U.S. patent application number 14/214854 was filed with the patent office on 2014-10-09 for nucleic acid amplification.
The applicant listed for this patent is Theranos, Inc.. Invention is credited to Kamila Belhocine, Josephine Lee, Pranav Patel, Aaron Richardson, Scott Tabakman.
Application Number | 20140302504 14/214854 |
Document ID | / |
Family ID | 51537942 |
Filed Date | 2014-10-09 |
United States Patent
Application |
20140302504 |
Kind Code |
A1 |
Belhocine; Kamila ; et
al. |
October 9, 2014 |
Nucleic Acid Amplification
Abstract
Methods and compositions for the amplification of nucleic acids
are disclosed. Amplification methods provided herein may be
performed under isothermal conditions. Methods and compositions may
include reagents such as nucleic acid polymerases, ligases, and
primers.
Inventors: |
Belhocine; Kamila; (Palo
Alto, CA) ; Lee; Josephine; (Palo Alto, CA) ;
Patel; Pranav; (Palo Alto, CA) ; Richardson;
Aaron; (Palo Alto, CA) ; Tabakman; Scott;
(Palo Alto, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Theranos, Inc. |
Palo Alto |
CA |
US |
|
|
Family ID: |
51537942 |
Appl. No.: |
14/214854 |
Filed: |
March 15, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61802241 |
Mar 15, 2013 |
|
|
|
Current U.S.
Class: |
435/6.12 ;
435/289.1; 435/91.2 |
Current CPC
Class: |
C12Q 1/6827 20130101;
C12Q 1/6827 20130101; C12Q 1/6844 20130101; C12Q 2527/101 20130101;
C12Q 2531/119 20130101; C12Q 2521/101 20130101; C12Q 2521/501
20130101; C12Q 2537/143 20130101; C12Q 2537/143 20130101; C12Q
2521/101 20130101; C12Q 2521/501 20130101; C12Q 2531/119 20130101;
C12Q 2527/101 20130101; C12P 19/34 20130101; C12Q 1/6844
20130101 |
Class at
Publication: |
435/6.12 ;
435/91.2; 435/289.1 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method for amplifying a double stranded nucleic acid template,
comprising, (A) preparing a reaction mixture comprising: (i) a
double-stranded nucleic acid comprising at least one copy of the
double-stranded nucleic acid template, wherein the double-stranded
nucleic acid comprises a first strand and a second strand, (ii) an
isolated nucleic acid polymerase, (iii) an isolated nucleic acid
ligase, (iv) a first primer, wherein the first primer is
complementary to the first strand of the double-stranded nucleic
acid template, (v) a second primer, wherein the second primer is
complementary to the second strand of the double-stranded nucleic
acid template, and (B) incubating the reaction mixture at a
temperature of no greater than 70 C for at least 5 minutes, wherein
a plurality of concatemers comprising at least two copies of the
double-stranded nucleic acid template are generated, and the
double-stranded nucleic acid template is amplified at least
100-fold within 60 minutes of initiation of the method.
2. The method of claim 1, wherein the reaction mixture is incubated
at a temperature of no greater than 65 C.
3. The method of claim 2, wherein the reaction mixture is incubated
at a temperature of no greater than 60 C.
4. A vessel, comprising in fluid communication therein: (A) an
isolated nucleic acid polymerase, (B) an isolated nucleic acid
ligase, (C) a double-stranded nucleic acid template comprising a
first strand and a second strand, (D) a first primer, wherein the
first primer is complementary to the first strand of the
double-stranded nucleic acid template, (E) a second primer, wherein
the second primer is complementary to the second strand of the
double-stranded nucleic acid template,
5. A kit for detecting a target nucleic acid of interest comprising
at least a first strand, the kit comprising two or more fluidically
isolated containers, the containers collectively comprising: (A) an
isolated nucleic acid polymerase, (B) an isolated nucleic acid
ligase, (C) a first primer, wherein the first primer is
complementary to the first strand of the target nucleic acid of
interest, (D) a second primer, wherein the second primer is
complementary to a sequence complementary the first strand of the
target nucleic acid of interest.
6. The kit of claim 5, further comprising the target nucleic acid
of interest.
7. The vessel of claim 4, further comprising a nucleic acid
dye.
8-9. (canceled)
10. The vessel of claim 4, wherein the vessel comprises both a DNA
polymerase and a reverse transcriptase.
11. (canceled)
12. The method of claim 1, further comprising treating one or more
of the reaction components with a nucleic acid dye.
13. The method of claim 1, wherein the first primer and second
primer each comprise at least 6 nucleotides.
14. The method of claim 13, wherein the first primer and second
primer each comprise at least 8 nucleotides.
15. The method of claim 1, wherein the first primer and second
primer each comprise no more than 30 nucleotides.
16. The kit of claim 5, further comprising a nucleic acid dye.
17. The kit of claim 5, wherein the kit comprises both a DNA
polymerase and a reverse transcriptase.
18. The vessel of claim 4, wherein the first primer and second
primer each comprise at least 6 nucleotides.
19. The vessel of claim 18, wherein the first primer and second
primer each comprise at least 8 nucleotides.
20. The kit of claim 5, wherein the first primer and second primer
each comprise at least 6 nucleotides.
21. The kit of claim 20, wherein the first primer and second primer
each comprise at least 8 nucleotides.
22. The vessel of claim 4, wherein the first primer and second
primer each comprise no more than 30 nucleotides.
23. The kit of claim 5, wherein the first primer and second primer
each comprise no more than 30 nucleotides.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This applications claims the benefit of, and priority to
U.S. Provisional Patent Application No. 61/802,241, filed Mar. 15,
2013, the content of which is hereby incorporated by reference in
its entirety for all purposes.
BACKGROUND
[0002] There is an increasing need for methods and reagents for the
amplification of nucleic acids. Generation of multiple copies of a
particular nucleic acid is often necessary or helpful in order for
the nucleic acid to be used for a given application. For example,
in order to analyze the nucleotide sequence of a nucleic acid of
interest, frequently, the nucleic acid is replicated to increase
its copy number before the sequence is analyzed. In another
example, in order to determine the presence or absence of a
particular nucleic acid in a sample, a sample may be treated under
conditions such that if the particular nucleic acid is present in
the sample, it may be amplified. In another example, a nucleic acid
for use as probe may be copied repeatedly to generate a large
number of nucleic acids containing the same sequence as the
original nucleic acid template, thereby generating many copies of
the nucleic acid which may be used as a probe.
[0003] A variety of methods for the amplification of nucleic acids
are known. For example, polymerase chain reaction ("PCR") (see,
e.g. U.S. Pat. No. 4,683,202) is a popular method for the
amplification of nucleic acids. To successfully perform a PCR
reaction, the reaction must be performed at multiple different
temperatures. This requires hardware or other mechanisms for
repeatedly changing the temperature of the PCR reaction. Another
method for amplification of nucleic acids is referred to as
loop-mediated isothermal amplification ("LAMP") (see, e.g. U.S.
Pat. No. 6,410,278). LAMP reactions may be performed isothermally,
but typically involve the use of four different primers which
recognize a total of six distinct sequences on the target nucleic
acid.
[0004] To facilitate the generation of amplified nucleic acids for
the many and growing number of applications which use amplified
nucleic acids, new methods and reagents for the amplification of
nucleic acids are desired.
INCORPORATION BY REFERENCE
[0005] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
SUMMARY
[0006] Provided herein are methods and compositions relating to the
amplification of nucleic acids and the generation of
concatemers.
[0007] In some embodiments, a method is provided herein for
amplifying a double stranded nucleic acid template, comprising, (A)
preparing a reaction mixture comprising: (i) a double-stranded
nucleic acid comprising at least one copy of the double-stranded
nucleic acid template, wherein the double-stranded nucleic acid
comprises a first strand and a second strand, (ii) an isolated
nucleic acid polymerase, (iii) an isolated nucleic acid ligase,
(iv) a first primer, wherein the first primer is complementary to
the first strand of the double-stranded nucleic acid template, (v)
a second primer, wherein the second primer is complementary to the
second strand of the double-stranded nucleic acid template, and (B)
incubating the reaction mixture at a temperature of no greater than
70 C for at least 5 minutes, wherein a plurality of concatemers
comprising at least two copies of the double-stranded nucleic acid
template are generated, and the double-stranded nucleic acid
template is amplified at least 100-fold within 60 minutes of
initiation of the method.
[0008] In some embodiments, a method is provided herein for
amplifying a double-stranded nucleic acid template, wherein a
reaction mixture is prepared such that a double-stranded nucleic
acid comprising two or more copies of the double-stranded nucleic
acid template may undergo a primer-based replication process, a
cross-over strand-based replication process, or an end-to-end
ligation process. In some embodiments, the reaction mixture is
prepared and incubated such that all three processes simultaneously
occur in the reaction mixture.
[0009] In some embodiments, a method is provided herein for
amplifying a double-stranded nucleic acid template, wherein a
reaction mixture is prepared such that a double-stranded nucleic
acid comprising a single copy of the double-stranded nucleic acid
template may optionally undergo any one or both of a primer-based
replication process or an end-to-end ligation process. In some
embodiments, a method is provided herein for amplifying a
double-stranded nucleic acid template, wherein a reaction mixture
is prepared such that a double-stranded nucleic acid comprising two
or more copies of the double-stranded nucleic acid template may
optionally undergo any one, two, or three of a primer-based
replication process, a cross-over strand-based replication process,
or an end-to-end ligation process. In some embodiments, a method is
provided herein for amplifying a double-stranded nucleic acid
template, wherein a reaction mixture is prepared such that a
double-stranded nucleic acid comprising a single copy of the
double-stranded nucleic acid template may optionally undergo any
one or both of a primer-based replication process or an end-to-end
ligation process and such that a double-stranded nucleic acid
comprising two or more copies of the double-stranded nucleic acid
template may optionally undergo any one, two, or three of a
primer-based replication process, a cross-over strand-based
replication process, or an end-to-end ligation process.
[0010] In some embodiments, in methods provided herein involving
the amplification of nucleic acid templates or the generation of
concatemers, a plurality of concatemers comprising at least 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 50, or 100
copies of a nucleic acid template are generated.
[0011] In some embodiments, provided herein is a vessel, comprising
in fluid communication therein: (A) an isolated nucleic acid
polymerase, (B) an isolated nucleic acid ligase, (C) a nucleic acid
template comprising at least a first strand, (D) a first primer,
wherein the first primer is complementary to a first strand of a
double-stranded nucleic acid template, and (E) a second primer,
wherein the second primer is complementary to a second strand of
the double-stranded nucleic acid template.
[0012] In some embodiments, provided herein is a kit for detecting
a target nucleic acid of interest comprising at least a first
strand, the kit comprising two or more fluidically isolated
containers, the containers collectively comprising: (A) an isolated
nucleic acid polymerase, (B) an isolated nucleic acid ligase, (C) a
first primer, wherein the first primer is complementary to the
first strand of the target nucleic acid of interest, (E) a second
primer, wherein the second primer is complementary to a sequence
complementary the first strand of the target nucleic acid of
interest. In some embodiments, the kit comprises the target nucleic
acid of interest.
[0013] In some embodiments, a target nucleic acid provided herein
is a double-stranded nucleic acid template.
