between the probe domain (the region
of the ligation probe that hybridizes to a target sequence) and the
primer sequence, such that the amplicon contains the variable
spacer sequence.
[0184] In further aspects, variable spacer sequences of the
invention include nucleic acids such as DNA or RNA. Variable spacer
sequences may also include a combination of both
deoxyribonucleotides and ribonucleotides. Variable spacer sequences
may further include nucleotide analogues, linkers, or any of the
additional moieties discussed herein as optionally present in
ligation and/or target capture probes of the invention.
[0185] In general, for multiplex assays where multiple target
sequences are to be detected simultaneously, variable spacer
sequences are designed to result in either or both of the ligation
product or (in the case where an amplification reaction follows the
ligation step) the resulting amplicons from each ligation product
to have a different nucleotide length from other ligation products
or amplicons in the sample. That is, the amplicon from one target
nucleic acid will be different from the length of the amplicon from
a different target nucleic acid. Similarly, when multiple ligation
probe sets are used for a single target as is shown in FIGS. 6 and
11, each ligation product and/or amplicon will have a different
length, allowing detection of the different products.
[0186] The differing lengths of the variable spacer sequences will
depend on the sensitivity of the detection system. For
sophisticated Capillary electrophoresis (CE) systems, such as
Genetic Analyzer systems made by Life Technologies or the PACE
systems by Beckman Coulter, ligation products and/or amplicons can
be separated and thus detected when they differ in length by as few
as one nucleotide. Other CE systems with lower size resolution, may
require length differences of 5 to 100 base pairs depending on the
resolution. In general, higher resolution CE systems require longer
separation channel lengths and often longer separation times.
Furthermore, denaturing capillary electrophoresis systems tend to
have better resolution than non-denaturing systems. In general, the
variable spacer sequences are designed to result in nucleotide
differences ranging from 1 to 100 bases with 5 to 20 bases being
preferred. Greater size differences can be used, but often require
additional cost or reduce the number of possible ligation probes
that can be combined in a single test.
[0187] In addition, variable spacer sequences can be used in
conjunction with other labels to expand the number of different
"barcodes" that can be used. That is, a variable spacer sequence
length can be "reused" by encoding it with a second label; for
example, one amplicon containing a variable spacer sequence of 20
nucleotides can use a fluorescent label of a first color, and
another amplicon containing the 20 mer spacer sequence can use a
fluorescent label of a different color. These amplicons of the same
size can be simultaneously detected using a multi-channel CE
instrument that can identify amplicons with different wavelength
(color) products like is commonly practiced in forensic
medicine.
[0188] Additional Functionalities of Ligation Probes
[0189] In addition to the target domains, ligation moieties, and
optional linkers, one or more of the ligation probes of the
invention can have additional functionalities, including, but not
limited to, promoter and primer sequences (or complements thereof,
depending on the assay), labels, including label probe binding
sequences, and capture or anchor sequences.
[0190] In many embodiments, the ligation probes are constructed so
as to contain the necessary priming site or sites for the
subsequent amplification scheme. In a preferred embodiment the
priming sites are universal priming sites. By "universal priming
site" or "universal priming sequences" herein is meant a sequence
of the probe that will bind a primer for amplification.
[0191] In a preferred embodiment, one universal priming sequence or
site is used. In this embodiment, a preferred universal priming
sequence is the RNA polymerase T7 sequence, that allows the T7 RNA
polymerase make RNA copies of the adapter sequence as outlined
below. Additional disclosure regarding the use of T7 RNA polymerase
is found in U.S. Pat. Nos. 6,291,170, 5,891,636, 5,716,785,
5,545,522, 5,922,553, 6,225,060 and 5,514,545, all of which are
expressly incorporated herein by reference.
[0192] In a preferred embodiment, for example when amplification
methods requiring two primers such as PCR are used, each probe
preferably comprises an upstream universal priming site (UUP) and a
downstream universal priming site (DUP). Again, "upstream" and
"downstream" are not meant to convey a particular 5'-3'
orientation, and will depend on the orientation of the system.
Preferably, only a single UUP sequence and a single DUP sequence is
used in a probe set, although as will be appreciated by those in
the art, different assays or different multiplexing analysis may
utilize a plurality of universal priming sequences. In some
embodiments probe sets may comprise different universal priming
sequences. In addition, the universal priming sites are preferably
located at the 5' and 3' termini of the target probe (or the
ligated probe), as only sequences flanked by priming sequences will
be amplified.
[0193] In addition, universal priming sequences are generally
chosen to be as unique as possible given the particular assays and
host genomes to ensure specificity of the assay. However, as will
be appreciated by those in the art, sets of priming
sequences/primers may be used; that is, one reaction may utilize
500 target probes with a first priming sequence or set of
sequences, and an additional 500 probes with a second sequence or
set of sequences.
[0194] As will be appreciated by those in the art, when two priming
sequences are used, the orientation of the two priming sites is
generally different. That is, one PCR primer will directly
hybridize to the first priming site, while the other PCR primer
will hybridize to the complement of the second priming site. Stated
differently, the first priming site is in sense orientation, and
the second priming site is in antisense orientation.
[0195] As will be appreciated by those in the art, in general,
highly multiplexed reactions can be performed, with all of the
universal priming sites being the same for all reactions.
Alternatively, "sets" of universal priming sites and corresponding
probes can be used, either simultaneously or sequentially. The
universal priming sites are used to amplify the modified probes to
form a plurality of amplicons that are then detected in a variety
of ways, as outlined herein. In preferred embodiments, one of the
universal priming sites is a T7 site. In some embodiments this
priming site serves as a template for the synthesis of RNA.
[0196] In a preferred embodiment, when detecting multiple targets
simultaneously, all of the oligonucleotide ligation probe sets in
the reaction are designed to contain identical promoter or primer
pair binding sites such that following ligation and purification,
if appropriate, all of the ligated products can be amplified
simultaneously using the same enzyme and/or same primers. In other
words, in embodiments involving the detection of multiple target
nucleic acids, different ligation probe sets containing ligation
probes directed to different target nucleic acid sequences can in
some embodiments possess identical promoter or primer pair binding
sites (e.g., "universal" primer binding sites) such that the
ligation products resulting from the hybridization and ligation of
these ligation probes can be amplified using the same enzyme and/or
primers.
[0197] In one embodiment, one or more of the ligation probes
comprise a promoter sequence. In embodiments that employ a promoter
sequence, the promoter sequence or its complement will be of
sufficient length to permit an appropriate polymerase to interact
with it. Detailed descriptions of sequences that are sufficiently
long for polymerase interaction can be found in, among other
places, Sambrook and Russell, which are hereby incorporated by
reference for all purposes and in particular for all teachings
related to promoter sequences for polymerases. In certain
embodiments, amplification methods comprise at least one cycle of
amplification, for example, but not limited to, the sequential
procedures of: interaction of a polymerase with a promoter;
synthesizing a strand of nucleotides in a template-dependent manner
using a polymerase; and denaturing the newly-formed nucleic acid
duplex to separate the strands.
[0198] In another embodiment, one or both of the ligation probes
comprise a primer sequence. As outlined below, the ligation
products of the present invention may be used in additional
reactions such as enzymatic amplification reactions. In one
embodiment, the ligation probes include primer sequences designed
to allow an additional level of amplification. As used herein, the
term "primer" refers to nucleotide sequence, whether occurring
naturally as in a purified restriction digest or produced
synthetically, which is capable of acting as a point of initiation
of nucleic acid sequence synthesis when placed under conditions in
which synthesis of a primer extension product which is
complementary to a nucleic acid strand is induced, i.e. in the
presence of different nucleotide triphosphates and a polymerase in
an appropriate buffer ("buffer" includes pH, ionic strength,
cofactors etc.) and at a suitable temperature. One or more of the
nucleotides of the primer can be modified, for instance by addition
of a methyl group, a biotin or digoxigenin moiety, a fluorescent
tag or by using radioactive nucleotides. A primer sequence need not
reflect the exact sequence of the template. For example, a
non-complementary nucleotide fragment may be attached to the 5' end
of the primer, with the remainder of the primer sequence being
substantially complementary to the target strand.
[0199] By using several priming sequences and primers, a first
ligation product can serve as the template for additional ligation
products. These primer sequences may serve as priming sites for PCR
reactions, which can be used to amplify the ligation products. In
addition to PCR reactions, other methods of amplification can
utilize the priming sequences, including but not limited to ligase
chain reactions, Invader.TM. positional amplification by nick
translation (NICK), primer extension/nick translation, and other
methods known in the art. As used herein, "amplification" refers to
an increase in the number of copies of a particular nucleic acid.
Copies of a particular nucleic acid made in vitro in an
amplification reaction are called "amplicons" or "amplification
products".
[0200] Amplification may also occur through a second ligation
reaction, in which the primer sites serve as hybridization sites
for a new set of ligation probes which may or may not comprise
sequences that are identical to the first set of ligation probes
that produced the original ligation products. The target sequence
is thus exponentially amplified through amplification of ligation
products in subsequent cycles of amplification.
[0201] In another embodiment of this aspect of the invention, the
primer sequences are used for nested ligation reactions. In such
nested ligation reactions, a first ligation reaction is
accomplished using methods described herein such that the ligation
product can be captured, for example by using biotinylated primers
to the desired strand and capture on beads (particularly magnetic
beads) coated with streptavidin. After the ligation products are
captured, a second ligation reaction is accomplished by
hybridization of ligation probes to primer sequences within a
section of the ligation product which is spatially removed from
(i.e., downstream from) the end of the ligation product which is
attached to the capture bead, probe, etc. At least one of the
primer sequences for the secondary ligation reaction will be
located within the region of the ligation product complementary to
the ligation probe which is not the ligation probe that included
the anchor or capture sequence. The ligation products from this
second ligation reaction will thus necessarily only result from
those sequences successfully formed from the first chemical
ligation, thus removing any "false positives" from the
amplification reaction. In another embodiment, the primer sequences
used in the secondary reaction may be primer sites for other types
of amplification reactions, such as PCR.
[0202] In one embodiment, one or more of the ligation probes
comprise an anchor sequence. By "anchor sequence" herein is meant a
component of a ligation probe that allows the attachment of a
ligation product to a support for the purposes of detection.
Suitable means for detection include a support having attached
thereto an appropriate capture moiety. Generally, such an
attachment will occur via hybridization of the anchor sequence with
a capture probe, which is substantially complementary to the anchor
sequence.
[0203] In a preferred embodiment, one of the probes further
comprises an "anchor" sequence, (sometimes referred to in the art
and herein as "zip codes" or "bar codes" or "adapters"). Adapters
facilitate immobilization of probes to allow the use of "universal
arrays". That is, arrays (either solid phase or liquid phase
arrays) are generated that contain capture probes that are not
target specific, but rather specific to individual (preferably)
artificial adapter sequences.
[0204] Thus, an "adapter sequence" is a nucleic acid that is
generally not native to the target sequence, i.e. is exogenous, but
is added or attached to the target sequence. It should be noted
that in this context, the "target sequence" can include the primary
sample target sequence, or can be a derivative target such as a
reactant or product of the reactions outlined herein; thus for
example, the target sequence can be a PCR product, a first ligation
probe or a ligated probe in an OLA reaction, etc. The terms
"barcodes", "adapters", "addresses", tags" and "zipcodes" have all
been used to describe artificial sequences that are added to
amplicons to allow separation of nucleic acid fragment pools. One
preferred form of adapters are hybridization adapters. In this
embodiment adapters are chosen so as to allow hybridization to the
complementary capture probes on a surface of an array. Adapters
serve as unique identifiers of the probe and thus of the target
sequence. In general, sets of adapters and the corresponding
capture probes on arrays are developed to minimize
cross-hybridization with both each other and other components of
the reaction mixtures, including the target sequences and sequences
on the larger nucleic acid sequences outside of the target
sequences (e.g. to sequences within genomic DNA). Other forms of
adapters are mass tags that can be separated using mass
spectroscopy, electrophoretic tags that can be separated based on
electrophoretic mobility, etc. Some adapter sequences are outlined
in U.S. Ser. No. 09/940,185, filed Aug. 27, 2001, hereby
incorporated by reference in its entirety. Preferred adapters are
those that meet the following criteria. They are not found in a
genome, preferably a human genome, and they do not have undesirable
structures, such as hairpin loops.
[0205] In one embodiment the use of adapter sequences allow the
creation of more "universal" surfaces; that is, one standard array,
comprising a finite set of capture probes can be made and used in
any application. The end-user can customize the array by designing
different soluble target probes, which, as will be appreciated by
those in the art, is generally simpler and less costly. In a
preferred embodiment, an array of different and usually artificial
capture probes are made; that is, the capture probes do not have
complementarity to known target sequences. The anchor sequences can
then be incorporated in the target probes.
[0206] As will be appreciated by those in the art, the length of
the anchor sequences will vary, depending on the desired "strength"
of binding and the number of different anchor desired. In a
preferred embodiment, adapter sequences range from about 6 to about
500 basepairs in length, with from about 8 to about 100 being
preferred, and from about 10 to about 25 being particularly
preferred.
[0207] In a preferred embodiment, the adapter sequence uniquely
identifies the target analyte to which the target probe binds. That
is, while the adapter sequence need not bind itself to the target
analyte, the system allows for identification of the target analyte
by detecting the presence of the adapter. Accordingly, following a
binding or hybridization assay and washing, the probes including
the adapters are amplified. Detection of the adapter then serves as
an indication of the presence of the target nucleic acid.
[0208] In one embodiment the adapter includes both an identifier
region and a region that is complementary to capture probes on a
universal array as described above. In this embodiment, the
amplicon hybridizes to capture probes on a universal array.
Detection of the adapter is accomplished following hybridization
with a probe that is complementary to the adapter sequence.
Preferably the probe is labeled as described herein.
[0209] In general, similar to variable spacer sequences, unique
adapter sequences are used for each unique target analyte. That is,
the elucidation or detection of a particular adapter sequence
allows the identification of the target analyte to which the target
probe containing that adapter sequence bound. However, as discussed
herein, in some cases, it is possible to "reuse" adapter sequences
and have more than one target analyte share an adapter
sequence.
[0210] In a preferred embodiment the adapters contain different
sequences or properties that are indicative of a particular target
molecule. That is, each adapter uniquely identifies a target
sequence. As described above, the adapters are amplified to form
amplicons. The adapter is detected as an indication of the presence
of the target analyte, i.e. the particular target nucleic acid.
[0211] In one embodiment of this aspect of the invention, the
upstream oligonucleotide is designed to have an additional
nucleotide segment that does not bind to the target of interest,
but is to be used to subsequently capture the ligated product on a
suitable solid support or device of some sort. In a preferred
embodiment of this aspect of the invention, the downstream
oligonucleotide has a detectable label attached to it, such that
following ligation, the resulting product will contain a capture
sequence for a solid support at its 3' end and a detectable label
at its 5' end, and only ligated products will contain both the
capture sequence and the label.
[0212] In another aspect of the invention pertaining in particular
to multiplex target detection, each upstream probe of a probe set
may be designed to have a unique sequence (also referred to herein
as an "anchor sequence) at is 3' end that corresponds to a
different position on a DNA array. Each downstream probe of a probe
set may optionally contain a detectable label that is identical to
the other downstream probes, but a unique target binding sequence
that corresponds to its respective targets. Following hybridization
with the DNA array, only ligated probes that have both an address
sequence (upstream probe) and a label (downstream probe) will be
observable. In another aspect of the invention, the detectable
label can be attached to the upstream probe and the capture
sequence can be a part of the downstream probe, such that the
ligated products will have the detectable label more towards the 3'
end and the capture sequence towards the 5' end of the ligated
product. The exact configuration is best determined via
consideration of the ease of synthesis as well as the
characteristics of the devices to be used to subsequently detect
the ligated reaction product.
[0213] The anchor sequence may have both nucleic and non-nucleic
acid portions. Thus, for example, flexible linkers such as alkyl
groups, including polyethylene glycol linkers, may be used to
provide space between the nucleic acid portion of the anchor
sequence and the support surface. This may be particularly useful
when the ligation products are large.
[0214] In addition, in some cases, sets of anchor sequences that
correspond to the capture probes of "universal arrays" can be used.
As is known in the art, arrays can be made with synthetic generic
sequences as capture probes, that are designed to non-complementary
to the target sequences of the sample being analyzed but to
complementary to the array binding sequences of the ligation probe
sets. These "universal arrays" can be used for multiple types of
samples and diagnostics tests because same array binding sequences
of the probes can be reused/paired with different target
recognition sequences.
[0215] In one embodiment, one or more of the ligation probes
comprise a label. By "label" or "labeled" herein is meant that a
compound has at least one element, isotope or chemical compound
attached to enable the detection of the compound, e.g. renders a
ligation probe or ligation or transfer product detectable using
known detection methods, e.g., electronic, spectroscopic,
photochemical, or electrochemiluminescent methods. In general,
labels fall into three classes: a) isotopic labels, which may be
radioactive or heavy isotopes; b) magnetic, electrical, thermal;
and c) colored or luminescent dyes; although labels include enzymes
and particles such as magnetic particles as well. The dyes may be
chromophores or phosphors but are preferably fluorescent dyes,
which due to their strong signals provide a good signal-to-noise
ratio. Suitable dyes for use in the invention include, but are not
limited to, fluorescent lanthanide complexes, including those of
Europium and Terbium, fluorescein, fluorescein isothiocyanate,
carboxyfluorescein (FAM), dichlorotriazinylamine fluorescein,
rhodamine, tetramethylrhodamine, umbelliferone, eosin, erythrosin,
coumarin, methyl-coumarins, pyrene, Malacite green, Cy3, Cy5,
stilbene, Lucifer Yellow, Cascade Blue.TM., Texas Red, alexa dyes,
dansyl chloride, phycoerythin, green fluorescent protein and its
wavelength shifted variants, bodipy, and others known in the art
such as those described in Haugland, Molecular Probes Handbook,
(Eugene, Oreg.) 6th Edition; The Synthegen catalog (Houston, Tex.),
Lakowicz, Principles of Fluorescence Spectroscopy, 2nd Ed., Plenum
Press New York (1999), and others described in the 6th Edition of
the Molecular Probes Handbook by Richard P. Haugland, herein
expressly incorporated by reference. Additional labels include
nanocrystals or Q-dots as described in U.S. Ser. No. 09/315,584,
herein expressly incorporated by reference.
[0216] In a preferred embodiment, the label is a secondary label
that part of a binding partner pair. For example, the label may be
a hapten or antigen, which will bind its binding partner. In a
preferred embodiment, the binding partner can be attached to a
solid support to allow separation of extended and non-extended
primers. For example, suitable binding partner pairs include, but
are not limited to: antigens (such as proteins (including
peptides)) and antibodies (including fragments thereof (FAbs,
etc.)); proteins and small molecules, including
biotin/streptavidin; enzymes and substrates or inhibitors; other
protein-protein interacting pairs; receptor-ligands; and
carbohydrates and their binding partners. Nucleic acid-nucleic acid
binding protein pairs are also useful. In general, the smaller of
the pair is attached to the NTP for incorporation into the primer.
Preferred binding partner pairs include, but are not limited to,
biotin (or imino-biotin) and streptavidin, digeoxinin and Abs, and
Prolinx.TM. reagents.
[0217] In a preferred embodiment, the binding partner pair
comprises biotin or imino-biotin and streptavidin. Imino-biotin is
particularly preferred as imino-biotin disassociates from
streptavidin in pH 4.0 buffer while biotin requires harsh
denaturants (e.g. 6 M guanidinium HCl, pH 1.5 or 90% formamide at
95.degree. C.).
[0218] In a preferred embodiment, the binding partner pair
comprises a primary detection label (for example, attached to a
ligation probe) and an antibody that will specifically bind to the
primary detection label. By "specifically bind" herein is meant
that the partners bind with specificity sufficient to differentiate
between the pair and other components or contaminants of the
system. The binding should be sufficient to remain bound under the
conditions of the assay, including wash steps to remove
non-specific binding. In some embodiments, the dissociation
constants of the pair will be less than about 10.sup.-4 to
10.sup.-6 M.sup.-1, with less than about 10.sup.-5 to 10.sup.-9
M.sup.-1 being preferred and 10.sup.-9 M.sup.-1 being particularly
preferred.
[0219] In a preferred embodiment, the secondary label is a
chemically modifiable moiety. In this embodiment, labels comprising
reactive functional groups are incorporated into the nucleic acid.
The functional group can then be subsequently labeled with a
primary label. Suitable functional groups include, but are not
limited to, amino groups, carboxy groups, maleimide groups, oxo
groups and thiol groups, with amino groups and thiol groups being
particularly preferred. For example, primary labels containing
amino groups can be attached to secondary labels comprising amino
groups, for example using linkers as are known in the art; for
example, homo- or hetero-bifunctional linkers as are well known
(see 1994 Pierce Chemical Company catalog, technical section on
cross-linkers, pages 155 200, incorporated herein by
reference).
[0220] In this embodiment, the label may also be a label probe
binding sequence or complement thereof. By "label probe" herein is
meant a nucleic acid that is substantially complementary to the
binding sequence and is labeled, generally directly.
[0221] The compositions of the invention are generally made using
known synthetic techniques. In general, methodologies based on
standard phosphoramidite chemistries find particular use in one
aspect of the present invention, although as is appreciated by
those skilled in the art, a wide variety of nucleic acid synthetic
reactions are known. The spacing of the addition of fluorophores
and quenchers is well known as well.
VI. Target Capture Probes
[0222] In certain aspects, and in accordance with any of the
description of ligation probes discussed herein, the present
invention further includes the use of target capture probes. These
target capture probes do not generally participate in the chemical
ligation reactions of the ligation probes, and are useful for
increasing the specificity of assays. As is described further
herein, the target capture probes are hybridized to the target
nucleic acid either upstream or downstream from the ligation
product formed from ligation probes as described herein.
Hybridizing the target capture probe to the target nucleic acid
forms a target complex that comprises the target nucleic acid, one
or more ligation products, and the target capture probe. The target
capture probe contains a capture moiety that can be used to capture
the target complex on a solid support or a bead. Following capture
on the support or bead, the non-bound species are removed. The
target complex (also referred to herein as a "capture complex")
and/or the ligation products is then either analyzed or subjected
to amplification (such as PCR).
[0223] In general, target capture probes of the invention contain a
domain that hybridizes to the target nucleic acid and an additional
capture moiety that can be used to selectively capture
complexes/molecules that are bound to the target capture through
hybridization or other interactions. In some embodiments, the
capture moiety can be a capture nucleic acid sequence, a bead, or a
binding partner of a binding partner pair (such as biotin, which
can then be captured with its binding partner streptavidin).
[0224] Target capture probes are designed to hybridize upstream or
downstream from ligation probes hybridized to target domains of a
nucleic acid. In certain embodiments, target capture probes will
hybridize within a predetermined distance to the ligation
probes.
[0225] As is discussed herein, methods of the invention include the
hybridization of two or more ligation probes to target domains of a
target nucleic acid under conditions such that the ligation probes
spontaneously undergo a chemical ligation without the addition of a
ligase enzyme. In embodiments of the invention that use target
capture probes along with ligation probes, it will be appreciated
that the target capture probes can be hybridized to the target
nucleic acid prior to, subsequent to or simultaneously with
hybridization of the one or more ligation probes. Similarly,
hybridization of the target capture probes may also occur prior to,
subsequent to or simultaneously with the spontaneous ligation of
the ligation probes.
[0226] In certain embodiments, multiple target capture probes are
applied to a target nucleic acid. As will be appreciated, these
multiple capture probes can be designed to hybridize to any point
along the length of the target. In embodiments in which a single
ligation product is formed on a target nucleic acid, multiple
ligation probes may be used to flank the ligation product on either
side. This embodiment is of particular use in quality control or
assessments of nucleic acid integrity, because there is an
increased likelihood that even a degraded target nucleic acid will
be captured when multiple target capture probes are used.
[0227] In embodiments in which multiple ligation products are
formed on a target nucleic acid, multiple target capture probes may
also be hybridized to the target nucleic acid. As discussed above,
these multiple target capture probes may be designed to hybridize
at different points along the length of the target nucleic acid. In
some embodiments, target capture probes are designed to hybridize
at or near the 3', 5' or both the 3' and 5' end of the target
nucleic acid. In further embodiments, the target capture probes are
designed to be hybridized to regions upstream, downstream, and/or
interleaved between the different ligation products formed on a
target nucleic acid.
VII. Assays and Detection Methods
[0228] In exemplary aspects, the present invention provides methods
for detecting a plurality of different target nucleic acids in a
sample. Such target nucleic acids include at least a first and a
second target domain, where the first and second target domains are
adjacent to each other. The target nucleic acids can also include a
third domain that is a target capture domain, and that target
capture domain is located upstream or downstream (i.e., 5' or 3')
to the first and second target domains.
[0229] In preferred embodiments, assays of the invention include
the steps of providing ligation substrates that include a target
nucleic acid comprising the target domains and target capture
domains discussed above. These ligation substrates each further
include a first set of ligation probes comprising a first and
second nucleic acid ligation probe. The first nucleic acid ligation
probe is hybridized to the first target domain of the target
nucleic acid sequence, and the second nucleic acid ligation probe
is hybridized to the second target domain of the target nucleic
acid sequence. In such embodiments, the first target domain is
upstream of the second target domain. The first nucleic acid
ligation probe further includes a 5' ligation moiety and the second
nucleic acid ligation probe further includes a 3' ligation moiety.
Since the first and second target domains are adjacent to each
other, the ligation moieties of the two ligation probes hybridized
to the target domains are adjacent to each other and are able to
undergo ligation without the use of a ligase enzyme to form a
ligation product.
[0230] Embodiments of the invention further include hybridizing a
target capture probe to the third target capture domain of the
target nucleic acid to form a target complex. The target complex
thus comprises the target nucleic acid, the first ligation probe
hybridized to the first target domain, the second ligation probe
hybridized to the second ligation domain and the target capture
probe hybridized to the third target capture domain. The target
capture probe comprises a capture moiety, enabling capture of the
target complex on a surface or substrate (such as a bead).
Capturing the target complex can be used to separate the target
complex from unbound target nucleic acids and ligation probes--in
other words, use of a target capture probes provides a way to
isolate those target nucleic acids on which a ligation product has
successfully been formed.
[0231] Assays of the invention can further include amplifying the
ligation product formed from the different ligation substrates to
form amplicons and then detecting the amplicons to detect the
target nucleic acids.
[0232] As will be appreciated, assays of the invention may utilize
multiple sets of ligation probes directed to multiple target
domains. For example, the nucleic acids can further include a
fourth target domain adjacent to a fifth target domain, and the
ligation substrates further include a second set of ligation probes
that include a third ligation probe hybridized to the fourth target
domain and a fourth ligation probe hybridized to the fifth target
domain. As with the first and second ligation probes discussed
above, the third and fourth ligation probes ligate without the use
of a ligase enzyme, and thus the target nucleic acid comprises
multiple ligation products. Target capture probes can again be
hybridized to the target nucleic acids to form target complexes
comprising the multiple ligation products, the target nucleic acid
and the target capture probes. As discussed above, these target
complexes can be captured on a surface and the multiple ligation
products can in some embodiments be amplified to form amplicons,
which can then be detected.
[0233] Similarly, the target nucleic acids can further include a
sixth target domain adjacent to a seventh target domain, and a
third set of ligation probes containing fifth and sixth ligation
probes can be hybridized to the sixth and seventh target domains
respectively. As with any of the ligation probes discussed herein,
the fifth and sixth ligation probes are ligated without the use of
a ligase enzyme, thus producing a target nucleic acid comprising
multiple ligation products (i.e., the ligation product formed from
the ligation of the first and second ligation probes and/or the
ligation product formed from the third and fourth ligation probes).