[0014] In some embodiments, in a method, kit, or vessel provided
herein comprising a first and second primer, the primers each
comprise at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, or 20 nucleotides. In some embodiments, in a method, kit,
or vessel provided herein comprising a first and second primer, the
primers each comprise no more than 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 30, 40, or 50 nucleotides.
[0015] In some embodiments, all of the processes of a method
provided herein are performed at a temperature of no greater than
85, 80, 75, 70, 65, 60, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15,
or 10 C. In some embodiments, some of the processes of a method
provided herein are performed at a temperature of no greater than
85, 80, 75, 70, 65, 60, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15,
or 10 C.
[0016] In some embodiments, two or more processes of a method
provided herein are performed simultaneously in the same reaction.
In some embodiments, all of the processes of a method provided
herein are performed simultaneously in the same reaction.
[0017] In some embodiments, in a method provided herein, a nucleic
acid template is amplified at least 10, 100, 1000, 10,000, 100,000,
or 1,000,000-fold within 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90,
120, or 180 minutes of initiation of the method.
[0018] In some embodiments, a kit provided herein comprises the
target nucleic acid of interest.
[0019] In some embodiments, a vessel or kit provided herein
comprises a nucleic acid dye.
[0020] In some embodiments, in a vessel or kit provided herein
comprising an isolated nucleic acid polymerase, the isolated
nucleic acid polymerase is a DNA polymerase. In some embodiments,
in a vessel or kit provided herein comprising an isolated nucleic
acid polymerase, the isolated nucleic acid polymerase is a reverse
transcriptase. In some embodiments, in a vessel or kit provided
herein comprising an isolated nucleic acid polymerase, the vessel
or kit comprises both a DNA polymerase and a reverse
transcriptase.
[0021] In some embodiments, in a method, vessel, or kit provided
herein comprising a nucleic acid polymerase, the nucleic acid
polymerase has strand displacement activity.
[0022] In some embodiments, a method provided herein comprises
treating one or more of the reaction components or steps of the
method with a nucleic acid dye.
[0023] In some embodiments, methods provided herein may include
generating a template linear double-stranded nucleic acid template
for use in a process provided herein from a single-strand target
molecule. A single-strand target molecule may be DNA or RNA (e.g.
mRNA).
[0024] In some embodiments, a method provided herein comprises
measuring a fluorescent signal from an assay comprising the
method.
[0025] In some embodiments, a nucleic acid ligase may be included
with a method or composition provided herein. In some embodiments,
a nucleic acid template may be amplified more rapidly with a method
provided herein when a ligase is included in a reaction mixture for
a method provided herein, as compared to if a nucleic acid ligase
is not included in the reaction.
[0026] In some embodiments, amplification methods provided herein
may be performed without thermocycling.
[0027] In some embodiments, amplification methods provided herein
may be performed at a constant temperature. In some embodiments,
amplification methods provided herein may be performed within a
temperature range of extending no more than 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 15, 20, 25, or 30 degrees C. above or below a set
temperature of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,
75, or 80 C.
[0028] In some embodiments, amplification methods provided herein
may comprise incubating a reaction mixture at two, three, four,
five or more different temperatures or temperature ranges. In some
embodiments, methods provided herein may comprise incubating a
reaction mixture at a first temperature, wherein the first
temperature is a temperature no greater than 30, 35, 40, 45, 50,
55, 60, 65, 70, or 75 C, and a second temperature, wherein the
second temperature is a temperature no greater than 35, 40, 45, 50,
55, 60, 65, 70, 75, or 80 C. In some embodiments, in a method
provided herein, a reaction mixture may be maintained at a
temperature of no greater than 35, 40, 45, 50, 55, 60, 65, 70, 75,
80, 85, 90, or 95 C during the method.
[0029] In some embodiments, methods and compositions provided
herein comprise, by volume, at least 10%, 20%, 30%, 40%, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% water. In some
embodiments, methods and compositions provided herein comprise, by
volume, collectively no more than 1, 2, 3, 4, 5, 10, 15, 20, 25,
30, 35, 40, 45, 50, 60, 70, or 80% glycerol or polyethylene
glycol.
[0030] In some embodiments, methods provided herein may comprise
assaying a sample for a pathogen. In some embodiments, methods
provided herein may comprise detecting a pathogen in a sample. In
some embodiments, methods provided herein may comprise measuring
the quantity of pathogen in a sample. Pathogens may include, for
example, bacteria, viruses, protists, and fungi.
[0031] In some embodiments, methods provided herein may be
performed without the use of a primer which contains
self-complementary regions. In some embodiments, methods and
compositions provided herein do not comprise a primer which
contains self-complementary regions.
[0032] In some embodiments, methods and compositions provided
herein may be performed or prepared in conjunction with methods and
compositions provided in U.S. Provisional Patent Application No.
61/800,606, filed Mar. 15, 2013, which is herein incorporated by
reference in its entirety.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] In the drawings,
[0034] FIG. 1 is a general schematic of a method and processes
provided herein.
[0035] FIG. 2 is a graph depicting results from reactions performed
according to a method provided herein.
[0036] FIG. 3 is a graph depicting results from reactions performed
according to a method provided herein.
[0037] FIG. 4 is a graph depicting results from reactions performed
according to a method provided herein.
[0038] It is noted that the drawings and elements therein are not
necessarily drawn to shape or scale. For example, the shape or
scale of elements of the drawings may be simplified or modified for
ease or clarity of presentation. It should further be understood
that the drawings and elements therein are for exemplary
illustrative purposes only, and not be construed as limiting in any
way.
DETAILED DESCRIPTION
[0039] While the invention includes various modifications and
alternative forms, specific embodiments thereof are shown by way of
example in the drawings and will herein be described in detail. It
should be understood, however, that there is no intent to limit the
invention to the particular forms disclosed, but on the contrary,
the invention is to cover all modifications, equivalents, and
alternatives falling within the spirit and scope of the invention
as defined by the claims.
[0040] In some embodiments, provided herein are methods and
compositions relating to the amplification of nucleic acids and the
generation of concatemers.
[0041] Generation of nucleic acid concatemers also amplifies the
number of copies of the nucleic acid template/particular nucleic
acid in the concatemer. Accordingly, references herein to methods
and compositions for the generation of concatemers also applies to
the amplification of nucleic acids, and vice versa.
[0042] As used herein, a "polynucleotide" refers to a polymeric
chain containing two or more nucleotides. "Polynucleotides"
includes primers, oligonucleotides, nucleic acid strands, etc. A
polynucleotide may contain standard or non-standard nucleotides.
Typically, a polynucleotide contains a 5' phosphate at one terminus
("5' terminus") and a 3' hydroxyl group at the other terminus ("3'
terminus) of the chain. The most 5' nucleotide of a polynucleotide
may be referred to herein as the "5' terminal nucleotide" of the
polynucleotide. The most 3' nucleotide of a polynucleotide may be
referred to herein as the "3' terminal nucleotide" of the
polynucleotide.
[0043] The term "downstream" as used herein in the context of a
polynucleotide containing a 5' terminal nucleotide and a 3'
terminal nucleotide refers to a position in the polynucleotide
which is closer to the 3' terminal nucleotide than a reference
position in the polynucleotide. For example, in a primer having the
sequence: 5' ATAAGC 3', the "G" is downstream from the "T" and all
of the "A"s.
[0044] The term "upstream" as used herein in the context of a
polynucleotide containing a 5' terminal nucleotide and a 3'
terminal nucleotide, refers to a position in the polynucleotide
which is closer to the 5' terminal nucleotide than a reference
position in the polynucleotide. For example, in a primer having the
sequence: 5' ATAAGC 3', the "T" is upstream from the "G", the "C",
and the two "A"s closest to the "G".
[0045] As used herein, "nucleic acid" includes both DNA and RNA,
including DNA and RNA containing non-standard nucleotides. A
"nucleic acid" contains at least one polynucleotide (a "nucleic
acid strand"). A "nucleic acid" may be single-stranded or
double-stranded.
[0046] As used herein, a "concatemer" refers to a nucleic acid
molecule which contains within it two or more copies of a
particular nucleic acid, wherein the copies are linked in series.
Within the concatemer, the copies of the particular nucleic acid
may be linked directly to each other, or they may be indirectly
linked (e.g. there may be nucleotides between the copies of the
particular nucleic acid). In an example, the particular nucleic
acid may be that of a double-stranded nucleic acid template, such
that a concatemer may contain two or more copies of the
double-stranded nucleic acid template. In another example, the
particular nucleic acid may be that of a polynucleotide template,
such that a concatemer may contain two or more copies of the
polynucleotide template.
[0047] As used herein, a "target" nucleic acid or molecule refers
to a nucleic acid of interest. A target nucleic acid/molecule may
be of any type, including single-stranded or double stranded DNA or
RNA (e.g. mRNA).
[0048] As used herein, "complementary" sequences refer to two
nucleotide sequences which, when aligned anti-parallel to each
other, contain multiple individual nucleotide bases which pair with
each other. It is not necessary for every nucleotide base in two
sequences to pair with each other for sequences to be considered
"complementary". Sequences may be considered complementary, for
example, if at least 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 98%, 99%, or 100% of the nucleotide bases in two
sequences pair with each other. In addition, sequences may still be
considered "complementary" when the total lengths of the two
sequences are significantly different from each other. For example,
a primer of 15 nucleotides may be considered "complementary" to a
longer polynucleotide containing hundreds of nucleotides if
multiple individual nucleotide bases of the primer pair with
nucleotide bases in the longer polynucleotide when the primer is
aligned anti-parallel to a particular region of the longer
polynucleotide.
[0049] As used herein, the term "isolated" as applied to proteins,
nucleic acids, or other biomolecules refers to a molecule that has
been purified or separated from a component of its
naturally-occurring environment (e.g. a protein purified from a
cell in which it was naturally produced). An "isolated" molecule
may be in contact with other molecules (for example, as part of a
reaction mixture). As used herein, "isolated" molecules also
include recombinantly-produced proteins or nucleic acids which have
an amino acid or nucleotide sequence which occurs naturally.
"Isolated" nucleic acids include polypeptide-encoding nucleic acid
molecules contained in cells that ordinarily express the
polypeptide where, for example, the nucleic acid molecule is at a
chromosomal location different from that of natural cells. In some
embodiments, "isolated" polypeptides are purified to at least 50%,
60%, 70%, 80%, 90%, 95%, 98%, or 100% homogeneity as evidenced by
SDS-PAGE of the polypeptides followed by Coomassie blue, silver, or
other protein staining method.
[0050] As used herein, a nucleic acid molecule which is described
as containing the "sequence" of a template or other nucleic acid
may also be considered to contain the template or other nucleic
acid itself (e.g. a molecule which is described as containing the
sequence of a template may also be described as containing the
template), unless the context clearly dictates otherwise.
[0051] Embodiments of methods and compositions provided herein may
be described with reference to FIG. 1. A single template linear
double-stranded nucleic acid 110 or multiple template linear
double-stranded nuclei acid 150 may be provided (FIG. 1A). A single
template linear double-stranded nucleic acid 110 contains a single
copy of a double-stranded nucleic acid template 100. A multiple
template linear double-stranded nucleic acid 150 contains two or
more copies of a double-stranded nucleic acid template 100a, 100b,
100c, etc. A double-stranded nucleic acid template comprises a
first template strand 101 and a second template strand 102.
[0052] A single template linear double-stranded nucleic acid 110
contains a first strand 111 and a second strand 112. The sequence
of the first template strand 101 is within the first strand 111 and
the sequence of the second template strand 102 is within the second
strand 112. A multiple template linear double-stranded nucleic acid
150 contains a first strand 151 and a second strand 152. The
sequences of the multiple copies of the first template strand 101a,
101b, 101c are within the first strand 151 and the sequences of the
multiple copies of the second template strand 102a, 102b, 102c are
within the second strand 152.
[0053] In some embodiments, a single template linear
double-stranded nucleic acid 110 or multiple template linear
double-stranded nuclei acid 150 may be treated under conditions
such that at least 1 or 2 processes may occur with the single
template linear double-stranded nucleic acid 110 (FIG. 1B) or at
least 1, 2, or 3, processes may occur with the multiple template
linear double-stranded nuclei acid 150 (FIG. 1C).
[0054] With reference to FIG. 1B, a single template linear
double-stranded nucleic acid 110 may be treated such that it may
undergo at least 1 or 2 different processes.
[0055] As a first alternative, the first 111 and second 112 strands
are separated, the first template strand 101 and second template
strand 102 are annealed with a first primer 105 or a second primer
106, respectively, and serve as a template for the nucleic acid
polymerase-based generation of new template strands complementary
to the respective template strand. Specifically, from the 3'
terminus of the first primer 105, a new second template strand 132
may be synthesized which is complementary to the first template
strand 101, and from the 3' terminus of the second primer 106, a
new first template strand 121 may be synthesized which is
complementary to the second template strand 102. This may result in
the formation of two new single template linear double-stranded
nucleic acids 120, 130, each of which contains a copy of the
double-stranded nucleic acid template 100a, 100b. The first new
single template linear double-stranded nucleic acid 120 contains
the second template strand 102 and the new first template strand
121. The second new single template linear double-stranded nucleic
acid 130 contains the first template strand 101 and the new second
template strand 132. The above general process may be referred to
herein as "primer-based replication".
[0056] As a second alternative, a single template linear
double-stranded nucleic acid 110 may be treated under conditions
such that it is ligated end-to-end with another double-stranded
nucleic acid 140. Preferably, the other double-stranded nucleic
acid is another copy of the single template linear double-stranded
nucleic acid or a multiple template linear double-stranded nucleic
acid. The conditions may include incubating the double-stranded
nucleic acids with a ligase. As an example, in FIG. 1B the single
template linear double-stranded nucleic acid 110 is shown ligated
end-to-end to another single template linear double-stranded
nucleic acid 140, which results in the formation of a new multiple
template linear double-stranded nucleic acid 145. The new multiple
template linear double-stranded nucleic acid 145 contains two
copies of the double-stranded nucleic acid template 100a, 100b--one
from each of single template linear double-stranded nucleic acid
110 and single template linear double-stranded nucleic acid 140.
The above general process may be referred to herein as "end-to-end
ligation".
[0057] With reference to FIG. 1C, a multiple template linear
double-stranded nucleic acid 150 may be treated such that it may
undergo at least 1, 2, or 3 different processes.
[0058] As a first alternative, a multiple template linear
double-stranded nucleic acid 150 may be treated to undergo
primer-based replication as described above. The reaction
conditions and reagents for primer-based replication with a
multiple template linear double-stranded nucleic acid 150 may be
the same for as with a single template linear double-stranded
nucleic acid 110. With primer-based replication of a multiple
template linear double-stranded nucleic acid, each new multiple
template linear double-stranded nucleic acid formed as a result of
the process may contain multiple copies of the double-stranded
nucleic acid template. In the case of the multiple template linear
double-stranded nucleic acid 150, each multiple template linear
double-stranded nucleic acid contains 3 copies of the
double-stranded nucleic acid template 100a, 100b, and 100c;
accordingly, each new multiple template linear double-stranded
nucleic acid generated from 150 may contain as many as 3 copies of
the double-stranded nucleic acid template. However, since in some
circumstances a primer may anneal to any copy of its complementary
sequence in a multiple template linear double-stranded nucleic
acid, new template strands which are complementary to a first or
second strand of a multiple template linear double-stranded nucleic
acid will not necessarily be as long as or contain as many copies
of a template strand as the first or second strand of a multiple
template linear double-stranded nucleic acid. For example, since
multiple template linear double-stranded nucleic acid 150 contains
3 copies of the double-stranded nucleic acid template 100a, 100b,
and 100c, if a primer anneals to a template strand of copy 100b, a
new template strand generated from that primer will only contain 2
copies of the template strand.
[0059] As a second alternative, a multiple template linear
double-stranded nucleic acid 150 may be treated to undergo
end-to-end ligation with another double-stranded nucleic acid, as
described above. Preferably, the other double-stranded nucleic acid
is another copy of the single template linear double-stranded
nucleic acid or a multiple template linear double-stranded nucleic
acid. A new multiple template linear double-stranded nucleic acid
formed by the end-to-end ligation of a multiple template linear
double-stranded nucleic acid may contain as many copies of the
double-stranded nucleic acid template as were collectively present
in double-stranded nucleic acids which formed the new multiple
template linear double-stranded nucleic acid.
[0060] As a third alternative, a multiple template linear
double-stranded nucleic acid 150 may be treated with another
multiple template linear double-stranded nucleic acid 160 under
conditions such that a cross-over structure 170 comprising a 3'
terminal region of a strand of the multiple template linear
double-stranded nucleic acid 150 annealed to a 3' terminal region
of a strand of the multiple template linear double-stranded nucleic
acid 160 is formed. Specifically, the multiple template linear
double-stranded nucleic acid 160 contains a first strand 161 and a
second strand 162, and three copies of the double-stranded nucleic
acid template 100a, 100b, 100c. Each copy of the double-stranded
nucleic acid template contains a first template strand 101 and a
second template strand 102. The multiple template linear
double-stranded nucleic acids 150, 160 may be treated under
conditions such that, for example, the sequence of the first
template strand 101a of the first strand of multiple template
linear double-stranded nucleic acid 161 anneals to the sequence of
second template strand 102c of the second strand of multiple
template linear double-stranded nucleic acid 152, to produce a
cross-over structure comprising these strands. In general, since
multiple template linear double-stranded nucleic acids each contain
at least two copies of the double-stranded nucleic acid template,
there are a plurality of regions within any given multiple template
linear double-stranded nucleic acids which may be complementary to
another multiple template linear double-stranded nucleic acids,
thus facilitating the formation of cross-over structures between
different multiple template linear double-stranded nucleic acids. A
cross-over structure 170 may be treated with a nucleic acid
polymerase under conditions such an extension product of the first
strand of multiple template linear double-stranded nucleic acid 161
and an extension product of the second strand of multiple template
linear double-stranded nucleic acid 152 are formed. The polymerase
may generate an extension product from the 3' terminus of the
nucleic acid strands 161, 152. The extension product of the first
strand of multiple template linear double-stranded nucleic acid 161
may be referred to herein as a "new concatemer first strand" 181.
The extension product of the second strand of multiple template
linear double-stranded nucleic acid 152 may be referred to herein
as a "new concatemer second strand" 182. Together, the new
concatemer first strand 181 and new concatemer second strand 182
may be referred to as a "new concatemer" 180. The new concatemer
180 may have a greater nucleotide length than either of the parent
molecules used to generated the new concatemer 180, and may contain
more copies of the double-stranded nucleic acid template. For
example, new concatemer 180 contains 5 copies of the
double-stranded nucleic acid template 100a, 100b, 100c, 100d, and
100e. A new concatemer may exclusively contain copies of the
double-stranded nucleic acid template, or it may contain
nucleotides in addition to copies of the double-stranded nucleic
acid template. The above general process may be referred to herein
as "cross-over strand replication".
[0061] In some embodiments, in methods provided herein, one or more
of the above processes may simultaneously occur in the same
reaction. In some embodiments, all of the above processes may
simultaneously occur in the same reaction. In addition, a molecule
generated according to a process provided herein may then be used
in the same or another process provided herein. For example, if a
multiple template linear double-stranded nucleic acid is generated
from a single template linear double-stranded nucleic acid, that
multiple template linear double-stranded nucleic acid may undergo
any of the processes described herein for multiple template linear
double-stranded nucleic acid. In another example, molecule formed
by an end-to-end ligation process provided herein may then be used
as a template for a primer-based replication process. In another
example, a new concatemer formed by a cross-over strand based
replication process may then be used as part of an end-to-end
ligation process. In another example, 2, 3, 4, 5, or more
double-stranded nucleic acids maybe simultaneously ligated into a
single molecule in an end-to-end ligation process provided
herein.
[0062] In some embodiments, methods provided herein may lead to an
increase in one or both of: 1) the number of nucleic acid molecules
containing a double-stranded nucleic acid template of interest, and
2) the number of copies of the double-stranded nucleic acid
template of interest present in nucleic acid molecules containing
double-stranded nucleic acid template of interest. Accordingly, in
some embodiments, double-stranded nucleic acid templates may be
rapidly amplified according to methods and processes provided
herein. For example, in some embodiments, a nucleic acid template
may be amplified at least 500-fold within 0.1, 0.5, 1, 3, 5, 10,
15, 20, 30, 40, 50, 60, 90, 120, or 180 minutes of starting the
method. In another example, in some embodiments, a nucleic acid
template may be amplified at least 10,000-fold within 0.1, 0.5, 1,
3, 5, 10, 15, 20, 30, 40, 50, 60, 90, 120, or 180 minutes of
starting the method. In another example, in some embodiments, a
nucleic acid template may be amplified at least 5, 10, 25, 50, 100,
250, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 500,000, or
1,000,000-fold over the original amount of the nucleic acid
template present in a reaction mixture at the start of the method
within 0.1 minute, 0.5 minute, 1 minute, 3 minutes, 5 minutes, 10
minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50
minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 6
hours, 8 hours, 12 hours, 16 hours, or 24 hours of initiation of
the method. In some embodiments, when a method is initiated, all of
the reagents for a process of the method are in a vessel containing
the reaction mixture for the method. In some embodiments, when a
method is initiated, all of the reagents for all of the processes
of the method are in a vessel containing the reaction mixture for
the method.
[0063] In some embodiments, in a method provided herein, a nucleic
acid template may be amplified at greater than a linear rate. In
some embodiments, in a method provided herein, a nucleic acid
template may be amplified exponentially. In some embodiments, in a
method provided herein, a nucleic acid template may at least double
in number every 1, 2, 3, 5, 10, 15, 20, 25, 30, 45, 60, 90, 120,
180, or 240 minutes after the initiation of the method. In some
embodiments, a nucleic acid template may amplified at least 5, 10,
25, 50, 100, 250, 500, 1,000, 5,000, 10,000, 50,000, 100,000,
500,000, 1,000,000, or 10,000,000-fold over the original amount of
the nucleic acid template present in the reaction at the start of
the method.