As will be appreciated, a target nucleic acid may contain any
number of target domains, and thus any number of ligation probe
sets can be used to form ligation products on that target nucleic
acid in accordance with the present invention.
[0234] Any of the above target nucleic acids comprising multiple
ligation products may further be hybridized with one or more target
capture probes to form target complexes. Those target complexes can
then be captured on a substrate. The ligation products of the
target complexes are then amplified using methods known in the art
and discussed herein to form amplicons, and those amplicons are
then detected to identify and/or quantify the presence of target
nucleic acids.
[0235] In certain embodiments, the ligation products formed in
accordance with any of the methods described herein are not
amplified but are instead directly detected using any of the
methods described herein.
[0236] Prior to detecting the ligation or transfer reaction
product, there may be additional amplification reactions. Secondary
amplification reactions can be used to increase the signal for
detection of the target sequence; e.g. by increasing the number of
ligated products produced per copy of target. In one embodiment,
any number of standard amplification reactions can be performed on
the ligation product, including, but not limited to, strand
displacement amplification (SDA), nucleic acid sequence based
amplification (NASBA), ligation amplification and the polymerase
chain reaction (PCR); including a number of variations of PCR,
including "quantitative competitive PCR" or "QC-PCR", "arbitrarily
primed PCR" or "AP-PCR", "immuno-PCR", "Alu-PCR", "PCR single
strand conformational polymorphism" or "PCR-SSCP", "reverse
transcriptase PCR" or "RT-PCR", "biotin capture PCR", "vectorette
PCR". "panhandle PCR", and "PCR select cDNA subtraction", among
others. In one embodiment, the amplification technique is not PCR.
According to certain embodiments, one may use ligation techniques
such as gap-filling ligation, including, without limitation,
gap-filling OLA and LCR, bridging oligonucleotide ligation,
FEN-LCR, and correction ligation. Descriptions of these techniques
can be found, among other places, in U.S. Pat. No. 5,185,243,
published European Patent Applications EP 320308 and EP 439182,
published PCT Patent Application WO 90/01069, published PCT Patent
Application WO 02/02823, and U.S. patent application Ser. No.
09/898,323.
[0237] In addition to standard enzymatic amplification reactions,
it is possible to design probe schemes where the ligated product
that is initially produced can itself be the target of a secondary
chemical ligation reaction.
[0238] Furthermore, "preamplification reactions" can be done on
starting sample nucleic acids to generate more target sequences for
the chemical reaction ligation. For example, whole genome
amplification can be done.
[0239] As will be appreciated by those skilled in the art, assays
utilizing methods and compositions of the invention can take on a
wide variety of configurations, depending on the desired
application, and can include in situ assays (similar to FISH),
solution based assays (e.g. transfer/removal of fluorophores and/or
quenchers), and heterogeneous assays (e.g. utilizing solid supports
for manipulation, removal and/or detection, such as the use of high
density arrays). In addition, assays can include additional
reactions, such as pre-amplification of target sequences and
secondary amplification reactions after ligation has occurred, as
is outlined herein.
[0240] Assays pertaining to this aspect of the invention, as
described herein, may rely on increases in a signal, e.g. the
generation of fluorescence or chemiluminescence. However, as will
be appreciated by those in the art, assays that rely on decreases
in such signals are also possible.
[0241] In one embodiment, assay reactions are performed "in situ"
(also referred to in various assay formats as "in vitro" and/or "ex
vivo" depending on the sample), similar to FISH reactions. Since no
exogeneous enzymes need be added, reagents can be added to cells
(living, electroporated, fixed, etc.) such as histological samples
for the determination of the presence of target sequences,
particularly those associated with disease states or other
pathologies.
[0242] In addition, "in vitro" assays can be done where target
sequences are extracted from samples. Samples can be processed
(e.g. for paraffin embedded samples, the sample can be prepared),
the reagents added and the reaction allowed to proceed, with
detection following as is done in the art.
[0243] In some embodiments, ligation products (also referred to
herein as "ligated products") are detected using solid supports.
For example, the ligated products are attached to beads, using
either anchor probe/capture probe hybridization or other binding
techniques, such as the use of a binding partner pair (e.g. biotin
and streptavidin). In one embodiment, a transfer reaction results
in a biotin moiety being transferred from the first ligation probe
to a second ligation probe comprising a label. Beads comprising
streptavidin are contacted with the sample, and the beads are
examined for the presence of the label, for example using FACS
technologies.
[0244] In other embodiments, ligated products are detected using
heterogeneous assays. That is, the reaction is done in solution and
the product is added to a solid support, such as an array or beads.
Generally, one ligation probe comprises an anchor sequence or a
binding pair partner (e.g. biotin, haptens, etc.) and the other
comprises a label (e.g. a fluorophore, a label probe binding
sequence, etc.). The ligated product is added to the solid support,
and the support optionally washed. In this embodiment, only the
ligated product will be captured and be labeled.
[0245] In another aspect of the invention, one of oligonucleotide
probes has an attached magnetic bead or some other label (biotin)
that allows for easy manipulation of the ligated product. The
magnetic bead or label can be attached to either the upstream or
the downstream probe using any number of configurations as outlined
herein.
[0246] As described herein, secondary reactions can also be done,
where additional functional moieties (e.g. anchor sequences,
primers, labels, etc.) are added. Similarly, secondary
amplification reactions can be done as described herein.
[0247] Detection systems are known in the art, and include optical
assays (including fluorescence and chemiluminescent assays),
enzymatic assays, radiolabelling, surface plasmon resonance,
magnetoresistance, cantilever deflection, sequencing, surface
plasmon resonance, etc. In some embodiments, the ligated product
can be used in additional assay technologies, for example, as
described in 2006/0068378, hereby incorporated by reference, the
ligated product can serve as a linker between light scattering
particles such as colloids, resulting in a color change in the
presence of the ligated product.
[0248] In some embodiments, the detection system can be included
within the sample collection tube; for example, blood collection
devices can have assays incorporated into the tubes or device to
allow detection of pathogens or diseases.
[0249] PCT applications WO 95/15971, PCT/US96/09769,
PCT/US97/09739, PCT US99/01705, WO96/40712 and WO98/20162, all of
which are expressly incorporated herein by reference in their
entirety, describe novel compositions comprising nucleic acids
containing electron transfer moieties, including electrodes, which
allow for novel detection methods of nucleic acid
hybridization.
[0250] One technology that has gained increased prominence involves
the use of DNA arrays (Marshall et al., Nat Biotechnol. (1998)
16(1):27-31), especially for applications involving simultaneous
measurement of numerous nucleic acid targets. DNA arrays are most
often used for gene expression monitoring where the relative
concentration of 1 to 100,000 nucleic acids targets (mRNA) is
measured simultaneously. DNA arrays are small devices in which
nucleic acid anchor probes are attached to a surface in a pattern
that is distinct and known at the time of manufacture (Marshall et
al., Nat Biotechnol. (1998) 16(1):27-31) or can be accurately
deciphered at a later time such as is the case for bead arrays
(Steemers et al., Nat Biotechnol. (2000) 18(1):91-4; and Yang et
al., Genome Res. (2001) 11(11):1888-98.). After a series of
upstream processing steps, the sample of interest is brought into
contact with the DNA array, the nucleic acid targets in the sample
hybridize to anchor oligonucleotides on the surface, and the
identity and often concentration of the target nucleic acids in the
sample are determined.
[0251] Many of the nucleic acid detection methods in current use
have characteristics and/or limitations that hinder their broad
applicability. For example, in the case of DNA microarrays, prior
to bringing a sample into contact with the microarray, there are
usually a series of processing steps that must be performed on the
sample. While these steps vary depending upon the manufacturer of
the array and/or the technology that is used to read the array
(fluorescence, electrochemistry, chemiluminescence,
magnetoresistance, cantilever deflection, surface plasmon
resonance), these processing steps usually fall into some general
categories: Nucleic acid isolation and purification, enzymatic
amplification, detectable label incorporation, and clean up
post-amplification. Other common steps are sample concentration,
amplified target fragmentation so as to reduce the average size of
the nucleic acid target, and exonuclease digestion to convert PCR
amplified targets to a single stranded species.
[0252] The requirement of many upstream processing steps prior to
contacting the DNA array with the sample can significantly increase
the time and cost of detecting a nucleic acid target(s) by these
methods. It can also have significant implications on the quality
of the data obtained. For instance, some amplification procedures
are very sensitive to target degradation and perform poorly if the
input nucleic acid material is not well preserved (Foss et al.,
Diagn Mol Pathol. (1994) 3(3):148-55). Technologies that can
eliminate or reduce the number and/or complexity of the upstream
processing steps could significantly reduce the cost and improve
the quality of results obtained from a DNA array test. One method
for reducing upstream processing steps involves using ligation
reactions to increase signal strength and improve specificity.
[0253] As outlined above, the assays can be run in a variety of
ways. In assays that utilize detection on solid supports, there are
a variety of solid supports, including arrays, that find use in the
invention.
[0254] In some embodiments, solid supports such as beads find use
in the present invention. For example, binding partner pairs (one
on the ligated product and one on the bead) can be used as outlined
above to remove non-ligated reactants. In this embodiment, magnetic
beads are particularly preferred.
[0255] In some embodiments of the invention, capture probes are
attached to solid supports for detection. For example, capture
probes can be attached to beads for subsequent analysis using any
suitable technique, e.g. FACS. Similarly, bead arrays as described
below may be used.
[0256] In one embodiment, the present invention provides arrays,
each array location comprising at a minimum a covalently attached
nucleic acid probe, generally referred to as a "capture probe". By
"array" herein is meant a plurality of nucleic acid probes in an
array format; the size of the array will depend on the composition
and end use of the array. Arrays containing from about 2 different
capture ligands to many thousands can be made. Generally, for
electrode-based assays, the array will comprise from two to as many
as 100,000 or more, depending on the size of the electrodes, as
well as the end use of the array. Preferred ranges are from about 2
to about 10,000, with from about 5 to about 1000 being preferred,
and from about 10 to about 100 being particularly preferred. In
some embodiments, the compositions of the invention may not be in
array format; that is, for some embodiments, compositions
comprising a single capture probe may be made as well. In addition,
in some arrays, multiple substrates may be used, either of
different or identical compositions. Thus, for example, large
arrays may comprise a plurality of smaller substrates. Nucleic acid
arrays are known in the art, and can be classified in a number of
ways; both ordered arrays (e.g. the ability to resolve chemistries
at discrete sites), and random arrays (e.g. bead arrays) are
included. Ordered arrays include, but are not limited to, those
made using photolithography techniques (e.g. Affymetrix
GeneChip.RTM.), spotting techniques (Synteni and others), printing
techniques (Hewlett Packard and Rosetta), electrode arrays, three
dimensional "gel pad" arrays and liquid arrays.
[0257] In a preferred embodiment, the arrays are present on a
substrate. By "substrate" or "solid support" or other grammatical
equivalents herein is meant any material that can be modified to
contain discrete individual sites appropriate for the attachment or
association of nucleic acids. The substrate can comprise a wide
variety of materials, as will be appreciated by those skilled in
the art, including, but not limited to glass, plastics, polymers,
metals, metalloids, ceramics, and organics. When the solid support
is a bead, a wide variety of substrates are possible, including but
not limited to magnetic materials, glass, silicon, dextrans, and
plastics.
[0258] As will be appreciated, and as is also discussed above, a
number of detection methods can be used to detect the presence of
and quantify the ligation products (or the amplicons of the
ligation products) formed in accordance with the present invention.
Such detection methods can be utilized with any of the ligation
products (or their amplicons) in any of the assays discussed
herein. Such detection methods include without limitation:
capillary electrophoresis, mass spectrometry, microarray analysis,
sequencing, real-time PCR, optical detection, fluorescence
detection, bioluminescence detection, chemiluminescence detection,
electrochemical detection, electrochemiluminescence detection and
lateral flow detection.
[0259] In addition to the above described-assays, specific assays
of use in the invention include chemical ligation dependent probe
amplification (CLPA) assays, which are discussed in further detail
below.
Chemical Ligation Dependent Probe Amplification (CLPA)
[0260] In some aspects, the invention relates to chemical ligation
dependent probe amplification (CLPA) technology. CLPA is based on
the chemical ligation of target specific ligation probes to form a
ligation product. This ligation product subsequently serves as a
template for an enzymatic amplification reaction to produce
amplicons which are subsequently analyzed using any suitable means,
including without limitation detection methods such as those
discussed above: capillary electrophoresis, mass spectrometry,
microarray analysis, sequencing, real-time PCR, optical detection,
fluorescence detection, bioluminescence detection,
chemiluminescence detection, electrochemical detection,
electrochemiluminescence detection and lateral flow detection. CLPA
can be used for a variety of purposes including but not limited to
analysis of complex gene signature patterns. Unlike other
techniques, such as DASL (Bibikova, M., et al., American Journal of
Pathology, (2004), 165:5, 1799-1807) and MLPA (Schouten, U.S. Pat.
No. 6,955,901), which utilize an enzymatic ligation reaction, CLPA
uses a chemical ligation reaction.
[0261] In one embodiment, the CLPA assay comprises the use of two
or more ligation probes that incorporate reactive moieties that can
self-ligate when properly positioned on a target sequence. In a
preferred embodiment, a 3'-phosphorothioate moiety on a first probe
reacts with a 5'-DABSYL leaving group on a second probe (See Scheme
I below).
##STR00001##
[0262] The 5'-DABSYL group reacts about four times faster than
other moieties, e.g. iodine, and also simplifies purification of
the probes during synthesis.
[0263] CLPA has several distinct advantages over other
sequence-based hybridization techniques. First, CLPA can be applied
directly to RNA analysis without the need to make a DNA copy
beforehand. Second, CLPA is relatively insensitive to sample
contaminants and can be applied directly to impure samples
including body samples such as blood, urine, saliva and feces.