[0064] The presence of multiple copies of a nucleic acid template
in a concatemer or other multiple template linear double-stranded
nucleic acid generated according to a method provided herein may
contribute to the rapid amplification of nucleic acid templates
according to methods provided herein. In particular, since multiple
copies of a strand of a nucleic acid template may be present in a
single concatemer/multiple template linear double-stranded nucleic
acid strand, the loading of a single polymerase onto a single
concatemer/multiple template linear double-stranded nucleic acid
strand may result in the generation of multiple copies of a strand
of the nucleic acid template. In some situations, the time required
for nucleic acid polymerases to encounter and load onto nucleic
acid strands may significantly impact the overall speed of an
amplification reaction. For example, if each nucleic acid strand
that a polymerase encounters during a replication reaction only
contains a single copy of a strand of a nucleic acid template, a
polymerase may need to encounter and load onto a new template
strand after each copy of the strand of the template is generated.
In contrast, with a concatemer, after the polymerase encounters and
loads on a concatemer strand, it may synthesize multiple copies of
a strand of the template without needing to leave the concatemer
strand or encounter and load onto another strand.
[0065] In methods provided herein, double-stranded nucleic acids
which do not contain a double-stranded template of interest may be
joined as part of an end-to-end ligation process provided herein.
However, since these molecules will generally not be specifically
amplified by a polymerase with the processes provided herein, these
molecules will not be specifically amplified with methods provided
herein. Accordingly, the methods provided herein permit the rapid
and specific amplification of double-stranded templates of
interest.
[0066] In some embodiments of methods provided herein, the various
processes described herein involving a double-stranded nucleic acid
template are equally likely to occur. In other embodiments, one or
more the processes are more or less likely to occur. In some
embodiments, under certain reaction conditions the various
processes described herein involving a double-stranded nucleic acid
template are equally likely to occur. In some embodiments, under
certain reaction conditions one or more the processes are more or
less likely to occur. For example, in a method provided herein
involving a nucleic acid polymerase and a ligase, the polymerase
and the ligase may have different optimal temperatures for
activity. If the reaction is incubated at a temperature closer to
the optimal temperature for the polymerase, than polymerase-based
processes described herein (primer-based replication and cross-over
strand-based replication) may be favored over end-to-end ligation.
In some examples, methods provided herein may be performed at two
or more temperatures, in order to promote the occurrence of
different processes provided herein in a reaction mixture (e.g.
based on different optimal temperatures for different enzymes).
[0067] In processes described herein involving a nucleic acid
polymerase, the polymerase may have strand displacement activity.
For example, in primer-based replication processes described
herein, as a polymerase generates an extension product from the
primer along one strand of the double-stranded nucleic acid
template, it may displace the other strand of the double-stranded
nucleic acid template from the one strand, as the two strands may
still be partially annealed at the start of the generation of the
extension product of the primer. In another example, in a
cross-over strand replication process described herein, as a
polymerase generates an extension product from the 3' end of a
strand of a multiple template linear double-stranded nucleic acid
in a cross-over structure, the polymerase may displace the other
original strand of the multiple template linear double-stranded
nucleic acid. During the generation of an extension product of a
polymerase, the extension product may become covalently linked to
the original molecule which served as the primer for the generation
of the extension product (e.g. a primer or larger nucleic acid
strand). In some situations, the molecule which served as the
primer for the generation of an extension product of a polymerase
may be considered to be part of the extension product of the
polymerase. In some embodiments, conditions such that an extension
product of a primer or other nucleic acid strand may be formed may
include may include any condition sufficient to support
polymerase-based nucleic acid synthesis. Example conditions for
polymerase-based nucleic acid synthesis are known in the art and
are provided, for example, in Molecular Cloning: A Laboratory
Manual, M. R. Green and J. Sambrook, Cold Spring Harbor Laboratory
Press (2012), which is herein incorporated by reference in its
entirety. The same or different types of polymerases may be used
for the different processes in methods provided herein.
[0068] Nucleic acid molecules generated according to methods and
compositions provided herein may be of any length of nucleotides.
In some embodiments, nucleic acid molecules generated herein may be
at least 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400,
500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000,
7000, 8000, 9000, 10,000, 15,000, 20,000, or 25,000 nucleotides in
length. In some embodiments, nucleic acid molecules generated
herein may be no more than 30, 40, 50, 60, 70, 80, 90, 100, 150,
200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000,
3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 15,000, 20,000,
or 25,000 nucleotides in length. In some embodiments, nucleic acid
molecules generated herein may have a length selected from a range
having a minimum value of 30, 40, 50, 60, 70, 80, 90, 100, 150,
200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000,
3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 15,000, or 20,000
nucleotides in length, and a maximum value of 40, 50, 60, 70, 80,
90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000,
1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000,
15,000, 20,000, or 25,000 nucleotides in length. In some
embodiments, at least some nucleic acid molecules generated
according to a method or composition provided herein have
characteristics described above. In some embodiments, at least 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of nucleic acid
molecules generated according to a method or composition provided
herein have characteristics described above.
[0069] Nucleic acid molecules generated according to methods and
compositions provided herein may contain any number of copies of a
double-stranded nucleic acid template. In some embodiments, nucleic
acid molecules generated herein may contain at least 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,
40, 45, 50, 60, 70, 80, 90, or 100 copies of a double-stranded
nucleic acid template. In some embodiments, nucleic acid molecules
generated herein may contain no more than 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50,
60, 70, 80, 90, or 100 copies of a double-stranded nucleic acid
template. In some embodiments, nucleic acid molecules generated
herein may have a number of copies of a double-stranded nucleic
acid template selected from a range having a minimum value of 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,
30, 35, 40, 45, 50, 60, 70, 80, or 90 copies, and a maximum value
of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 copies. In some
embodiments, at least some nucleic acid generated according to a
method or composition provided herein have characteristics
described above. In some embodiments, at least 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, or 95% of nucleic acid generated according
to a method or composition provided herein have characteristics
described above.
[0070] Detection of Reactions
[0071] Progress of a method provided herein may be monitored in
multiple different ways. In one embodiment, a reaction may be
assayed for a nucleic acid amplification product (e.g. for the
level of the product or the rate of its generation). In another
embodiment, a reaction may be assayed for the activity of a
polymerase along a nucleic acid template (e.g. for movement of a
polymerase along a template strand). Thus, in some embodiments,
events of a method provided herein may observed due to the
accumulation of product from a method (which may be during or after
completion of steps of the method), or due to detectable events
occurring during the steps of a method.
[0072] The presence of amplified nucleic acids can be assayed, for
example, by detection of reaction products (amplified nucleic acids
or reaction by-products) or by detection of probes associated with
the reaction progress.
[0073] In some embodiments, reaction products may be identified by
staining the products with a dye. In some embodiments, a dye may
have greater fluorescence when bound to a nucleic acid than when
not bound to a nucleic acid. In some embodiments, a dye may
intercalate with a double-stranded nucleic acid or it may bind to
an external region of a nucleic acid. Nucleic acid dyes that may be
used with methods and compositions provided herein include, for
example, cyanine dyes, PicoGreen.RTM., OliGreen.RTM.,
RiboGreen.RTM., SYBR.RTM. dyes, SYBR.RTM. Gold, SYBR.RTM. Green I,
SYBR.RTM. Green II, ethidium bromide, dihydroethidium,
BlueView.TM., TOTO.RTM. dyes, TO-PRO.RTM. dyes, POPO.RTM. dyes,
YOYO.RTM. dyes, BOBO.RTM. dyes, JOJO.RTM. dyes, LOLO.RTM. dyes,
SYTOX.RTM. dyes, SYTO.RTM. dyes, propidium iodide, hexidium iodide,
methylene blue, DAPI, acridine orange, quinacrine, acridine dimers,
9-amino-6-chloro-2-methoxyacridine, bisbenzimide dyes, Hoechst
dyes, 7-aminoactinomycin D, actinomycin D, hydroxystilbamidine,
pyronin Y, Diamond.TM. dye, GelRed.TM., GelGreen.TM. and LDS
751.
[0074] In some embodiments, reaction products may be identified by
analysis of turbidity of amplification reactions [or example, where
increased turbidity is correlated with formation of reaction
products and reaction by-products (e.g. pyrophosphate complexed
with magnesium)].
[0075] In some embodiments, reaction products may be identified by
separating a reaction performed according to a method herein by gel
electrophoresis, followed by staining of the gel with a dye for
nucleic acids. The dye may be any nucleic acid dye disclosed herein
or otherwise known in the art.
[0076] In some embodiments, any method or composition known in the
art for the detection of nucleic acids or processes associated with
the generation of nucleic acids may be used with methods and
compositions provided herein.
[0077] In some embodiments, a nucleic acid probe which contains a
nucleotide sequence complementary to a portion of a nucleic acid
template strand (or strand having a similar or identical sequence)
and which contains one or both of a fluorescent reporter
(fluorophore) and a quencher are included in a reaction provided
herein.
[0078] In an example, a nucleic acid probe may contain a
fluorescent reporter at its 5' or 3' terminus, and a quencher at
the other terminus. The probe may further have a nucleotide
sequence containing, in order, at least a first, second, and third
region, where the first and third regions are complementary to each
other, and where at least a portion of the second region is
complementary to a portion of a strand of the nucleic acid template
(the probe "detection sequence"). In some embodiments, the length
of the second region may be greater than the length of the first or
third regions. In some embodiments, the length of the second region
may be between 10 and 40 nucleotides, and the length of first and
third regions may be between 4 and 10 nucleotides. The probe may
have at least two different conformations: (A) a conformation where
the probe is not annealed to its detection sequence and where the
first and third regions are annealed to each other; this
conformation may be a "stem-loop" structure, where the first and
third regions form the stem and the second region forms the loop,
and (B) a conformation where the probe is annealed to its detection
sequence; in this conformation, the second region or a portion
thereof is annealed to its detection sequence and the first and
third regions are not annealed to each other. In conformation (A)
of the probe, the fluorescent reporter and quencher (which are
located at opposite termini of the probe/at the outer ends of the
first and third regions) may be in close proximity to each other
(both being at the end of the stem structure formed by the
annealing of the first and third regions), such that the
fluorescent reporter is quenched. In conformation (B) of the probe,
the fluorescent reporter and quencher may not be in close proximity
to each other, such that the fluorescent reporter is not quenched.
The probe may be used to monitor accumulation of a selected
reaction product, for example, under reaction conditions where the
probe may either form a stem-loop structure or anneal to its
detection sequence. In some embodiments, if the detection sequence
is present, the probe may anneal to the detection sequence, and the
probe may fluoresce in response to light of a wavelength of the
fluorphore's excitation spectrum. In contrast, if the detection
sequence is not present, the probe may form a stem-loop structure,
and not fluoresce in response to light of a wavelength of the
fluorphore's excitation spectrum.
[0079] In another example, a nucleic acid probe may contain a
fluorescent reporter at its 5' or 3' terminus, and it may be
annealed to a nucleic acid primer containing a quencher. The
nucleic acid primer containing a quencher may contain the quencher
at a position in the primer such that when the nucleic acid probe
is annealed to the primer, the fluorescent reporter is quenched.
The probe may be used to monitor accumulation of a selected
reaction product, for example, under reaction conditions where the
probe may either anneal to the primer or anneal to its detection
sequence. In some embodiments, if the detection sequence is
present, the probe may anneal to the detection sequence, and the
probe may fluoresce in response to light of a wavelength of the
fluorphore's excitation spectrum. In contrast, if the detection
sequence is not present, the probe may remain paired with the
primer, and not fluoresce in response to light of a wavelength of
the fluorphore's excitation spectrum.
[0080] In probes containing a fluorescent reporter and quencher
pair, the fluorescent reporter and quencher may be selected so that
the quencher can effectively quench the reporter. In some
embodiments, a fluorescent reporter is paired with a quencher where
the emission maximum of the fluorescent reporter is similar to the
absorption maximum of the quencher. Fluorphores that may be used as
the fluorescent reporter include, for example, CAL Fluor Gold, CAL
Fluor Orange, Quasar 570, CAL Fluor Red 590, CAL Fluor Red 610, CAL
Fluor Red 610, CAL Fluor Red 635, Quasar 670 (Biosearch
Technologies), VIC, NED (Life Technologies), Cy3, Cy5, Cy5.5 (GE
Healthcare Life Sciences), Oyster 556, Oyster 645 (Integrated DNA
Technologies), LC red 610, LC red 610, LC red 640, LC red 670, LC
red 705 (Roche Applies Science), Texas red, FAM, TET, HEX, JOE,
TMR, and ROX. Quenchers that may be used include, for example,
DDQ-I, DDQ-II (Eurogentec), Eclipse (Epoch Biosciences), Iowa Black
FQ, Iowa Black RQ (Integrated DNA Technologies), BHQ-1, BHQ-2,
BHQ-3 (Biosearch Technologies), QSY-7, QSY-21 (Molecular Probes),
and Dabcyl.
[0081] In some embodiments, a method provided herein may be
monitored in an apparatus containing a light source and an optical
sensor. In some situations, the reaction may be positioned in the
path of light from the light source, and light absorbed by the
sample (e.g. in the case of a turbid reaction), scattered by the
sample (e.g. in the case of a turbid reaction), or emitted by the
sample (e.g. in the case of a reaction containing a fluorescent
molecule) may be measured. In some embodiments, a method provided
herein may be performed or monitored in a device or module therein
as disclosed in U.S. patent application Ser. No. 13/769,779, filed
Feb. 18, 2013, which is herein incorporated by reference in its
entirety.
[0082] Using methods provided herein, specific amplification
products of a nucleic acid template of interest may be identified
within, for example, 30 seconds, 1 minute, 3 minutes, 5 minutes, 10
minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 60
minutes, 90 minutes, 120 minutes, 180 minutes, or 240 minutes of
initiation of an amplification reaction. In other examples, using
methods provided herein, amplification reactions which are positive
for a nucleic acid template of interest may be identified when as
few as 10, 50, 100, 500, 1000, 5000, 10,000, 50,000, 100,000,
500,000, or 1,000,000 copies of the template are generated. In
other examples, using methods provided herein, the presence of a
nucleic acid template of interest in a sample containing as few as
1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 100, 200, 500, 1000,
5000, or 10,000 copies of the template of interest may be
identified.
[0083] Methods provided herein may be performed for any length of
time. Typically, the method will be performed for a length of time
sufficient to monitor, for example, the rate of nucleic acid
replication, the occurrence of polymerase activity, or the
accumulation of amplification product. In some embodiments, a
method provided herein may be performed for a total of less than 10
seconds, 30 seconds, 1 minute, 5 minutes, 10 minutes, 20 minutes,
30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours,
8 hours, 12 hours, 16 hours, or 24 hours, by which time the rate of
nucleic acid replication, the occurrence of polymerase activity, or
the accumulation of amplification product was measured.
[0084] Methods provided herein may be terminated in various ways.
In one embodiment, steps of a method may end upon the reduction in
concentration or complete consumption of one or more reagents
involved in one or more steps of the method (e.g. dNTPs). In
another embodiment, steps of a method may end upon inactivation of
one or more enzymes involved in one or more steps of the method
(e.g. polymerases). Enzymes may be inactivated by various ways. For
example, enzymes may gradually lose enzymatic activity over time
due to random events that affect the structure of the enzyme, or
enzymes may be exposed to a condition to accelerate the
inactivation of the enzyme activity (e.g. high heat, extreme pH,
etc.).
[0085] A single template linear double-stranded nucleic acid
comprises a single copy of a double-stranded nucleic acid template.
In some embodiments, the entirety of the single template linear
double-stranded nucleic acid is a double-stranded nucleic acid
template. In some embodiments, a single template linear
double-stranded nucleic acid contains nucleotides in addition to
the double-stranded nucleic acid template. In some embodiments, a
multiple template linear double-stranded nucleic acid comprises
only copies of the double-stranded nucleic acid template. In some
embodiments, a multiple template linear double-stranded nucleic
acid contains nucleotides in addition to the double-stranded
nucleic acid templates. Typically, template linear double-stranded
nucleic acids provided herein have blunt ends. In some embodiments,
template linear double-stranded nucleic acids may have sticky ends.
A template linear double-stranded nucleic acids nucleic acid may
contain DNA, RNA, or a mixture thereof.
[0086] A single template linear double-stranded nucleic acid may be
generated by any process wherein a double-stranded nucleic acid
containing a single copy of a double-stranded nucleic template may
be formed. For example, a single template linear double-stranded
nucleic acid may be generated from a previous single template
linear double-stranded nucleic acid according to a primer-based
replication process described herein. In another example, a single
template linear double-stranded nucleic acid may be generated from
a circular nucleic acid containing the sequence of the
double-stranded nucleic template, by a primer-based replication
process. In another example, a single template linear
double-stranded nucleic acid may be generated from a
single-stranded RNA molecule, through a process involving a reverse
transcriptase. In another example, a single template linear
double-stranded nucleic acid may be generated by the end-to-end
ligation of double-stranded nucleic acid that does not contain a
copy of the double-stranded nucleic acid template with a previous
single template linear double-stranded nucleic acid. A multiple
template linear double-stranded nucleic acid may be generated by
any process wherein a double-stranded nucleic acid containing a two
or more copies of a double-stranded nucleic template may be formed,
such as by the processes described herein. As used herein, the term
"template linear double-stranded nucleic acid" includes both single
template linear double-stranded nucleic acids and multiple template
linear double-stranded nucleic acids.
[0087] A template linear double-stranded nucleic acid may be of any
length of nucleotides. For example, a template linear
double-stranded nucleic acid may be at least 2, 3, 4, 5, 6, 7, 8,
9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90,
100, 150, 200, 250, 300, 350, 400, 500, 750, 1000, or 1500
nucleotides in length. In another example, a template linear
double-stranded nucleic acid may be between 2 and 100,000, between
5 and 100,000, between 10 and 100,000, between 15 and 100,000,
between 20 and 100,000, between 25 and 100,000, between 30 and
100,000, between 50 and 100,000, between 70 and 100,000, between
100 and 100,000, between 200 and 100,000, between 2 and 10,000,
between 5 and 10,000, between 10 and 10,000, between 15 and 10,000,
between 20 and 10,000, between 25 and 10,000, between 30 and
10,000, between 50 and 10,000, between 70 and 10,000, between 100
and 10,000, between 200 and 10,000, between 2 and 5,000, between 5
and 5,000, between 10 and 5,000, between 15 and 5,000, between 20
and 5,000, between 25 and 5,000, between 30 and 5,000, between 50
and 5,000, between 70 and 5,000, between 100 and 5,000, between 200
and 5,000, between 2 and 3,000, between 5 and 3,000, between 10 and
3,000, between 15 and 3,000, between 20 and 3,000, between 25 and
3,000, between 30 and 3,000, between 50 and 3,000, between 70 and
3,000, between 100 and 3,000, between 200 and 3,000, between 2 and
1,000, between 5 and 1,000, between 10 and 1,000, between 15 and
1,000, between 20 and 1,000, between 25 and 1,000, between 30 and
1,000, between 50 and 1,000, between 70 and 1,000, between 100 and
1,000, between 200 and 1,000, between 2 and 500, between 5 and 500,
between 10 and 500, between 15 and 500, between 20 and 500, between
25 and 500, between 30 and 500, between 50 and 500, between 70 and
500, between 100 and 500, or between 200 and 500 nucleotide bases
in length.
[0088] A double-stranded nucleic acid template may be generated by
any method provided herein for the generation of a template linear
double-stranded nucleic acid provided herein.
[0089] In some embodiments, a double-stranded nucleic acid template
is a double-stranded DNA molecule that was generated from an RNA
molecule (e.g. a single stranded RNA molecule, such as mRNA). A
double-stranded DNA molecule may be generated from an RNA molecule
through techniques that are well-known in the art, for example,
through reverse transcription. Example conditions for generating a
double-stranded DNA molecule from an RNA molecule are provided, for
example, in RNA: A Laboratory Manual, D. Rio et al., Cold Spring
Harbor Laboratory Press (2011), which is herein incorporated by
reference in its entirety. Briefly, in some examples, a primer
which is complementary to an mRNA sequence of interest may be
incubated with: reverse transcriptase enzyme (e.g. AMV reverse
transcriptase, M-MLV reverse transcriptase, Superscript II.TM.
reverse transcriptase, Superscript III.TM. reverse transcriptase,
or ThermoScript.TM. reverse transcriptase), dNTPs, and the mRNA
sequence of interest. The primer may anneal to the mRNA, and then,
starting from the 3' end of the primer, the reverse transcriptase
may synthesize a strand of DNA complementary to the mRNA (cDNA). In
some embodiments, the mRNA annealed to the cDNA may be degraded
(e.g. with an RNase; the RNase may be the reverse transcriptase,
which may also have RNase activity), and the cDNA may then be
incubated with: a different primer which is complementary to the
strand of cDNA, dNTPs, and a DNA polymerase (e.g. any DNA
polymerase discussed elsewhere herein). Then, starting from the 3'
end of the different primer, the DNA polymerase may synthesize a
strand of DNA complementary to cDNA, thereby generating a linear
double-stranded DNA molecule.
[0090] In some embodiments, a double-stranded nucleic acid template
may be generated from a single-stranded RNA molecule in the same
reaction mixture in which the double-stranded nucleic acid template
is amplified according to a method provided herein. In some
embodiments, the same primer may be used for both A) generation of
a cDNA strand from an RNA molecule, and B) as a first or second
primer in primer-based replication process provided herein.
[0091] A double-stranded nucleic acid template may be of any length
of nucleotides. For example, a double-stranded nucleic acid
template may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16,
18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250,
300, 350, 400, 500, 750, 1000, or 1500 nucleotides in length. In
another example, a double-stranded nucleic acid template may be
between 2 and 100,000, between 5 and 100,000, between 10 and
100,000, between 15 and 100,000, between 20 and 100,000, between 25
and 100,000, between 30 and 100,000, between 50 and 100,000,
between 70 and 100,000, between 100 and 100,000, between 200 and
100,000, between 2 and 10,000, between 5 and 10,000, between 10 and
10,000, between 15 and 10,000, between 20 and 10,000, between 25
and 10,000, between 30 and 10,000, between 50 and 10,000, between
70 and 10,000, between 100 and 10,000, between 200 and 10,000,
between 2 and 5,000, between 5 and 5,000, between 10 and 5,000,
between 15 and 5,000, between 20 and 5,000, between 25 and 5,000,
between 30 and 5,000, between 50 and 5,000, between 70 and 5,000,
between 100 and 5,000, between 200 and 5,000, between 2 and 3,000,
between 5 and 3,000, between 10 and 3,000, between 15 and 3,000,
between 20 and 3,000, between 25 and 3,000, between 30 and 3,000,
between 50 and 3,000, between 70 and 3,000, between 100 and 3,000,
between 200 and 3,000, between 2 and 1,000, between 5 and 1,000,
between 10 and 1,000, between 15 and 1,000, between 20 and 1,000,
between 25 and 1,000, between 30 and 1,000, between 50 and 1,000,
between 70 and 1,000, between 100 and 1,000, between 200 and 1,000,
between 2 and 500, between 5 and 500, between 10 and 500, between
15 and 500, between 20 and 500, between 25 and 500, between 30 and
500, between 50 and 500, between 70 and 500, between 100 and 500,
or between 200 and 500 nucleotide bases in length.