Third, CLPA involves fewer steps than other known methods, thereby
reducing the time required to gain a result. Moreover, CLPA probes
can be stored dry, and properly designed systems will spontaneously
react to join two or more oligonucleotides in the presence of a
complementary target sequence. Chemical ligation reactions show
excellent sequence selectivity and can be used to discriminate
single nucleotide polymorphisms.
[0264] Significantly, unlike enzymatic ligation methods, CLPA shows
nearly identical reactivity on DNA and RNA targets which, as
described more fully below, renders CLPA more efficient that other
known systems, and expands the scope of applications to which CLPA
can be utilized.
[0265] Advantageously, the CLPA assay reduces the number of steps
required to detect a target nucleic acid, which provides the
potential to achieve results in significantly shorter time periods.
For example, the general process flow for a standard reverse
transcriptase (RT)-multiplex ligase-dependent probe ligation (MLPA)
involves the following steps: [0266] 1. Isolate total RNA. [0267]
2. Use Reverse Transcriptase to make cDNA copy. [0268] 3. Hybridize
MLPA probe sets to the cDNA target overnight. [0269] 4. Add DNA
Ligase to join target-bound probes. [0270] 5. Amplify ligated
probes, e.g. PCR amplification using Taq polymerase and
fluorescently labeled PCR primers. [0271] 6. Analyze the sample,
for example, by CE.
[0272] Unlike standard RT-MLPA, CLPA enables analysis to be carried
out directly on cell and blood lysates and on RNA targets without a
need for steps involving isolating the RNA and performing a reverse
transcription reaction to make a cDNA copy prior to ligation. This
shortens the time for achieving a result and provides a means to
achieve faster analysis.
[0273] In further embodiments, CLPA methods of the present
invention are conducted on formalin fixed paraffin embedded (FFPE)
tissues. In still further embodiments, the FFPE tissues are first
subjected to a chemical denaturant to lyse the tissues, and then
are subjected to any of the CLPA methods described herein. In yet
further embodiments, proteinase K and/or sonication are used to
further treat the FFPE tissues prior to application of CLPA
methods.
[0274] In further embodiments of the invention, faster reaction
times are further facilitated by driving the hybridization reaction
with higher probe concentrations. Thus, for example, input probe
sets may be incorporated in the CLPA reaction at relatively high
concentrations, for example, approximately 100-fold higher than
those typically used in an MLPA reaction. Elevating the probe
concentration significantly reduces the time required for the
hybridization step, typically from overnight to between about 15
minutes to about 1 hour.
[0275] CLPA probes are generally incorporated in a reaction at a
concentration of 250 nanomolar (nM) to 0.01 pM (picomolar) for each
probe. Generally, the concentration is between about 1 nM to about
1 pM. Factors to consider when choosing probe concentration include
the particular assay and the target being analyzed. The S- or
phosphorothioate or Nucleophile probe and L- or leaving group or
DABSYL containing probes are incorporated at a concentration that
equals or exceeds the concentration of the target. Total
concentration of S- and L-probes can reach as high as 10 micromolar
(uM). As a non-limiting example, 1 nM for each S and L
probe.times.250 CLPA probe pairs would equal 500 nm (1 nm per
probe.times.2 probes per pair.times.250 targets) at 10 nM for each
probe would mean a total concentration of 5 uM.
[0276] The target concentration usually ranges from about 10
micrograms of total RNA to about 10 nanograms, but it can be a
little as a single copy of a gene.
[0277] When higher probe concentrations are used it is generally
preferred to incorporate a purification step prior to
amplification, especially for high multiplex analysis (e.g. greater
than about 5 targets). In one embodiment of this aspect of this
invention, a solid support based capture methodology can be
employed including membrane capture, magnetic bead capture and/or
particle capture. In a preferred embodiment, a biotin/streptavidin
magnetic bead purification protocol is employed after ligation and
prior to enzymatic amplification. In some instances, the magnetic
particles can be directly added to the amplification master mix
without interfering with the subsequent amplification reaction. In
other instances, it is preferable to release the captured
oligonucleotide from the beads and the released oligonucleotide
solution is subsequently amplified without the capture particle or
surface being present.
[0278] In a preferred embodiment, CLPA involves hybridization of a
set of probes to a nucleic acid target sequences such that the
probes can undergo self-ligation without addition of a ligase.
After a ligation product is produced, amplification is generally
used to facilitate detection and analysis of the product. For this
purpose, probes are designed to incorporate PCR primers such as,
e.g. universal PCR primers. In a preferred embodiment, the
universal primers are not added until after the ligation portion of
the reaction is complete, and the primers are added after surface
capture purification (such as with the use of target capture
probes, which are described in further detail herein) along with
the polymerase, often as part of a PCR master mix.
[0279] The CLPA probes possess reactive moieties positioned such
that when the CLPA probes are bound to the nucleic acid target, the
reactive moieties are in close spatial orientation and able to
undergo a ligation reaction without the addition of ligase enzyme.
In a preferred embodiment, the chemical ligation moieties are
chosen so as to yield a ligated reaction product that can be
efficiently amplified by an amplification enzyme, which is in
preferred embodiments a DNA polymerase, but can include other
enzymes such as RNA polymerase. Without being bound by theory,
chemical ligation chemistries and probe set designs that produce
reaction products that more closely resemble substrates that are
known as being able to be amplified by DNA and RNA polymerases are
more likely to yield efficient probe sets that can be used in the
CLPA assay. Especially preferred reaction chemistries are chemical
moieties that yield reaction products that closely resemble native
DNA such as illustrated in Scheme 1 involving a reaction between a
3'-phosphorothioate and a 5' DABSYL leaving group. In another
preferred embodiment, probes sets comprise a 3'-diphosphorothioate
(Miller, G. P. et al, Bioorganic and Medicinal Chemistry, (2008)
16:56-64, which is hereby incorporated by reference in its entirety
and in particular for all teachings related to probes and
3'-diphosphorothioate chemistries) and a 5'-DABSYL leaving
group.
[0280] The CLPA probes of the present invention may also
incorporate a stuffer sequence (also referred to herein as a
variable spacer sequence) to adjust the length of the ligation
product. The length of the stuffer sequence can be specific to the
target sequence to which the ligation probe is directed, thus
providing a convenient means to facilitate analysis of ligation
product(s). The stuffer sequence can be located on either or both
probes. In embodiments utilizing a 3'-phosphorothioate probe, the
stuffer sequence is in preferred embodiments incorporated on this
probe (also referred to herein as the "S probe"). As is also
discussed above, the stuffer sequence may be located in a region of
the ligation probe that is non-complementary to the target nucleic
acid, or the stuffer sequence may be located in a region that at
least partially overlaps with a region that is complementary to a
domain of the target nucleic acid. In further embodiments, the
stuffer sequence is located between the primer sequence and the
probe domain.
[0281] In further embodiments of the invention, CLPA-CE, the
stuffer sequence is varied in length in order to produce one or
more variable length ligation products which provide the basis for
detection and identification of specific target sequences based on
length variation. In a preferred embodiment, variable length
ligation products are analyzed by capillary electrophoresis (CE).
Generally, stuffer sequences are included such that the length of
different ligation products varies in a range of at least 1 base
pair to about 10 base pairs; preferably from 1 base pair to 4 base
pairs. In a preferred embodiment, the length of the different
ligation products vary from approximately 80 bp to about 400 bp;
preferably in a range of about 100 bp to about 300 bp; more
preferably in a range of about 100 bp to about 200 bp. In further
embodiments, the stuffer sequences are of a length such that the
length of the ligation product is from about 5-1000, 10-950,
15-900, 20-850, 25-800, 30-750, 35-700, 40-650, 50-600, 55-550,
60-500, 65-450, 70-400, 75-350, 80-300, 85-250, 90-200, 95-150 base
pairs.
[0282] In some embodiments, CLPA probes may further contain other
optional element(s) to facilitate analysis and detection of a
ligated product. Such elements include any of the elements
discussed above in the section on ligation probe pairs. For
example, for embodiments referred to herein as CLPA-MDM (and
discussed in further detail below), it is preferred that at least
one of the ligation probes for incorporate an array binding
sequence to bind to an appropriate capture sequence on a microarray
platform. For CLPA-MDM, the different CLPA reaction products are
not separated by size differences but by the differences in the
array binding sequence. In this embodiment, the sequence of the
array binding sequence is varied so that each CLPA probe will bind
to a unique site on a DNA microarray. The length of the array
binding sequence in CLPA-MDM usually varies from 15 to 150 bases,
more specifically from 20 to 80 bases, and most specifically from
25 to 50 bases.
[0283] In further embodiments, CLPA probes preferably also include
other elements to facilitate purification and/or analysis including
but not limited to labels such as fluorescent labels and hapten
moieties such as, for example, biotin for purifying or detecting
ligation product(s). For example, probes and/or ligation product(s)
that incorporate biotin can be purified on any suitable
avidin/streptavidin platform including beads. While biotin/avidin
capture systems are preferred, other hapten systems (e.g.
Digoxigenin (DIG) labeling) can be used, as can
hybridization/oligonucleotide capture.
Hybridization/oligonucleotide capture is a preferred method when it
is desirable to release the capture product from the beads at a
later stage. In addition to magnetic beads, anti-hapten labeled
supports (filter paper, porous filters, surface capture) can be
used.
[0284] CLPA probe-labeling can be on either probe, either at the
end or internally. Preferably biotin is incorporated at the 5' end
on the phosphorothioate (S-probe).
[0285] In a preferred embodiment, a CLPA probe set consists of 2
oligonucleotide probes with complementary reactive groups (FIGS. 1
and 2). In another embodiment, the CLPA probe set may consist of 3
or more probes that bind adjacent to each other on a target. In a
preferred embodiment of the 3-probe CLPA reaction, the outer probes
are designed to contain the enzymatic amplification primer binding
sites, and the inner probe is designed to span the region of the
target between the other probes. In a more preferred embodiment,
the outer probes have non-complementary reactive groups such that
they are unable to react with each other in the absence of the
internal (middle) probe (FIG. 3). In some instances, both outer
probes may have similar reactive moieties except that one group is
at the 5' end of one probe and the 3'-end of the other probe, and
the L-probe chemistries may also be similar to each other except
for positioning on the probe. As is known to one who is skilled in
the art, different chemical reagents and processes may be needed to
manufacture the probes for the 3-probe CLPA reaction compared to
the probes for the 2-probe CLPA system.
[0286] In a preferred embodiment of the 3-probe CLPA system, one
outer probe contains a 3' phosphorothioate (3'S-probe), the other
outer probe contains a 5'-phosphorothioate (5'-S-probe) and the
center probe contains both a 3'- and a 5'-DABSYL leaving group. The
manufacture of a 5'-DABSYL leaving group probes has been reported
previously (Sando et al, J. Am. Chem. Soc., (2002), 124(10)
2096-2097). We recently developed a new DNA synthesis reagent that
allows for the routine incorporation of a 3'-DABSYL leaving group
(see FIG. 4).
[0287] In further embodiments and in accordance with any of the
CLPA methods described herein, the assays may be conducted in the
presence of a buffer of the invention. Such buffers are described
in detail above, and any combination of buffer components may be of
use in the CLPA methods of the invention. In preferred embodiments,
CLPA methods of the invention, including the target capture CLPA,
CLPA-CE, and CLPA-MDM methods described below, are conducted in a
buffer of the invention, including buffers comprising a denaturant
comprising a chaotropic cation such as guanidinium
hydrochloride.
[0288] Target Capture CLPA
[0289] In further embodiments, CLPA assays utilizing two or more
ligation probes can be combined with a target capture probe that
binds upstream or downstream to the CLPA probe set on the same
nucleic acid target. This target capture probe contains a capture
moiety (which can without limitation include a hapten, a bead, an
oligonucleotide capture sequence, and so forth) that can be used to
selectively capture complexes/molecules that are bound to the
target capture through hybridization or other interactions (FIG.
10).
[0290] The target capture probe can be used with any embodiment of
CLPA discussed herein.
[0291] As is also discussed in further detail above, more than one
target capture probe can be applied to a target nucleic acid. These
multiple target capture probes can be designed to flank one or more
ligation products, either by binding at or near both the 3' and 5'
ends of the target nucleic acids or by interleaving the target
capture probes between multiple ligation products. As is discussed
in further detail below, multiple target capture probes can be used
in methods for assessing the quality/integrity of target nucleic
acids, particularly RNA.
[0292] CLPA-CE
[0293] In one aspect, the present invention provides methods for
using ligation probes containing variable spacer sequences to
produce ligation products, and those ligation products are then
detected using capillary electrophoresis.
[0294] In certain embodiments, the ligation products are first
amplified using any method known in the art (including PCR) to
produce amplicons, and it is then the amplicons that are detected
using capillary electrophoresis. In further embodiments, the
ligation products and/or amplicons are detected by size
differentiation capillary electrophoresis (CE) on a sieving matrix,
or by slab gel electrophoresis.
[0295] A schematic representation for CLPA-CE is provided in FIG.
1. In the embodiment depicted in this figure, a blood sample is
subjected to cell lysis by any appropriate means, including without
limitation chemical, mechanical or osmotic cell lysis. Preferably,
chemical lysis is used.
[0296] Following chemical lysis, probes directed to target nucleic
acids are applied. FIG. 6 provides a general schematic
representation of the design of a probe set for CLPA-CE analysis.
In this example, the S probe is designed to include a universal PCR
primer for subsequent amplification of ligation product(s). The S
probe also includes a stuffer sequence designed with a length that
correlates with a specific target sequence. The S probe also
includes a target binding sequence (also referred to herein as a
"probe domain") that is complementary to a target domain of the
target nucleic acid comprising the target sequence to which the
stuffer sequence correlates. As will be appreciated, a set of
probes may contain a plurality of S probes that are all directed to
the same target domain, or the set may contain a mixture of S
probes directed to different target domains. Likewise, the L-probe
includes a target binding sequence that is complementary to a
target domain adjacent to the target domain to which the S probe
binds. In this embodiments, the L-probe also includes a universal
primer. In further embodiments, one or both of the probes are
labeled with a fluorophore (FAM, Cy3, Cy5, etc), however they can
also be detected without fluorescence labeling. The labeling is in
some embodiments accomplished by using a fluorescently labeled PCR
primer.