[0092] A "primer" as used herein may refer to a polynucleotide
which is i) capable of hybridizing to an original nucleic acid
strand and ii) acting as a point of initiation for the synthesis of
a new nucleic acid strand, wherein the new nucleic acid strand is
an extension product of the primer and is complementary to the
original strand. A primer may have a free --OH group at its 3'
terminus, which may serve as the origin of synthesis for the
extension product.
[0093] A primer may contain standard nucleotides [e.g. standard DNA
deoxyribonucleotides (deoxyadenosine monophosphate, deoxyguanosine
monophosphate, thymidine monophosphate, deoxycytidine
monophosphate) or standard RNA ribonucleotides (adenosine
monophosphate, guanosine monophosphate, uridine monophosphate,
cytidine monophosphate)], alternative nucleotides (e.g. inosine),
modified nucleotides, nucleotide analogs, or a combination thereof.
For example, an oligonucleotide primer may include peptide nucleic
acids, morpholinos (e.g. phosphorodiamidate morpholino oligos),
locked nucleic acids [see, for example, Kaur, H, et. al,
Biochemistry 45 (23), 7347-55 (2006)], glycol nucleic acids, or
threose nucleic acids. A primer may have a backbone, including, for
example, phosphodiester linkages, phosphorothioate linkages (a
non-bridging O is replaced with sulfur), or peptide linkages (as
part of a peptide nucleic acid). Alternative nucleotides, modified
nucleotides, and nucleotide analogs may be referred to collectively
herein as "non-standard nucleotides."
[0094] The presence of a non-standard nucleotide in a primer may
affect various properties of the primer. In some embodiments,
inclusion of a non-standard nucleotide in a primer may increase or
decrease the thermodynamic stability of a primer to a complementary
sequence thereof. For example, a primer having increased
thermodynamic stability may contain a locked nucleic acid. A primer
having decreased thermodynamic stability may contain, for example,
inosine (described by Auer et al., Nucl. Acids Res. 24; 5021-5025
(1996)) or a negatively charged chemical group, such as a
carboxylic acid.
[0095] A primer provided herein may be of any length. In some
embodiments, a primer may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250,
300, 350, 400, 500, 750, 1000, or 1500 nucleotides in length. In
some embodiments, a primer may be no more than 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200,
250, 300, 350, 400, 500, 750, 1000, or 1500 nucleotides in length.
In some embodiments, a primer may have a length selected from a
range having a minimum value of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350,
400, 500, 750, or 1000 nucleotides in length, and a maximum value
of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80,
90, 100, 150, 200, 250, 300, 350, 400, 500, 750, 1000, or 1500
nucleotides in length.
[0096] In some embodiments, a primer may be of any length and
contain any nucleotide sequence which permits sufficiently stable
and specific annealing of the primer to its complement at the
temperature being used for a method or step thereof involving the
primer. The exact length desired of a primer may depend on a
variety of factors, including the temperature of a reaction, the
chemical composition of the primer, and the reaction involving the
primer. In some embodiments, the template-binding region of a
primer may be of any length and contain any nucleotide sequence
which permits sufficiently stable and specific annealing of the
template-binding region of the primer to its complement at the
temperature being used for a method or step thereof involving the
primer. The exact length desired of the template-binding region of
a primer may depend on a variety of factors, including the
temperature of a reaction, the chemical composition of the
template-binding region of the primer, and the reaction involving
the primer. The inclusion of one or more non-standard nucleotides
in the primer may change the desired length of the primer for use
in a method provided herein, as compared to the length of a
corresponding primer lacking a non-standard nucleotide. For
example, if with a method provided herein it is desired to have a
primer with a certain Tm, in some embodiments, a primer with the
selected Tm may be of a shorter length if the primer contains at
least some non-standard nucleotides, as compared to if the primer
contains only standard nucleotides.
[0097] A primer provided herein may be prepared by any suitable
method. For example, a primer may be chemically synthesized. In
another example, a naturally occurring nucleic acid may be
isolated, cleaved (e.g. with restriction enzymes), and/or modified
to generate or to become part of a primer described herein.
[0098] In some embodiments, a label may be attached to a primer.
Labels include, for example, binding ligands (e.g. digoxin or
biotin), enzymes, fluorescent molecules/fluorphores, luminescent
molecules, quencher molecules, or radioisotopes. In other
embodiments, a base of an oligonucleotide may be replaced with a
fluorescent analog, such as 2-aminopurine (see, for example, Proc.
Acad. Sci. USA, 91, 6644-6648 (1994), which is herein incorporated
by reference in its entirety).
[0099] In some embodiments, conditions such that: i) a primer
anneals to a template strand in a primer-based replication process
provided herein, or ii) a 3' terminal region of a first strand of a
first multiple template linear double-stranded nucleic acid anneals
with a 3' terminal region of a second strand of a second multiple
template linear double-stranded nucleic acid, to produce a
cross-over structure comprising these strands, may each include
incubating the nucleic acids at a temperature such that the strands
of double-stranded nucleic acid molecules "breathe" (i.e. undergo
brief periods of localized rupture of hydrogen bonds connecting
base pairs) to a degree sufficient to facilitate the entry of a
primer or different nucleic acid strand between the strands of a
double-stranded molecule, and the annealing of the primer or
different nucleic acid strand to one of the strands of the opened
double-stranded nucleic acid molecule. In some embodiments, methods
or processes provided herein may be performed or incubated at a
temperature of at least 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,
63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,
80, 81, 82, 83, 84, 85, 90, or 95 C. In some embodiments, methods
or processes provided herein may be performed or incubated at a
temperature of no greater than 10, 15, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,
44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,
61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,
78, 79, 80, 81, 82, 83, 84, 85, 90, or 95 C. In some embodiments,
methods or processes provided herein may be performed or incubated
at a temperature between 10, 15, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,
62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,
79, 80, 81, 82, 83, 84, 85, or 90 C and 15, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,
60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,
77, 78, 79, 80, 81, 82, 83, 84, 85, 90 or 95 C.
[0100] In some embodiments, for a method or process provided
herein, the step or process is performed at a temperature below the
melting temperature (Tm) of the relevant potentially paired
nucleotide strands, or regions thereof (e.g. a primer to a nucleic
acid template strand, or a 3' terminal region of a first strand of
a first multiple template linear double-stranded nucleic acid
anneals with a 3' terminal region of a second strand of a second
multiple template linear double-stranded nucleic acid, etc.) In
some embodiments, for a method or process provided herein, the
process or method is performed at a temperature above the Tm of the
relevant potentially paired nucleotide strands, or regions thereof.
Generally, melting temperature is regarding as the temperature at
which 50% of nucleic acids having a given mutually complementary
nucleotide sequence are base-paired.
[0101] In some embodiments, a nucleic acid polymerase is included
with a method or composition provided herein. A polymerase may
generate an extension product of a primer. The primer and extension
product thereof may be complementary to a template nucleic acid
strand. Generally, a nucleic acid polymerase will initiate
synthesis of an extension product of a primer at the 3' end of the
primer. In some embodiments, a DNA polymerase is included with a
method or composition provided herein. As used herein, a "DNA
polymerase" refers to a nucleic acid polymerase which has primary
or exclusive polymerase activity on DNA templates. In some
embodiments, a reverse transcriptase is included with a method or
composition provided herein. As used herein, a "reverse
transcriptase" refers to a nucleic acid polymerase which can
synthesize a DNA strand from an RNA template.
[0102] In some embodiments, a polymerase provided herein may have
strand displacement activity. Polymerases having strand
displacement activity include, for example, exo-Bca DNA polymerase,
phi29 DNA polymerase, Klenow Fragment of E. coli DNA Polymerase I,
Vent.sub.R DNA polymerase, Deep Vent.sub.R DNA polymerase,
9.degree. N.sub.m DNA polymerase, and Large Fragment of Bst DNA
Polymerase.
[0103] Modified versions of polymerases may also be used with the
methods and compositions provided herein, provided that the
modified polymerase has sequence-dependent nucleic acid synthesis
activity. A modified version of a polymerase ("modified
polymerase") may have, for example, 100 or fewer, 70 or fewer, 50
or fewer, 40 or fewer, 30 or fewer, 20 or fewer, 10 or fewer, 5 or
fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 different amino
acid from the sequence of the parent version of the polymerase. In
some embodiments, a modified polymerase may contain no more than
1000, 700, 500, 400, 300, 200, 100, 50, 40, 30, 20, 10, or 5
greater or fewer amino acids than the parent polymerase. In some
embodiments, a modified polymerase may comprise a fragment of a
parent polymerase. In some embodiments, a modified polymerase may
comprise a chimeric polypeptide with a portion derived from a
polymerase and a portion derived from a non-polymerase protein. In
some embodiments, a modified polymerase may have, for example,
increased catalytic activity, increased stability, or increased
thermostability as compared to the parent polymerase.
[0104] In some embodiments, a polymerase provided herein is
thermostable. A thermostable polymerase may have, for example, a
half-life of at least 5, 10, 15, 20, 30, 40, 50, 60, 90, 120, or
180 minutes at a temperature of at up to 25, 30, 35 40, 45, 50, 55,
60, 65, 70, 75, 80, 85, 90, or 95 C. In some embodiments, a
modified polymerase may be thermostable.
[0105] In some embodiments, methods and processes provided herein
include or are performed under conditions sufficient to support
polymerase-based nucleic acid synthesis. Example conditions for
polymerase-based nucleic acid synthesis are known in the art and
are provided, for example, in Green and Sambrook, supra.
Non-limiting components for a polymerase-based nucleic acid
synthesis reaction may include one or more of: polymerase enzyme
(at a concentration between, for example, 0.01 and 10 units enzyme
per 50 microliters reaction volume, or any range therein including,
for example, between 0.01-1, 0.1-10, 0.1-5, 0.5-10, 0.5-5, 0.5-2,
1-10, or 1-5 units enzyme per 50 microliters reaction volume, where
1 unit of enzyme will incorporate 15 nmol of dNTPs into
polymerization product in 30 minutes at 75 C); template (at a
concentration of at least, for example, 1, 10, 100, 1,000, 10,000,
or 100,000 copies per reaction); primer (at a concentration
between, for example, 0.01 and 10 micromolar, or any range therein
including, for example, between 0.01-1, 0.1-10, 0.1-5, 0.5-5, or
0.5-2 micromolar); dNTPs (e.g. dATP, dTTP, dGTP, and dCTP, at a
concentration between, for example, 50 and 500 micromolar each of
dATP, dTTP, dGTP, and dCTP, or any range therein including, for
example, between 50-350, 100-500, 100-300, 200-500, or 300-400
micromolar each of dATP, dTTP, dGTP, and dCTP); salt (e.g. KCl or
potassium acetate, at a concentration between, for example, 1 and
200 millimolar, or any range therein including, for example,
between 1-100, 1-50, 1-20, 1-10, 10-20, 10-50, or 10-200
millimolar); buffer (e.g. Tris-HCl or Tris-acetate, pH 7.8-8.5, at
a concentration between, for example, 1 and 100 millimolar, or any
range therein including, for example, between 1-50, 1-20, 1-10,
1-5, 10-100, 20-100, or 50-100 millimolar); and magnesium ions (at
a concentration between, for example 0.1 and 10 millimolar, or any
range therein, including, for example, between 0.1-5, 0.1-1,
0.5-10, 0.5-5, or 0.5-2.5 millimolar). Additional non-limiting
components for a polymerase-based nucleic acid synthesis reaction
may increase the speed of the reaction, increase the fidelity of
the reaction, or increase the stability of enzymes or DNA in the
reaction, and may include one or more of: gelatin (at a
concentration between, for example, 0.0001% and 0.1% w/v), BSA (at
a concentration between, for example, 0.01 and 1 microgram per
microliter), sucrose (at a concentration between, for example 0.01
molar and 0.8 molar), trehalose (at a concentration between, for
example 0.01 molar and 0.8 molar), DMSO (at a concentration
between, for example, 0.01 and 10% v/v), betaine (at a
concentration between, for example, 0.1 and 10 molar), formamide
(at a concentration between, for example, 0.1 and 10% v/v),
glycerol (at a concentration between, for example, 0.1 and 20%
v/v), polyethylene glycol (at a concentration between, for example,
0.1 and 20% v/v), non-ionic detergents [e.g. NP-40 (at a
concentration between, for example, 0.01 and 1% v/v), Tween-20 (at
a concentration between, for example, 0.01 and 1% v/v), and Triton
X-100 (at a concentration between, for example, 0.01 and 1% v/v)],
ammonium ions [e.g. ammonium sulfate (at a concentration between,
for example, 1 and 100 millimolar)], and EDTA (at a concentration
between, for example, 0.001 and 0.1 millimolar). Other reagents may
also be present in a polymerase-based nucleic acid synthesis
reaction provided herein. For example, reagents to sufficient to
synthesize RNA reaction products or reaction products containing
non-standard nucleotides may be used. Conditions sufficient to
support polymerase-based nucleic acid synthesis may include a
variety of temperatures and pH values. For example, the pH of a of
a polymerase-based nucleic acid synthesis reaction be between, for
example pH 6.0 and pH 10.0, such as 6.5, 7, 7.5, 7.8, 7.9, 8, 8.1,
8.2, 8.5, 9, or 9.5. The temperature of a polymerase-based nucleic
acid synthesis reaction may be constant or varied. A constant
temperature may be between, for example, 10 C and 95 C, such as 20,
25, 30, 35, 37, 40, 42, 45, 50, 55, 60, 65, 70, 75, 80, or 85 C. A
varied temperature may at two or more different temperatures
between, for example 10C and 95 C, such two or more temperatures
selected from 20, 25, 30, 35, 37, 40, 42, 45, 50, 55, 60, 65, 70,
75, 80, or 85 C.
[0106] In some embodiments, a nucleic acid ligase is included with
a method or composition provided herein. Ligases catalyze the
formation of phosphodiester bonds between nucleotides, typically
between the 5' phosphate of one nucleotide, and the 3' hydroxyl
group of another nucleotide.
[0107] Nucleic acid ligases include E. coli DNA ligase, Taq DNA
ligase, T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Ampligase.TM.,
T4 RNA ligase 1, and T4 RNA ligase 2.
[0108] In order to catalyze the ligation reaction, certain ligases
require ATP (e.g. T4 DNA ligase) or NAD+ (E. coli DNA ligase). In
some embodiments, a ligase may ligate nucleic acids having blunt
ends. In some embodiments, a ligase may ligate nucleic acids having
sticky ends. In some embodiments, a ligase may ligate nucleic acids
having both blunt and sticky ends.
[0109] Modified versions ligases may also be used with the methods
and compositions provided herein, provided that the modified ligase
has the ability to catalyze the formation of phosphodiester bonds
between nucleotides. A modified version of a ligase ("modified
ligase") may have, for example, 100 or fewer, 70 or fewer, 50 or
fewer, 40 or fewer, 30 or fewer, 20 or fewer, 10 or fewer, 5 or
fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 different amino
acid from the sequence of the parent, naturally occurring version
of the ligase. In some embodiments, a modified ligase may contain
no more than 1000, 700, 500, 400, 300, 200, 100, 50, 40, 30, 20,
10, or 5 greater or fewer amino acids than the parent ligase. In
some embodiments, a modified ligase may comprise a fragment of a
parent ligase. In some embodiments, a modified ligase may comprise
a chimeric polypeptide with a portion derived from a ligase and a
portion derived from a non-ligase protein. In some embodiments, a
modified ligase may have, for example, increased catalytic
activity, increased stability, or increased thermostability as
compared to the parent ligase.
[0110] In some embodiments, a ligase provided herein is
thermostable. A thermostable ligase may have, for example, a
half-life of at least 5, 10, 15, 20, 30, 40, 50, 60, 90, 120, or
180 minutes at a temperature of at up to 25, 30, 35 40, 45, 50, 55,
60, 65, 70, 75, 80, 85, 90, or 95 C. In some embodiments, a
modified ligase may be thermostable.
[0111] In some embodiments, a ligase used with methods and
compositions provided herein may be a modified ligase referred to
herein as "p50-Tth", which has the amino acid sequence:
TABLE-US-00001 mghhhhhhhhhhssghiegrasadgpylqileqpkqrgfrfryvce
gpshgglpgasknihlhahslvgkhcedgictvtagpkdmvvgfan
lgilhvtkkkvfetlearmteacirgynpgllvhpdlaylqaeggg
drqlgdrekelirqaalqqtkemdlsvvrlmftaflpdstgsftrr
lepvvsdaiydskapnasnlkivrmdrtagcvtggeeiyllcdkvq
kddiqirfyeeeenggvwegfgdfsptdvhrqfaivfktpkykdin
itkpasvfvqlrrksdletsepkpflyypeikdkeevqrkrqkgss
gtsgggsgggmtleearkrvnelrdliryhnyryyvladpeisdae
ydrllrelkeleerfpelkspdsptlqvgarpleatfrpvrhptrm
ysldnafnldelkafeerieralgrkgpfaytvehkvdglsvnlyy
eegvlvygatrgdgevgeevtqnlltiptiprrlkgvperlevrge
vympieaflrlneeleergerifknprnaaagslrqkdpritakrg
lratfyalglgleeveregvatqfallhwlkekgfpvehgyaravga
egveavyqdwlkkrralpfeadgvvvkldelalwrelgytaraprf
aiaykfpaeeketrlldvvfqvgrtgrvtpvgilepvflegsevsr
vtlhnesyieeldirigdwvlvhkaggvipevlrvlkerrtgeerp
irwpetcpecghrllkegkvhrcpnplcpakrfeairhfasrkamd
iqglgeklierllekglvkdvadlyrlrkedlvglermgeksaqnl
lrqieeskkrglerllyalglpgvgevlarnlaarfgnmdrlleas
leelleveevgeltarailetlkdpafrdlvrrlkeagvemeakek
ggealkgltfvitgelsrpreevkallrrlgakvtdsvsrktsylv
vgenpgsklekaralgvptlteeelyrlleartgkkaeelv.
Ligase p50-Tth has thermostable blunt-end ligation activity at
temperatures of at least 60 C. Ligase p50-Tth is a chimeric protein
which comprises a His10-containing leader sequence, a p50 sequence
from the human NF-kappa-B protein accession number NP.sub.--003989
amino acids 40-366 (indicated in italics), a flexible glycine rich
sequence, and a Tth DNA ligase, from Thermus Thermophilus HB8,
accession YP.sub.--144363 (indicated with underlining). In some
embodiments, a modified version of p50-Tth ligase may be used with
methods and compositions provided herein (e.g. with 100 or fewer,
70 or fewer, 50 or fewer, 40 or fewer, 30 or fewer, 20 or fewer, 10
or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1
different amino acids from p50-Tth ligase).
[0112] Methods provided herein may be performed at a variety of
temperatures. In some embodiments, all steps of a method are
performed at the same temperature. Thus, temperature cycling such
as in PCR is not necessary with methods disclosed herein. In some
embodiments, methods provided herein may be performed at two or
more different temperatures. In some embodiments, a reaction
mixture containing reagents for a method provided herein is
incubated at two or more different temperatures. In some examples,
different temperatures may be selected to optimize the rate,
accuracy, or other feature of different processes of a method
provided herein. For example, a temperature may be selected to
increase the enzymatic activity of a polymerase. In some examples,
different temperatures may be selected to increase the binding
specificity of a primer to a template or to increase the
accessibility of a template to a primer (e.g. higher temperatures
may promote the separation of duplex template nucleic acids, or may
promote the sequence-specific binding of primers). In some
embodiments, all of the steps of a method provided herein are
performed at a temperature of no greater than 80, 70, 60, 50, 40,
30, 20 or 10.degree. C. In some embodiments, a method provided
herein is performed at a temperature between 20-60, 30-70, 40-80,
30-40, 35-45, 40-50, 45-55, 50-60, or 55-65.degree. C. Methods
disclosed herein may be performed with or without a
thermocycler.
[0113] As one consideration, the temperature used for a method or
process provided herein may be selected to be appropriate for the
enzyme(s) being used in the method. In some embodiments, for
methods in which a polymerase or ligase is used, the temperature(s)
of the reaction is selected such that it does not significantly
impair the activity of the polymerase or ligase (e.g. the
temperature of the reaction may be selected such that polymerase
and ligase have a half-life of at least 24, 12, 6, 4, 3, 2, 1,
0.75, 0.5, 0.25, or 0.1 hours). Alternatively, methods may be
performed at a temperature that impairs the activity of the
enzyme(s) being used in the method (e.g. the temperature of the
reaction may be selected such that an enzyme in the reaction has a
half-life of no more than 24, 12, 6, 4, 3, 2, 1, 0.75, 0.5, 0.25,
or 0.1 hours). In some embodiments, if a method is performed at a
temperature or other condition (e.g. pH) that impairs the activity
of one or more the enzymes, additional enzyme may be added to the
reaction at one or more intervals after the initiation of the
method to supplement the activity of the impaired enzyme(s).
[0114] In some embodiments, one or more processes of a method
provided herein occur in the same reaction vessel (e.g. tube, tip,
container, etc.). In some embodiments, all of the processes of a
method occur in the same reaction vessel.
[0115] Reagents for methods provided herein can all be provided
together at the start of a reaction, or they may be added
sequentially, where after one, two, or more steps new reagents are
added to a reaction. In some circumstances, new reagents (e.g.
enzymes, primers) may be added to a reaction vessel during the
course of the reaction, to increase the amount of reagents
available to act on substrates or to replace the function of
reagents that have become inactivated (e.g. enzymes). New reagents
may be added to a reaction at one or more selected time intervals
after the initiation of a reaction of a method provided herein (for
example, at 1, 3, 5, 7, 10, 15, 20, 30, 45, or 60 minutes after the
initiation of a reaction).