[0297] In the embodiment of CLPA-CE probes pictured in FIG. 6, the
S probe also includes a biotin moiety at the 5' end to facilitate
purification and removal of unligated probe.
[0298] As shown in FIG. 1, after the S- and L-probes are hybridized
to the target nucleic acid, the probes undergo a spontaneous
chemical ligation to produce a ligation product. In some
embodiments in accordance with FIG. 1, a bead purification step is
used to remove unligated probes, although the removal of unligated
probes is not a required step for all embodiments of CLPA-CE
methods. After ligation and the optional removal of unligated
probes, the ligation products are amplified. In embodiments in
which the ligation probes contain universal primer sequences,
universal PCR primer pairs are used to produce amplicons. In
embodiments that utilize other types of primer sequences,
complementary primer pairs can be used to generate amplicons.
[0299] Due to the presence of the variable spacer sequence in one
(or in some embodiments both) of the ligation probes, the amplicons
have a unique length that corresponds to a specific target nucleic
acid. Those amplicons are then analyzed using capillary
electrophoresis analysis, and the relative intensity of each peak
corresponds to relative expression. (FIG. 1 and FIG. 7). In
alternative embodiments, other suitable size separation techniques
can be used to determine target nucleic acid expression in the
sample.
[0300] In further embodiments and in accordance with any of the
above, the CLPA-CE method includes a step of hybridizing a target
capture probe to the target nucleic acid. The resulting target
complex comprising the target capture probe, the target nucleic
acid and the ligation product is then, prior to amplification of
the ligation product, captured on a surface or a substrate through
the capture moiety of the target capture probe. In further
embodiments, unbound target nucleic acids and ligation probes are
removed, leaving only the target complexes and the ligation
products. The ligation products can then be amplified and analyzed
according to any of the methods described herein. In some
embodiments, the ligation products are removed from the target
complex prior to amplification using methods known in the art,
including heating and the addition of denaturants or other agents
to change the hybridization conditions.
[0301] CLPA-MDM
[0302] In another embodiment of this aspect of the invention, CLPA
ligation products are analyzed/detected by microarray analysis
(CLPA-MDM). A schematic representation of CLPA-MDM is provided in
FIG. 2. CLPA-MDM differs from CLPA-CE in at least the following
respects. First, the probe sets differ in design. For example, a
general representation of a CLPA-MDM probe set is depicted in FIG.
2. As with CLPA-CE probes, CLPA-MDM probe sets can include
universal primers for amplification of ligation product(s). They
also include target specific sequences, as well as ligation
moieties for enzyme-independent ligation. Additionally, CLPA-MDM
probes also may include a stuffer sequence, however the purpose of
this stuffer sequence is to adjust the size of the CLPA-MDM to the
same length in an effort to standardize enzymatic amplification
efficiency. Normalization of amplicon size is not a requirement but
can provide advantages for amplification efficiency. A second
difference between the design of CLPA-CE and CLPA-MDM probe sets is
that the latter include a unique array binding sequence for use
with an appropriate microarray platform.
[0303] In respect of the CLPA-MDM aspect of the invention, a
microarray binding site (ABS sequence) is incorporated into the
probe designs for use with a "universal" microarray platform for
the detection. Similar to the CLPA-CE system, probes are preferably
labeled with a fluorophore, for example by using a fluorescently
labeled PCR primer. Alternatively, for example, a sandwich assay
labeling technique can be used for the final read-out. Sandwich
assays involve designing the probes with a common (generic) label
binding site (LBS) in place or in addition to the stuffer sequence
and using a secondary probe that will bind to this site during the
array hybridization step. This methodology is particularly useful
when it is desirable to label the arrays with a chemiluminescent
system like a horse radish peroxidase (HRP) labeled
oligonucleotide, or with an electrochemical detection system.
Generally, planar microarrays are employed (e.g. microarrays
spotted on glass slides or circuit board) for the read-out.
However, bead microarrays such as those available from Luminex and
Illumina can also be used (e.g. Luminex xMAP/xtag).
[0304] Nucleic acid Quality Assessment
[0305] In certain aspects, multiple CLPA probe sets that differ in
binding position on a nucleic acid target relative to a target
capture probe are used (FIG. 11) in methods to assess nucleic acid
quality. In the embodiment pictured in FIG. 11, a target capture
probe is hybridized to one end of the target nucleic acid, and
multiple ligation products produced from different probe sets
directed to different target domains of the target nucleic acid are
used to produce a target complex comprising the target capture
probe and the multiple ligation products.
[0306] In such aspects, differences in the signals from the
different ligation products provide an assessment of the quality of
the nucleic acid target. For example, by measuring the differences
in capture efficiency/signal from the unique CLPA probe set located
closest to the target capture probe (e.g., 1-100 bp away from the
target capture probe) compared to a CLPA probe set further from the
target capture probe (e.g., 300-1000 bp away), it is possible to
infer the fragmentation or level of degradation of the nucleic acid
target. In further exemplary embodiments, the relative ratio of
signals generated for the different CLPA probe sets provides a
measure of the level of degradation for a particular nucleic acid
target. In further embodiments, the ligation probe sets are
designed such that they are directed to target domains at known
distances from the domain to which the target capture probe is
hybridized. In such embodiments, the target complex comprising the
target capture probe, the ligation products and the target nucleic
acid are captured on a surface or a substrate through a capture
moiety on the target capture probe, and unbound target nucleic
acids and ligation probes are separated from the captured target
complexes. The relative ratio of signals associated with the
multiple different probe sets then provides an indication of the
amount of degradation present in the target nucleic acids in the
sample, and the known distances between the ligation products and
the target capture probe provides a kind of "molecular ruler" for
quantifying the degradation in the target nucleic acids. For
example, in reference to FIG. 11, if in an exemplary embodiment the
majority of the target nucleic acids in a sample are degraded in a
domain located between the domains to which CLPA set 2 and CLPA set
3 are hybridized, then the signal from CLPA sets 1 and 2 will be
relatively larger than the signal from CLPA set 3. This relative
ratio between the signals provides an indication of the length and
integrity of the target nucleic acids in the sample.
[0307] As will be appreciated by those in the art, the spacing of
the different probe sets will depend on the size of the original
target and the desired information. The spacing can be relatively
equidistant (e.g. the use of 3 probe sets spaced roughly 30% of the
total length apart), or can be clustered as desired.
[0308] In further embodiments, multiple target capture probes are
used for assessment of the quality of the nucleic acid target. As
is further discussed herein, the different target capture probes
can be designed to hybridize near or at the ends of the target
nucleic acid and may also or in the alternative be designed to
hybridize between different ligation products. In some embodiments,
two target capture probes are designed to hybridize to domains both
upstream and downstream of one or more ligation products. Use of
the two different capture probes can help ensure that even in
highly degraded samples, all target complexes comprising ligation
products are efficiently captured on a surface or a substrate and
separated from unbound target nucleic acids and ligation probes
prior to amplification of the ligation products.
[0309] Information on degradation is useful in assessing the
quality of the nucleic acid and more specifically RNA contained in
samples. This technique is particularly useful in assessing the
quality of RNA in formalin fixed paraffin embedded (FFPE) tissue
samples and other samples where the risk of sample degradation is
high.
[0310] As discussed above, the difference in capture efficiency can
be indirectly determined by measuring the relative ratio of
signals. This methodology can be combined with microarray, CE,
sequencing, real time PCR and other methods of nucleic acid
analysis to further assess the target nucleic acids in accordance
with any of the methods described above.
VIII. Hardware
[0311] In one aspect of the invention, a fluidic device similar to
those described by Liu (2006) is used to automate the methodology
described in this invention. See for example U.S. Pat. No.
6,942,771, herein incorporated by reference for components
including but not limited to cartridges, devices, pumps, wells,
reaction chambers, and detection chambers. The fluidic device may
also include zones for capture of magnetic particles, separation
filters and resins, including membranes for cell separation (i.e.
Leukotrap.TM. from Pall). The device may include detection chambers
for in-cartridge imaging of fluorescence signal generated during
Real-Time PCR amplification (i.e. SYBR green, Taqman, Molecular
Beacons), as well as capillary electrophoresis channels for
on-device separation and detection of reactions products (amplicons
and ligation products). In a preferred embodiment, the capillary
electrophoresis channel can be molded in a plastic substrate and
filled with a sieving polymer matrix (POP-7.TM. from Applied
Biosystems). Channels containing non-sieving matrix can also be
used with properly designed probe sets.
[0312] In a preferred embodiment, the devices of the invention
comprise liquid handling components, including components for
loading and unloading fluids at each station or sets of stations.
The liquid handling systems can include robotic systems comprising
any number of components. In addition, any or all of the steps
outlined herein may be automated; thus, for example, the systems
may be completely or partially automated.
[0313] As will be appreciated by those in the art, there are a wide
variety of components which can be used, including, but not limited
to, one or more robotic arms; plate handlers for the positioning of
microplates; holders with cartridges and/or caps; automated lid or
cap handlers to remove and replace lids for wells on non-cross
contamination plates; tip assemblies for sample distribution with
disposable tips; washable tip assemblies for sample distribution;
96 well loading blocks; cooled reagent racks; microtiter plate
pipette positions (optionally cooled); stacking towers for plates
and tips; and computer systems.
[0314] Fully robotic or microfluidic systems include automated
liquid-, particle-, cell- and organism-handling including high
throughput pipetting to perform all steps of screening
applications. This includes liquid, particle, cell, and organism
manipulations such as aspiration, dispensing, mixing, diluting,
washing, accurate volumetric transfers; retrieving, and discarding
of pipet tips; and repetitive pipetting of identical volumes for
multiple deliveries from a single sample aspiration. These
manipulations are cross-contamination-free liquid, particle, cell,
and organism transfers. This instrument performs automated
replication of microplate samples to filters, membranes, and/or
daughter plates, high-density transfers, full-plate serial
dilutions, and high capacity operation.
[0315] In a preferred embodiment, chemically derivatized particles,
plates, cartridges, tubes, magnetic particles, or other solid phase
matrix with specificity to the assay components are used. The
binding surfaces of microplates, tubes or any solid phase matrices
include non-polar surfaces, highly polar surfaces, modified dextran
coating to promote covalent binding, antibody coating, affinity
media to bind fusion proteins or peptides, surface-fixed proteins
such as recombinant protein A or G, nucleotide resins or coatings,
and other affinity matrix are useful in this invention.
[0316] In a preferred embodiment, platforms for multi-well plates,
multi-tubes, holders, cartridges, minitubes, deep-well plates,
microfuge tubes, cryovials, square well plates, filters, chips,
optic fibers, beads, and other solid-phase matrices or platform
with various volumes are accommodated on an upgradable modular
platform for additional capacity. This modular platform includes a
variable speed orbital shaker, and multi-position work decks for
source samples, sample and reagent dilution, assay plates, sample
and reagent reservoirs, pipette tips, and an active wash
station.
[0317] In a preferred embodiment, thermocycler and thermoregulating
systems are used for stabilizing the temperature of heat exchangers
such as controlled blocks or platforms to provide accurate
temperature control of incubating samples from 0.degree. C. to
100.degree. C.; this is in addition to or in place of the station
thermocontrollers.
[0318] In a preferred embodiment, interchangeable pipet heads
(single or multi-channel) with single or multiple magnetic probes,
affinity probes, or pipetters robotically manipulate the liquid,
particles, cells, and organisms. Multi-well or multi-tube magnetic
separators or platforms manipulate liquid, particles, cells, and
organisms in single or multiple sample formats.
[0319] In some embodiments, the instrumentation will include a
detector, which can be a wide variety of different detectors,
depending on the labels and assay. In a preferred embodiment,
useful detectors include a microscope(s) with multiple channels of
fluorescence; plate readers to provide fluorescent, electrochemical
and/or electrical impedance analyzers, ultraviolet and visible
spectrophotometric detection with single and dual wavelength
endpoint and kinetics capability, fluorescence resonance energy
transfer (FRET), luminescence, quenching, two-photon excitation,
and intensity redistribution; CCD cameras to capture and transform
data and images into quantifiable formats; capillary
electrophoresis systems, mass spectrometers and a computer
workstation.
[0320] These instruments can fit in a sterile laminar flow or fume
hood, or are enclosed, self-contained systems, for cell culture
growth and transformation in multi-well plates or tubes and for
hazardous operations. The living cells may be grown under
controlled growth conditions, with controls for temperature,
humidity, and gas for time series of the live cell assays.
Automated transformation of cells and automated colony pickers may
facilitate rapid screening of desired cells.
[0321] Flow cytometry or capillary electrophoresis formats can be
used for individual capture of magnetic and other beads, particles,
cells, and organisms.
[0322] The flexible hardware and software allow instrument
adaptability for multiple applications. The software program
modules allow creation, modification, and running of methods. The
system diagnostic modules allow instrument alignment, correct
connections, and motor operations. The customized tools, labware,
and liquid, particle, cell and organism transfer patterns allow
different applications to be performed. The database allows method
and parameter storage. Robotic and computer interfaces allow
communication between instruments.
[0323] In a preferred embodiment, the robotic apparatus includes a
central processing unit which communicates with a memory and a set
of input/output devices (e.g., keyboard, mouse, monitor, printer,
etc.) through a bus. Again, as outlined below, this may be in
addition to or in place of the CPU for the multiplexing devices of
the invention. The general interaction between a central processing
unit, a memory, input/output devices, and a bus is known in the
art. Thus, a variety of different procedures, depending on the
experiments to be run, are stored in the CPU memory.
[0324] These robotic fluid handling systems can utilize any number
of different reagents, including buffers, reagents, samples,
washes, assay components such as label probes, etc.
IX. Overall Process
[0325] One distinct advantage of aspects of the present invention
is the ability to collect samples, particularly for RNA detection,
into a stabilization buffer that allows both for a) long time
periods before testing without sample degradation (of particular
use in the assay of RNA, as RNA is degraded extremely rapidly under
traditional conditions) and b) the ability to run the assay in the
collection buffer without having to do further purification steps
to remove components from the mixture that prevent enzymatic assays
from being run, such as high denaturing salt concentrations. The
ability to collect samples such as blood directly into a buffer
that simultaneously lyses the cells and stabilizes the RNA for long
periods of time (e.g. days to weeks or longer) allows remote
collection and a time lag before assay processing. In addition,
these methods thus avoid any special conditions for handling, such
as avoidance of heat exposure and/or cold chain handling. For
example, samples can be collected and mailed using regular mail and
be tested without additional steps.
[0326] Furthermore, the ability to assay directly in the collected
sample is a significant benefit over the use of sample preparation
steps to purify the samples for traditional enzyme based
assays.
[0327] In further embodiments, a sample is collected into a
stabilization buffer in one geographic location and can then be
transported to a different geographic location prior to conducting
assays on the sample in accordance with any of the aspects and
embodiments of the invention discussed herein. In other words, as
is discussed above, the present invention provides methods and
compositions that result in a stabilized sample that can be
collected in one geographic location and then subjected to chemical
ligation methods and assays as discussed herein in a different
geographic location.
X. Kits
[0328] In another aspect of the invention, a kit for the routine
detection of a predetermined set of nucleic acid targets is
produced that utilizes probes, techniques, methods, and a chemical
ligation reaction as described herein as part of the detection
process. The kit can comprise probes, target sequences,
instructions, buffers, and/or other assay components.
[0329] In a further aspect the invention provides for kits for
stabilizing and detecting or quantifying RNA in a sample comprising
a buffer solution comprising a denaturant, a first ligation probe,
and a second ligation probe. In a preferred embodiment, the
denaturant is chosen from the group consisting of guanidinium
hydrochloride and guanidinium isothiocyanate. In a further
preferred embodiment the buffer solution further comprises EDTA, a
reducing agent, a surfactant and a pH buffer. In particularly
preferred embodiment, the reducing agent is chosen from the group
consisting of DTT and mercaptoethanol. In another particularly
preferred embodiment, the surfactant is chosen from the group
consisting of Triton X-100 and sodium N-lauroylsarcosine.
[0330] In still further aspects, the present invention provides
kits for detecting a target nucleic acid sequence where that target
sequence comprises an adjacent first and second target domain. Such
kits may include: a 2.times. lysis buffer comprising 6 M GuHCl and
at least one set of ligation probes, where that at least one set of
ligation probes includes a first ligation probe that includes a
first probe domain complementary to the first target domain of the
target nucleic acid sequence and a second ligation probe that
includes a second probe domain complementary to the second target
domain of the target nucleic acid sequence. As is further discussed
above, the ligation probes included in kits of the invention may
include further functionalities, including stuffer sequences,
primer sequences, and anchor sequences, and any combination
thereof.
EXAMPLES
Example 1: Quantitative Multiplex Detection of Five Targets
[0331] Multiplex CLPA reactions were performed using five (5) DNA
target mimics (corresponding to portions of the MOAP1 (SEQ ID
NO:5), PCNA (SEQ ID NO:9), DDB2 (SEQ ID NO:12), BBC3 (SEQ ID NO:16)
and BAX (SEQ ID NO:19) genes) combined in one reaction in the
presence of their respective CLPA probes (Table 1) (S and L probes
at 1 nM each). The target mimics were pooled in different
concentration as shown in Table 2. The target mimics, S probes and
L probes were incubated in PCR buffer (1.times.PCR buffer is 1.5 mM
MgCL.sub.2, 50 mM KCl, 10 mM Tris-HCl pH 8.3) for 1 hour at
50.degree. C. A 1 .mu.l aliquot of each reaction mixture was used
as template for PCR amplification using Dynamo SYBR green PCR mix
in the presence of Universal Primers (SEQ ID NOS 1 and 2, 300 nM).
The samples were PCR cycled for 27 cycles (95.degree. C. 15 min
followed by 27 cycles of 95.degree. C. (10 s), 60.degree. C. (24
s), 72.degree. C. (10 s). After PCR amplification, the samples were
denatured and injected into an ABI 3130 DNA sequencer (capillary
electrophoresis instrument). The CE trace from the ABI for the 3
samples as well as a plot of the peak versus target mimic
concentration of PCNA is shown in FIG. 7 and a plot of the linear
response of the signal of PCNA as a function on input concentration
is shown in FIG. 8.
TABLE-US-00001 TABLE 1 Probe and target sequence information. SEQ
Amplicon ID Name Sequence Detail Size 1 Forward PCR Primer
FAM-GGGTTCCCTAAGGGTTGGA 2 Reverse PCR Primer
GTGCCAGCAAGATCCAATCTAGA 3 MOAP1-L
LTACATCCTTCCTAGTCAATTACACTCTAGATTGGA 47 TCTTGCTGGCAC 4 MOAP1-S
5'-Biotin-GGGTTCCCTAAGGGTTGGATAGGTAA 41 ATGGCAGTGTAGAACS Ligated
MOAP1 Amplicon 5 MOAP1-Target GTGTAATTGACTAGGAAGGATGTAGTTCTACACTGC
mimic CATTTACCTA 6 MOAP1-RNA Target
GUGUAAUUGACUAGGAAGGAUGUAGUUCUACACUGC mimic CAUUUACCUA 7 PCNA-L
LTGGTTTGGTGCTTCAAATACTCTCTAGATTGGATC 45 TTGCTGGCAC 8 PCNA-S
Biotin-GGGTTCCCTAAGGGTTGGATCGAGTCTAC 63
AGATCCCCAACTTTCATAGTCTGAAACTTTCTCCS Ligated PCNA Amplicon 108 9
PCNA-Target Mimic AGTATTTGAAGCACCAAACCAGGAGAAAGTTTCAGA CTATGA 10
DDB2-L LTAGCAGACACATCCAGGCTCTAGATTGGATCTTGC 51 TGGCAC 11 DDB2-S
Biotin-GGGTTCCCTAAGGGTTGGATCGAGTCTAC 49 TCCAACTTTGACCACCATTCGGCTACS
Ligated DDB2 Amplicon 12 DDB2-Target Mimic
GCCTGGATGTGTCTGCTAGTAGCCGAATGGTGGTCA 13 DDB2-RNA Target
GCCUGGAUGUGUCUGCUAGUAGCCGAAUGGUGGUCA Mimic 14 BBC3-L
LTCCGAGATTTCCCCCTCTAGATTGGATCTTGCTGG 38 CAC 15 BBC3-S
Biotin-GGGTTCCCTAAGGGTTGGATCCCAGACTC 37 CTCCCTCTS Ligated BBC3
Amplicon 16 BBC3-Target Mimic GGG GGA AAT CTC GGA AGA GGG AGG AGT
CTG GG 17 BAX-L LTCACGGTCTGCCACGCTCTAGATTGGATCTTGCTG 39 GCAC 18
BAX-S Biotin-GGGTTCCCTAAGGGTTGGA TGA GTC 53 TAC ATGA TC CT
TCCCGCCACAAAGATGGS Ligated BAX Amplicon 92 19 BAX-Target Mimic
CGTGGCAGACCGTGACCATCTTTGTGGCGGGA 20 3-phosphorothioate
Biotin-GGGTTCCCTAAGGGTTGGACGGACGCCTG 51 GAPDH
CTTCACCACCTTCTTGATGTCAS 21 Middle 2L probe
LTCATATTTGGCAGGTTTTTCTAGACGGCAGGTL 32 GAPDH 22 5'-phosphorothioate
SCAGGTCCACCACTGACACGTTGGCAGTTCTAGATT 50 GAPDH GGATCTTGCTGGCAC
Ligated 3-probe amplicon 133 23 ReversePrimer
Biotin-ATTAACCCTCACTAAAGGGA A3632-p 24 GAPDH Target ACT GCC AAC GTG
TCA GTG GTG GAC CTG Mimic ACC TGC CGT CTA GAA AAA CCT GCC AAA TAT
GAT GAC ATC AAG AAG GTG GTG AAG CAG GCG TC 25 GAPDH 3-L
LTTTTCTAGACGGCAGGTCAGGTCCACCAGATGATC
GACGAGACACTCTCGCCATCTAGATTGGATCTTGCT GGCAC 26 GAPDH 3-S
GGGTTCCCTAAGGGTTGGACGGACCAACTCCTCGCC
ATATCATCTGTACACCTTCTTGATGTCATCATATTT GGCAGGTS 27 GAPDH-3-FAM/BHQ-1
(FAM)ccaactcctcgccatatcatctgtacacctt Taqman Probe cttg(BHQ-1) 28
GAPDH 4-L LTGCTGATGATCTTGAGGCTGTTGTCATACTGATGA
TCGACGAGACACTCTCGCCATCTAGATTGGATCTTG CTGGCAC 29 GAPDH-4-S
GGGTTCCCTAAGGGTTGGACGATGGAGTTGATGCTG
ACGGAAGTCATAGTAAGCAGTTGGTGGTGCAGGAGG CATS 30 GAPDH-4-QUASAR (Quasar
670)tgctgacggaagtcatagtaagca 670/BHQ-2 Taqman gttggt(BHQ-2) Probe
31 PCNA 2-L LTCCTTGAGTGCCTCCAACACCTTCTTGAGGATGAT
CGACGAGACACTCTCGCCATCTAGATTGGATCTTGC TGGCAC 32 PCNA 2-S
GGGTTCCCTAAGGGTTGGACGGTACAACAAGACCCA
GCTGACGACTCTTAATATCCCAGCAGGCCTCGTTGA TGAGGS 33 PCNA 2-Cal Fluor
(CAL Red 610)ctgacgactcttaatatcccagc Orange 560/BHQ-1
aggcctcgtt(BHQ-2) 34 DDB2-2-L LTTAGTTCCAAGATAACCTTGGTTCCAGGCTGATGA
TCGACGAGACACTCTCGCCATCTAGATTGGATCTTG CTGGCAC 35 DDB2-2-S
BiotinGGGTTCCCTAAGGGTTGGACGTTAGACGCC
AATAGGAGTTTCACTGGTGGCTACCACCCACTGAGA GGAGAAAAGTCATS 36 DDB2-2-(CAL
Fluor (Cal Orange 560)cgccaataggagtttcactg Orange 560/BHQ-1
gtggctacca(BHQ-2) 37 MPRS5-TC [BIOTIN]GCCAGAGAGGTTACGTGGCGGCTCTCTT
30 CA 38 MRPS5-S GGATGCTATGAGCGATCTGCAGCGTGCAGTCTTCAC 58
ATCTTCCCAGTCCAGTTTGACGS 39 MRPS5-L
LTCTGGAACCTCATCTTCTGGCTCTGGATCCTTCCT 79
AAGTGAATGTTGACAGGATGCTCTAGATTGGATCTT GCTGGCAC Ligated MRPS5
Amplicon 137 40 PCNA-TC [BIOTIN]TCTTCATCCTCGATCTTGGGAGCCAAGT 30 AG
41 PCNA-S GGATGCTATGAGCGATCTGCAGCCACTATACATCTT 76
ACTATACTTTACTCTACAACAAGGGGTACATCTGCA GACAS 42 PCNA-L
LTACTGAGTGTCACCGTTGAAGAGAGTGGAGTGGCT 70
TTTGTAAAGTCTTCTAGATTGGATCTTGCTGGCAC Ligated PCNA Amplicon 146 43
CDR2-TC [BIOTON]AGAGTGATCGGTATTTTGTTCTCTGTTC 29 A 44 CDR2-S
GGATGCTATGAGCGATCTGCAGCGCAATTCATTTCA 86
TTCACAATCAATCTAAAGATCTCCTTAAACAACGCT TTGTATTCTGGAGGS 45 CDR2-L
LTGTTGTAGGGGAACTCACGGGCTCTGGGTTGACAG 101
AGGCCAGTTAGGATGTTACCACCAGTGAATGTTGAC AGGATCCTCTAGATTGGATCTTGCTGGCAC
Ligated CDR2 Amplicon 187 *Luminex Bead # 46 GAPDH-L
LTCCATTGATGACAAGCTTCCCGTTCTCAGCTCGCG TTCTAAGCTTCCCTTTAGTGAGGGTTAAT
47 GAPDH-S Biotin-TAATACGACTCACTATAGGGCGAGTAGAA 12
AGTTGAAATTGATTATGATCTCGCTCCTGGAAGATG GTGATGGGATTS 48 ACTG2-L
LTTCTCCCAGTGACTGAGGGCTGGTGTGTCTTTGGC TCCCTTTAGTGAGGGTTAAT 49
ACTG2-S Biotin-TAATACGACTCACTATAGGGCGAATTGAG 72
AAAGAGATAAATGATAGGGACTGGAGCACCGAGGGT ATGAGAGGTTCS 50 ACPP-L
LTTCAACTCCTTGGCTAGTACACTTCGGTCTAGCGC TCCCTTTAGTGAGGGTTAAT 51 ACPP-S
Biotin-TAATACGACTCACTATAGGGCGGTAAGAG 66
TATTGAAATTAGTAAGAGGTCTCCATGCCGAAACAC CAAAGTCACAAACS 52 RDH11-L
LTGTGCATCTCAAAGCCATCTGCTGTCTTCGGCTCC CTTTAGTGAGGGTTAAT 53 RDH11-S
Biotin-TAATACGACTCACTATAGGGCGTTTGTTG 63
TTAAGTATGTGATTTAGGGAGGAAGTGACCCAAGTG GTTGACTCCTAS 54 DES-L
LTGTTCTCTGCTTCTTCCTTCAACTGAATCTCCTCC TGCTTCCCTTTAGTGAGGGTTAAT 55
DES-S Biotin-TAATACGACTCACTATAGGGTATTTGATA 68
AGAGAATGAAGAAGTATCCACGTCCGCTCGGAAGGC AGCCAAATS 56 Primer A3534-p
p-TAATACGACTCACTATAGGG *Luminex Product Insert Sheet MagTag-Plex
Microspheres. L = DABSYL ligation moiety S = phosphorothioate
moiety
TABLE-US-00002 TABLE 2 Sample Concentrations Sample Target Mimic
Concentrations 1 All Target mimics at 10 pM final Concentration 2
MOAP1, DDB2 and BBC3 at 10 pM, PCNA at 5 pM and BAX at 2 pM 3
MOAP1, DDB2 and BBC3 at 10 pM, PCNA at 1 pM and BAX at 0.5 pM
Example 2: CLPA Reactions Using MOAP1 and DDB2 DNA and RNA Target
Mimics
[0332] Reactions were prepared in duplicate as presented in Table 3
using DNA or RNA target mimics for the MOAP1 and DDB2 genes and
CLPA probes sets designed to target the sequences. The probe
numbers refer to the SEQ ID NOs in Table 1. The reagents were added
in the concentrations and volumes shown in Table 4. The respective
S-probe, L-probe and target mimic were heated to 50.degree. C. for
60 minutes in a 0.2 mL PCR tube, after which 2.5 .mu.l of the CLPA
reaction was used as template in a real-time PCR reaction with 40
amplification cycles. Real-time PCR data was averaged for the
duplicate samples and is presented in Table 3 (Ct value column).
Minimal differences in Ct value between RNA and DNA target mimics
were observed indicating similar probe ligation efficiency on RNA
and DNA substrates.
TABLE-US-00003 TABLE 3 CLPA Probe Sets. Target L-Probe S-Probe
Mimic (1 nM) (1 nM) (10 pM) Ct Sample Identifier SEQ ID NO SEQ ID
NO SEQ ID NO value 1 MOAP-1 3 4 5 19.5 DNA 2 MOAP-1 3 4 6 20 RNA 3
DDB2 DNA 10 11 12 21 4 DDB2 RNA 10 11 13 21
TABLE-US-00004 TABLE 4 Reagent table-Example 1 1X PCR Buffer
Buffer* 12.5 ul S-Probe (1 nM) & L-Probe (1 nM) 2.5 ul each
Target Mimic (100 pM) 2.5 ul Water 5.0 ul Heat at 50 C. for 1 hour
*1.times. PCR buffer is 1.5 mM MgCL2, 50 mM KCl, 10 mM Tris-HCl pH
8.3
Example 3: Direct Analysis of DDB2 RNA Transcripts in Lysis Buffer
and Lysed Blood
[0333] DDB2 messenger RNA (mRNA) was prepared using an in-vitro
transcription kit from Ambion and a cDNA vector plasmid from
Origene (SC122547). The concentration of mRNA was determined using
PicoGreen RNA assay kit from Invitrogen. The DDB2 probe sets (Table
5) were tested with different concentrations of DDB2 mRNA
transcript spiked into either water or whole blood. The reactions
mixture components are listed in Table 5. Samples 1-4 consisted of
DDB2 transcript at 10 ng, 1 ng, 0.1 ng and 0.01 ng in water, and
samples 5-8 consisted of the same concentration range spiked into
whole blood. Similar reactions protocols were followed with the
exception of adding Proteinase K to the blood samples so as to
reduce protein coagulation. The procedure is as follows: The
reagents were added in the concentrations and volumes in Table 5
and Table 6.
[0334] The RNA transcripts were stable once combined with the
buffer prior to the subsequent heat step and could be stored in
this buffer solution for several days without observing any
significant degradation of the RNA. The S-probes, mRNA transcript,
Guanidine hydrochloride lysis buffer and either water (samples 1-4)
or whole blood (samples 5-8) were heated to 80.degree. C. for 5
minutes and then they were moved to a 55.degree. C. heat block. The
L-probe, wash buffer, streptavidin beads and proteinase K were
added, and the reaction was incubated at 55.degree. C. for 60
minutes. The samples were removed from the heat block and the
magnetic beads were captured using a dynal MPC 96S magnetic capture
plate. The supernatant was removed and the beads were washed 3
times with wash buffer. DyNamo SYBR green PCR master mix (25 ul,
1.times.) and universal primers (SEQ ID NOS 1 and 2, 300 nM) were
added to the beads and samples were heat cycled using a Stratagene
MX4000 realtime PCR instrument for 30 cycles (95.degree. C. for 15
minutes, 30 cycles 95.degree. C. (10 s), 60.degree. C. (24 s),
72.degree. C. (10 s)). The Ct values were recorded and the
amplified samples were injected into an Agilent Bioanalyzer 2100 so
as to verify the length of the amplicons. All amplicons showed the
correct size (.about.96 bp) and the performance was comparable for
the blood and water samples demonstrating the ability to directly
analyze RNA in lysed blood. The results are summarizes in Table 7
below. In additional experiments it was shown that the length of
time (from a few minutes to several hours and even up to several
days) that the RNA transcripts were stored in the buffer solution
prior to the initial heat step did not significantly alter the
results summarized in Table 7.
TABLE-US-00005 TABLE 5 CLPA Probe Sets. Sample Identifier L-Probe
(1 nM) S-Probe (1 nM) RNA Transcript 1-8 DDB2 SEQ ID NO: SEQ ID NO:
Origene Plasmid 10 11 SC122547
TABLE-US-00006 TABLE 6 DDB2 reaction mixture. Samples 1-4 5-8 GuHCL
Lysis Buffer (2X) 12.5 .mu.l 12.5 .mu.l S-Probe (5 nM) 1 .mu.l 1
.mu.l RNA Transcript (10 ng/ul to 1 .mu.l 1 .mu.l 0.01 ng/ul) Whole
Blood 0 .mu.l 12.5 .mu.l Water 12.5 ul 0 .mu.l Heat 80.degree. C.
for 5 min, chill on ice Wash Buffer 20 .mu.l 15 .mu.l L-Probe (5
nM) 1 .mu.l 1 .mu.l Dynal M-270 Beads 2 .mu.l 2 .mu.l Proteinase K
(10 mg/ml) 0 .mu.l 5 .mu.l Total 50 .mu.l 50 .mu.l Incubate
55.degree. C. for 60 min.
[0335] a) GuHCL lysis buffer (1.times.) is 3M GUHCL, 20 mM EDTA, 5
mM DTT, 1.5% Triton, 30 mM Tris pH 7.2).
[0336] b) Wash Buffer is 100 mM Tris (pH 7.4), 0.01% Triton.
TABLE-US-00007 TABLE 7 Summary results of water versus blood Assay
DDB2 Conc Ct value Sample 1 10 ng 13.5 Water 2 1 ng 17 Water 3 0.1
ng 20.2 Water 4 0.01 ng 24 Water 5 10 ng 13.5 Blood 6 1 ng 16 Blood
7 0.1 ng 19.2 Blood 8 0.01 ng 23.5 Blood
Example 4: 3-Probe CLPA-CE Assay
[0337] Reactions were prepared in duplicate as presented in Table 8
using DNA target mimic probe SEQ ID NO 24 and the 3-probe CLPA
probe set (SEQ ID NOS 20, 21 and 22). The probe numbers refer to
the SEQ ID NOS in Table 1. The reagents were added in the
concentrations and volumes in Table 9. The S-probes, L-probe and
target mimics were immediately heated to 50.degree. C. for 60
minutes in a 0.2 mL PCR tube, after which 2.5 .mu.l of the CLPA
reaction was used as template in a Dynamo SYBR green PCR reaction
with 25 amplification cycles. Real-time PCR data was averaged for
the duplicate samples and is presented in Table 8 (Ct value
column). A 1 .mu.l sample of each reaction was then analyzed via
Agilent Bioanalyzer 2100 to determine the size of the reaction
product.
TABLE-US-00008 TABLE 8 CLPA Probe Sets. 3'-S probe 2L-Probe 5'-S
Probe Target Mimic Amplicon Samples Identifier SEQ ID NO SEQ ID NO
SEQ ID NO SEQ ID NO size Ct value 1&2 GAPDH 20 21 22 24 About
135 bp 16.3 3&4 Negative 20 21 22 24 None No CT observed
[0338] Probes at 1 nM concentration; target mimic at 10 pM
concentration.
TABLE-US-00009 TABLE 9 Reagent table-Example 4 1X PCR Buffer
Buffer* 12.5 .mu.l 3 and 5' S-Probe (10 nM) & 2L- 2.5 .mu.l
Probe (10 nM) each Target Mimic (1 nM) 2.5 .mu.l Water 2.5 .mu.l
Heat at 50.degree. C. for 1 hour *1.times. PCR buffer is 1.5 mM
MgCL2, 50 mM KCl, 10 mM Tris-HCl pH 8.3
Example 5: Multiplex Real-Time CLPA Detection of mRNA
[0339] In a 0.2 ml PCR tube were added 4 sets of CLPA reagents that
were engineered to possess unique binding sites for different color
dual labeled probes. The reactions were prepared as indicated in
Table 10 and Table 11. The CLPA probes sets and dual labeled probes
correspond to SEQ ID NOS 25 through 36 in Table 1. The S and
run-off transcript mRNA (GAPDH, PCNA and DDB2) were added to
2.times. lysis buffer (GuHCL lysis buffer (1.times.) is 3M GUHCL,
20 mM EDTA, 5 mM DTT, 1.5% Triton, 30 mM Tris pH 7.2). The mRNA
transcripts were stable once combined with the buffer prior to the
subsequent heat step and could be stored in this buffer solution
for several days without observing any significant degradation of
the RNA. The solution was then heated to 80.degree. C. for 5 min.
The samples were cooled on ice and streptavidin coated magnetic
beads (DYNAL M-270) and L-probe were added. The samples were heated
at 50.degree. C. for 1 hour. The magnetic beads were captured on a
DYNAL MPC plate and washed twice with wash buffer. The beads were
recaptured and dynamo PCR 1.times. mastermix was added with the 4
different dual labeled probes and universal PCR primers (25 ul
total volume). The samples were heat cycled using a Stratagene
MX4000 realtime PCR instrument for 30 cycles (95.degree. C. for 15
minutes, 30 cycles 95.degree. C. (10 s), 60.degree. C. (24 s),
72.degree. C. (10 s)) with proper filters for monitoring the
fluorescence in the FAM, Cal Fluor orange 560, Cal Fluor Red 610,
and Quasar 670 channels. The Ct values observed for each channel
were recorded and are indicated in Table 10.
TABLE-US-00010 TABLE 10 Multiplex reagents used in Example 5. S
Probes (25 pM) L Probes (25 pM) Ct(FAM)- Ct(560)- Ct(610)- Ct(670)-
Samples SEQ ID NOs SEQ ID NOs Targets GAPDH3 DDB2 PCNA GAPDH4 1
& 2 26, 29, 32, 35 25, 28 31, 34 250 ng yeast tRNA; 40 25.5
24.5 24.8 25.8 pg GAPDH(Origene SC118869), 40 pg PCNA (SC118528),
40 pg DDB2 (SC122547) mRNA 3 & 4 26, 29, 32, 35 25, 28 31, 34
250 ng yeast tRNA No Ct No Ct No Ct No Ct (negative) 5 & 6 26,
29, 32, 35 25, 28 31, 34 250 ng yeast tRNA; 40 22.1 24.5 22.1 22.2
pg GAPDH(Origene SC118869), 40 pg PCNA (SC118528), 40 pg DDB2 (SC1
22547) mRNA 7 & 8 26, 29, 32, 35 25, 28 31, 34 250 ng yeast
tRNA No Ct No Ct No Ct No Ct (negative)
TABLE-US-00011 TABLE 11 Additional reagents used in Example 5.
GuHCL Lysis Buffer (2X) 12.5 .mu.l S-Probes (0.25 nM Stock of each)
5 .mu.l mRNAs (250 ng tRNA +/- mRNAs) 5 .mu.l Water 2.5 .mu.l Heat
80.degree. C. for 5 min, chill on ice Water 18 .mu.l L-Probes (0.25
nm stock of each) 5 .mu.l Beads 2 .mu.l Total 50 .mu.l
[0340] Incubate 50.degree. C. 1 Hour
Example 6: Formalin-Fixed, Paraffin-Embedded (FFPE) CLPA Assay
[0341] Protocol: [0342] 1. 25 ul CLPA Lysis Buffer (6M GuHCl, 40 mM
EDTA, 10 mM DTT, 3% Triton X-100, 100 mM Tris pH 7.5) containing
400 pM of each S probe (Sequence IDs 47, 49, 51, 53, and 55) and 25
ul of TE buffer were added to a 200 ul PCR tube. [0343] 2.
Formalin-Fixed, Paraffin-Embedded (FFPE) tissue blocks were
sectioned using a microtome blade (Leica, RM 2155) to a thickness
of 5 microns and fixed on pathology glass slides. FFPE sample with
an approximate size of 2 mm.times.5 mm.times.5 microns were scraped
off the glass slide and added to PCR tubes. [0344] 3. The PCR tube
containing the FFPE sample and lysis buffer is sonicated at 50 Hz
in a water bath sonicator (Branson Ultrasonics) for 5 minutes at
55.degree. C. [0345] 4. The tube is removed from the sonication
bath and 40 ul of Proteinase K solution (2.5 mg/ml) and 10 ul of
L-probe solution (1 nM in each probe, Seq. ID 46, 48, 50, 52, and
54) were added with mixing. [0346] 5. The tubes were incubated at
55.degree. C. for 3 hours. [0347] 6. The samples were removed from
the heat block, and the samples were centrifuged for 1 min at 3000
rpm using a benchtop swing-rotor centrifuge. [0348] 7. Only the
supernatant were transferred to a fresh PCR tube. [0349] 8. 2.0 ul
on Invitrogen/Dynal M-270 streptavidin coated magnetic beads were
added, mixed and the samples were incubated for 5 minutes. [0350]
9. The beads were captured using a Dynal MPC 96-S magnetic plate
and the supernatant was removed. [0351] 10. The beads were washed 2
times with 200 ul of wash buffer (100 mM tris buffer, pH 7.0)
[0352] 11. The final wash was discarded and the beads were
resuspended in 10 ul of Dynamo F-450 Taq polymerase master mix
(Finnzymes) and 10 ul of PCR primer set (600 nM each of sequence ID
56 and 23). [0353] 12. The samples were then thermally cycled
according to the protocol: 95.degree. C. 10 min ramp; 28 cycle PCR:
94.degree. C. for 10 seconds; 60.degree. C. for 20 seconds;
72.degree. C. for 20 seconds. [0354] 13. At the thermal cycling was
complete, 1 ul Exonuclease was added to each PCR reaction tube and
the samples were incubated for 20 min at 37.degree. C. followed by
95 C for 2 min. [0355] 14. 2.5 ul of PCR reaction was removed from
each well and added to 22.5 ul of Luminex Bead Mix. The Luminex
Bead Mix was prepared as follows. Combine 2500 microspheres for
each set per reaction (Beads 66, 63, 68, 12 and 72).
Dilute/concentrate the MagPlex-TAG microsphere mixture to 111 of
each microsphere set per uL in 1.1.times. Tm Hybridization Buffer
by vortex and sonication for approximately 20 seconds. [0356] 15.
The samples are then incubated at 95.degree. C. for 1.5 min and
then 37.degree. C. for 1 hour with agitation using a Thermomixer.
[0357] 16. Prepare Reporter Mix by diluting SAPE to 10 ug/mL in
1.times. Tm Buffer (0.2M NaCl, 0.1 M Tris, 0.08% Triton X-100, pH
8.0). [0358] 17. Add 100 ul Reporter Mix to each well. Mix gently.
[0359] 18. Incubate at 37.degree. C. for 15 minutes. [0360] 19. The
MagTag assay was then run on the Luminex instrument according to
manufacturer's protocols The results are shown in FIG. 9.
Example 7: Target Capture CLPA Blood Assay
[0361] In a 0.2 ml PCR tube, 25 ul of whole blood was mixed with 25
ul of 2.times.GuHCl Lysis buffer. The sample was vortexed briefly.
To this tube was added 25 ul of a solution containing 2.0 nM target
capture probes (TC) and 2.0 nM S-probe probes. The solution was
mixed and heated to 80.degree. C. for 5 min and then cooled to
55.degree. C. While at 55.degree. C., 50 ul of a solution
containing 1.0 nM L-probes and 2 mg/ml of proteinase K were added.
The S-probe, L-probe and TC probe sequences are listed in Table 1,
Sequence ID 37-45. The solution was mixed and heated to 55.degree.
C. for 30 minutes. After 30 minutes, 2 ul of M-270 streptavidin
coated magnetic beads were added. The samples were mixed by gentle
pipetting followed by incubation for 5 minutes longer at 55.degree.
C. The samples were removed from the heat blood and immediately
placed onto a 96-well magnetic concentrator plate (Dynal). After 15
seconds, the supernatant was removed completely. The beads were
washed 3 times with 180 ul of wash buffer (100 mM tris buffer, pH
7.4, 0.01% triton). The was buffer was removed and the PCR mix
containing 300 nM each of primer 1 and 2 were added. The samples
were thermal cycled for 28 cycles (95.degree. C. 2 min, 28 cycles
94.degree. C. (10 s), 60 C (20 s) and 72.degree. C. (20 S)). After
thermal cycling, the 2.0 ul of PCR product was mixed with 1 ul of
ABI Genescan 600 V2 size standard and 17 ul of formamide. The
samples were heated to 95.degree. C. for 5 minutes and then placed
on an ABI 3500 Capillary electrophoresis instrument for analysis.
Only 3 peaks were observed within the range of 110-200 base pairs
on the CE trace.
TABLE-US-00012 TABLE 13 Example 7 results Gene Size Peak Height
(RFU) MRPS5 137 30000 PCNA 146 28000 CDR2 187 4000
[0362] The present specification provides a complete description of
the methodologies, systems and/or structures and uses thereof in
example aspects of the presently-described technology. Although
various aspects of this technology have been described above with a
certain degree of particularity, or with reference to one or more
individual aspects, those skilled in the art could make numerous
alterations to the disclosed aspects without departing from the
spirit or scope of the technology hereof. Since many aspects can be
made without departing from the spirit and scope of the presently
described technology, the appropriate scope resides in the claims
hereinafter appended. Other aspects are therefore contemplated.
Furthermore, it should be understood that any operations may be
performed in any order, unless explicitly claimed otherwise or a
specific order is inherently necessitated by the claim language. It
is intended that all matter contained in the above description and
shown in the accompanying drawings shall be interpreted as
illustrative only of particular aspects and are not limiting to the
embodiments shown. Unless otherwise clear from the context or
expressly stated, any concentration values provided herein are
generally given in terms of admixture values or percentages without
regard to any conversion that occurs upon or following addition of
the particular component of the mixture. To the extent not already
expressly incorporated herein, all published references and patent
documents referred to in this disclosure are incorporated herein by
reference in their entirety for all purposes. Changes in detail or
structure may be made without departing from the basic elements of
the present technology as defined in the following claims.
Sequence CWU 1
1
56119DNAArtificial SequenceSynthetic Primer 1gggttcccta agggttgga
19223DNAArtificial SequenceSynthetic Primer 2gtgccagcaa gatccaatct
aga 23347DNAArtificial SequenceSynthetic Probe 3tacatccttc
ctagtcaatt acactctaga ttggatcttg ctggcac 47441DNAArtificial
SequenceSynthetic Probe 4gggttcccta agggttggat aggtaaatgg
cagtgtagaa c 41546DNAArtificial SequenceSynthetic Oligonucleotide
5gtgtaattga ctaggaagga tgtagttcta cactgccatt taccta
46646RNAArtificial SequenceSynthetic Probe 6guguaauuga cuaggaagga
uguaguucua cacugccauu uaccua 46745DNAArtificial SequenceSynthetic
Probe 7tggtttggtg cttcaaatac tctctagatt ggatcttgct ggcac
45863DNAArtificial SequenceSynthetic Probe 8gggttcccta agggttggat
cgagtctaca gatccccaac tttcatagtc tgaaactttc 60tcc
63942DNAArtificial SequenceSynthetic Oligonucleotide 9agtatttgaa
gcaccaaacc aggagaaagt ttcagactat ga 421041DNAArtificial
SequenceSynthetic Probe 10tagcagacac atccaggctc tagattggat
cttgctggca c 411155DNAArtificial SequenceSynthetic Probe
11gggttcccta agggttggat cgagtctact ccaactttga ccaccattcg gctac
551236DNAArtificial SequenceSynthetic Oligonucleotide 12gcctggatgt
gtctgctagt agccgaatgg tggtca 361336RNAArtificial SequenceSynthetic
Oliogonucleotide 13gccuggaugu gucugcuagu agccgaaugg ugguca
361438DNAArtificial SequenceSynthetic Probe 14tccgagattt ccccctctag
attggatctt gctggcac 381537DNAArtificial SequenceSynthetic Probe
15gggttcccta agggttggat cccagactcc tccctct 371632DNAArtificial
SequenceSynthetic Oliogonucleotide 16gggggaaatc tcggaagagg
gaggagtctg gg 321739DNAArtificial SequenceSynthetic Probe
17tcacggtctg ccacgctcta gattggatct tgctggcac 391853DNAArtificial
SequenceSynthetic Probe 18gggttcccta agggttggat gagtctacat
gatccttccc gccacaaaga tgg 531932DNAArtificial SequenceSynthetic
Oliogonucleotide 19cgtggcagac cgtgaccatc tttgtggcgg ga
322051DNAArtificial SequenceSynthetic Probe 20gggttcccta agggttggac
ggacgcctgc ttcaccacct tcttgatgtc a 512132DNAArtificial
SequenceSynthetic Probe 21tcatatttgg caggtttttc tagacggcag gt
322250DNAArtificial SequenceSynthetic Probe 22caggtccacc actgacacgt
tggcagttct agattggatc ttgctggcac 502320DNAArtificial
SequenceSynthetic Primer 23attaaccctc actaaaggga
202489DNAArtificial SequenceSynthetic Oligonucleotide 24actgccaacg
tgtcagtggt ggacctgacc tgccgtctag aaaaacctgc caaatatgat 60gacatcaaga
aggtggtgaa gcaggcgtc 892576DNAArtificial SequenceSynthetic Probe
25ttttctagac ggcaggtcag gtccaccaga tgatcgacga gacactctcg ccatctagat
60tggatcttgc tggcac 762679DNAArtificial SequenceSynthetic Probe
26gggttcccta agggttggac ggaccaactc ctcgccatat catctgtaca ccttcttgat
60gtcatcatat ttggcaggt 792735DNAArtificial SequenceSynthetic Probe
27ccaactcctc gccatatcat ctgtacacct tcttg 352878DNAArtificial
SequenceSynthetic Probe 28tgctgatgat cttgaggctg ttgtcatact
gatgatcgac gagacactct cgccatctag 60attggatctt gctggcac
782975DNAArtificial SequenceSynthetic Probe 29gggttcccta agggttggac
gatggagttg atgctgacgg aagtcatagt aagcagttgg 60tggtgcagga ggcat
753030DNAArtificial SequenceSynthetic Probe 30tgctgacgga agtcatagta
agcagttggt 303177DNAArtificial SequenceSynthetic Probe 31tccttgagtg
cctccaacac cttcttgagg atgatcgacg agacactctc gccatctaga 60ttggatcttg
ctggcac 773277DNAArtificial SequenceSynthetic Probe 32tccttgagtg
cctccaacac cttcttgagg atgatcgacg agacactctc gccatctaga 60ttggatcttg
ctggcac 773333DNAArtificial SequenceSynthetic Probe 33ctgacgactc
ttaatatccc agcaggcctc gtt 333478DNAArtificial SequenceSynthetic
Probe 34ttagttccaa gataaccttg gttccaggct gatgatcgac gagacactct
cgccatctag 60attggatctt gctggcac 783579DNAArtificial
SequenceSynthetic Probe 35gggttcccta agggttggac gttagacgcc
aataggagtt tcactggtgg ctaccaccca 60ctgagaggag aaaagtcat
793630DNAArtificial SequenceSynthetic Probe 36cgccaatagg agtttcactg
gtggctacca 303730DNAArtificial SequenceSynthetic Probe 37gccagagagg
ttacgtggcg gctctcttca 303858DNAArtificial SequenceSynthetic Probe
38ggatgctatg agcgatctgc agcgtgcagt cttcacatct tcccagtcca gtttgacg
583979DNAArtificial SequenceSynthetic Probe 39tctggaacct catcttctgg
ctctggatcc ttcctaagtg aatgttgaca ggatgctcta 60gattggatct tgctggcac
794030DNAArtificial SequenceSynthetic Probe 40tcttcatcct cgatcttggg
agccaagtag 304176DNAArtificial SequenceSynthetic Probe 41ggatgctatg
agcgatctgc agccactata catcttacta tactttactc tacaacaagg 60ggtacatctg
cagaca 764270DNAArtificial SequenceSynthetic Probe 42tactgagtgt
caccgttgaa gagagtggag tggcttttgt aaagtcttct agattggatc 60ttgctggcac
704329DNAArtificial SequenceSynthetic Probe 43agagtgatcg gtattttgtt
ctctgttca 294486DNAArtificial SequenceSynthetic Probe 44ggatgctatg
agcgatctgc agcgcaattc atttcattca caatcaatct aaagatctcc 60ttaaacaacg
ctttgtattc tggagg 8645101DNAArtificial SequenceSynthetic Probe
45tgttgtaggg gaactcacgg gctctgggtt gacagaggcc agttaggatg ttaccaccag
60tgaatgttga caggatcctc tagattggat cttgctggca c
1014664DNAArtificial SequenceSynthetic Probe 46tccattgatg
acaagcttcc cgttctcagc tcgcgttcta agcttccctt tagtgagggt 60taat
644776DNAArtificial SequenceSynthetic Probe 47taatacgact cactataggg
cgagtagaaa gttgaaattg attatgatct cgctcctgga 60agatggtgat gggatt
764855DNAArtificial SequenceSynthetic Probe 48ttctcccagt gactgagggc
tggtgtgtct ttggctccct ttagtgaggg ttaat 554976DNAArtificial
SequenceSynthetic Probe 49taatacgact cactataggg cgaattgaga
aagagataaa tgatagggac tggagcaccg 60agggtatgag aggttc
765055DNAArtificial SequenceSynthetic Probe 50ttcaactcct tggctagtac
acttcggtct agcgctccct ttagtgaggg ttaat 555178DNAArtificial
SequenceSynthetic Probe 51taatacgact cactataggg cggtaagagt
attgaaatta gtaagaggtc tccatgccga 60aacaccaaag tcacaaac
785252DNAArtificial SequenceSynthetic Probe 52tgtgcatctc aaagccatct
gctgtcttcg gctcccttta gtgagggtta at 525376DNAArtificial
SequenceSynthetic Probe 53taatacgact cactataggg cgtttgttgt
taagtatgtg atttagggag gaagtgaccc 60aagtggttga ctccta
765459DNAArtificial SequenceSynthetic Probe 54tgttctctgc ttcttccttc
aactgaatct cctcctgctt ccctttagtg agggttaat 595573DNAArtificial
SequenceSynthetic Probe 55taatacgact cactataggg tatttgataa
gagaatgaag aagtatccac gtccgctcgg 60aaggcagcca aat
735620DNAArtificial SequenceSynthetic Primer 56taatacgact
cactataggg 20
* * * * *