[0116] In some embodiments, two or more sets of first and second
primers are provided in a method provided herein, where each set
contains a first primer and a second primer, and where different
primer sets are complementary to different nucleic acid templates.
Both the first and second primers in a set are complementary to
different strands of the same nucleic acid template. Inclusion of
two or more primer sets in a method provided herein may support the
simultaneous amplification of multiple different nucleic acid
templates in the same reaction vessel. This may be useful, for
example, for amplifying multiple templates of interest in a sample,
or for assaying for the presence of multiple different templates in
a sample. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,
20, 25, 30, 40, 50, 60, 70, 100, 200, 500 or more sets of first and
second primers are provided in a method provided herein, in order
to amplify or assay for the presence of 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 15, 20, 25, 30, 40, 50, 60, 70, 100, 200, 500 or more different
nucleic acid templates.
[0117] In some embodiments, provided herein is a vessel containing
one or more enzymes, primers, or other reagents provided herein.
Vessels may include any structure capable of supporting or
containing a liquid or solid material and may include, tubes,
containers, tips, etc. In some embodiments, a wall of a vessel may
permit the transmission of light through the wall. A vessel may be
optically clear. A vessel may contain, for example, any one or more
of an isolated nucleic acid polymerase, an isolated DNA polymerase,
an isolated reverse transcriptase, an isolated ligase, a first
primer, a second primer, a nucleic acid dye, or a nucleic acid
probe, as described elsewhere herein. Any number of copies of any
of the contents of a vessel may be provided (e.g. a first copy, a
second copy, a third copy, etc.) The contents of a vessel may be in
fluid communication. In some embodiments, a vessel may further
contain a nucleic acid template. In some embodiments, a vessel may
further contain nucleotides, buffers, salts, water, or other
reagents provided herein for the amplification of nucleic acids. In
some embodiments, a vessel may contain two or more sets of primers,
wherein each primer set comprises a first and second primer, and
the different primer sets are complementary to different nucleic
acid templates.
[0118] Two or more reagents useful for a method provided herein may
be packaged and provided as a kit. For example, a kit may include
any two or more of: a nucleic acid template, a first primer, a
second primer, a nucleic acid polymerase, a DNA polymerase, a
reverse transcriptase, an isolated ligase, buffers, a nucleic acid
dyes, a nucleic acid probe, or dNTPs, as described elsewhere
herein. Within the kit, the two or more reagents may be packaged in
separate vessels or the same vessel. In some embodiments, a kit may
further contain nucleotides, buffers, salts, water, or other
reagents provided herein for the amplification of nucleic
acids.
[0119] Applications
[0120] The various methods and compositions provided herein for the
amplification of a nucleic acid template can fulfill many of the
functions that have previously been carried out by other methods
and compositions for isothermal and thermocycler-dependent nucleic
acid amplification. A nucleic acid template for amplification
according to methods provided herein may also be referred to herein
as a "target nucleic acid". Methods and compositions provided
herein may be used, for example, for isolation and cloning of
nucleic acids of interest, gene expression analysis, diagnostic
identification of nucleic acids, synthesis of novel nucleic acids,
nucleic acid probe synthesis and labeling, forensic identification
of a subject, allele identification from a subject, genetic
screening, nucleic acid sequencing, and related applications. A
target nucleic acid molecule may be of any type, including
single-stranded or double stranded and DNA or RNA (e.g. mRNA).). A
target nucleic acid may be of any type or function (e.g. a
protein-coding sequence, a regulatory sequence, an intron, etc.). A
target nucleic acid may be the entirety of a gene, or a portion
thereof. Methods provided herein may include conversion of a single
strand nucleic acid target molecule to a template linear
double-stranded nucleic acid by methods disclosed herein or
otherwise known in the art.
[0121] In some embodiments, a method or composition provided herein
may be used to detect the amount of a target nucleic acid in a
sample (including the presence or absence of the target), to
measure the amount of an amplification product of a target formed
from a sample in a selected period of time, or to determine the
amount of time necessary to generate a certain number of copies of
a template from a sample. Samples which may be used with methods
and compositions provided herein are described elsewhere herein,
and may include, for example, a bodily fluid, a secretion, or a
tissue of a subject.
[0122] In some embodiments, a method provided herein may be
performed to simultaneously assay for at least 2, 3, 4, 5, 6, 7, 8,
9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, or more
different target nucleic acids in the same reaction vessel.
Typically, for each target nucleic acid of interest, a first primer
and a second primer are provided, each being complementary to a
strand of the nucleic acid target, or a complement thereof. The
amplification of the different target nucleic acids in the same
vessel may be monitored, for example, by the use of nucleic acid
probes having sequence specificity for detection sequences in the
different target nucleic acids, and different fluorophores.
[0123] In some embodiments, a method or composition provided herein
may be used to detect the presence or absence of a particular
nucleotide of interest in a target nucleic acid (e.g. in the case
of a mutation or SNP). For example, a first or second primer may be
selected which selectively binds to a region in a target nucleic
acid which includes or is adjacent to the nucleotide of interest.
The primer may be designed such that it selectively either: i)
binds to the region when the region contains the nucleotide of
interest, or ii) does not bind to the region when the region
contains the nucleotide of interest. A method as described herein
may be performed with the selected primer, and the outcome of the
amplification reaction may provide information regarding the
presence or absence of the nucleotide of interest in the target
nucleic acid. For example, if a first primer is designed to have a
nucleotide sequence which is complementary to a sequence in the
target nucleic acid which includes a particular nucleotide of
interest (e.g. a mutation), successful amplification of the target
nucleic acid with the selected primer from a sample may indicate
that the sample contains a target nucleic acid having the
particular nucleotide of interest. In some embodiments, a primer
used for analysis of a nucleotide of interest in a target nucleic
acid may contain a critical nucleotide at the 3' terminus of the
primer. In such a case, the annealing of the 3' terminal nucleotide
of the primer may be dependent on the presence of the nucleotide of
interest in the target nucleic acid. If the 3' terminal nucleotide
of the primer does not anneal with a nucleotide in the target
nucleic acid (e.g. due to a mismatch between the nucleotides), the
mismatch may significantly impair a nucleic acid polymerase from
synthesizing an extension product from the primer. Accordingly, in
some embodiments, a primer having a 3' terminal nucleotide which
corresponds to a nucleotide of interest may be useful for
determining the presence or absence of a particular nucleotide in a
target nucleic acid.
[0124] Methods and compositions provided herein may be used to
amplify a nucleic acid from any sample which may contain nucleic
acids. Examples of samples may include various fluid samples. In
some instances, the sample may be a bodily fluid sample from a
subject. The sample may include one or more fluid component. In
some instances, solid or semi-solid samples may be provided. The
sample may include tissue collected from the subject. The sample
may include a bodily fluid, secretion, or tissue of a subject. The
sample may be a biological sample. The biological sample may be a
bodily fluid, a secretion, or a tissue sample. Examples of
biological samples may include but are not limited to, blood,
serum, saliva, urine, gastric and digestive fluid, tears, stool,
semen, vaginal fluid, interstitial fluids derived from tumorous
tissue, ocular fluids, sweat, mucus, earwax, oil, glandular
secretions, breath, spinal fluid, hair, fingernails, skin cells,
plasma, nasal swab or nasopharyngeal wash, spinal fluid, cerebral
spinal fluid, tissue, throat swab, biopsy, placental fluid,
amniotic fluid, cord blood, emphatic fluids, cavity fluids, sputum,
pus, microbiota, meconium, breast milk or other excretions. The
sample may be provided from a human or animal. Samples may be from
a plant, microorganism (e.g. virus, bacteria), or other biological
material.
[0125] In some embodiments, methods and compositions provided
herein may be performed at or used at point of service locations
(e.g. a subject's home or work, grocery stores, drug stores,
clinics, schools, etc.). Methods and compositions provided herein
may permit the rapid amplification of nucleic acids in a sample
from a subject, in order to aid in the diagnosis or treatment of a
subject.
[0126] The assays and methods disclosed herein may be performed on
a device, or on a system, for processing a sample. The assays and
methods disclosed herein can be readily incorporated into and used
in device for processing a sample, or a system for processing a
sample, which may be an automated assay device, or may be an
automated assay system. Such a device, and such a system, may be
useful for the practice of the methods disclosed herein. For
example, a device may be useful for receiving a sample. A device
may be useful for preparing, or for processing a sample. A device
may be useful for performing an assay on a sample. A device may be
useful for obtaining data from a sample. A device may be useful for
transmitting data obtained from a sample. A device may be useful
for disposing of a sample following processing or assaying of a
sample.
[0127] A device may be part of a system, a component of which may
be a sample processing device. A device may be a sample processing
device. A sample processing device may be configured to facilitate
collection of a sample, prepare a sample for a clinical test, or
perform a method with one or more reagents, as disclosed herein. A
sample processing device may be configured to obtain data from a
sample. A sample processing device may be configured to transmit
data obtained from a sample. A sample processing device may be
configured to analyze data from a sample. A sample processing
device may be configured to communicate with another device, or a
laboratory, or an individual affiliated with a laboratory, to
analyze data obtained from a sample.
[0128] A sample processing device may be configured to be placed in
or on a subject. A sample processing device may be configured to
accept a sample from a subject, either directly or indirectly. A
sample may be, for example, a blood sample (e.g., a sample obtained
from a fingerstick, or from venipuncture, or an arterial blood
sample), a urine sample, a biopsy sample, a tissue slice, stool
sample, or other biological sample; a water sample, a soil sample,
a food sample, an air sample; or other sample. A blood sample may
comprise, e.g., whole blood, plasma, or serum. A sample processing
device may receive a sample from the subject through a housing of
the device. The sample collection may occur at a sample collection
site, or elsewhere. The sample may be provided to the device at a
sample collection site.
[0129] In some embodiments, a sample processing device may be
configured to accept or hold a cartridge. In some embodiments, a
sample processing device may comprise a cartridge. The cartridge
may be removable from the sample processing device. In some
embodiments, a sample may be provided to the cartridge of the
sample processing device. Alternatively, a sample may be provided
to another portion of a sample processing device. The cartridge
and/or device may comprise a sample collection unit that may be
configured to accept a sample.
[0130] A cartridge may include a sample, and may include reagents
for use in processing or testing a sample, disposables for use in
processing or testing a sample, or other materials. A cartridge may
contain reagents disclosed herein for the performing a method
disclosed herein. Following placement of a cartridge on, or
insertion of a cartridge into, a sample processing device, one or
more components of the cartridge may be brought into fluid
communication with other components of the sample processing
device. For example, if a sample is collected at a cartridge, the
sample may be transferred to other portions of the sample
processing device. Similarly, if one or more reagents are provided
on a cartridge, the reagents may be transferred to other portions
of the sample processing device, or other components of the sample
processing device may be brought to the reagents. In some
embodiments, the reagents or components of a cartridge may remain
on-board the cartridge. In some embodiments, no fluidics are
included that require tubing or that require maintenance (e.g.,
manual or automated maintenance).
[0131] A sample or reagent may be transferred to a device, such as
a sample processing device. A sample or reagent may be transferred
within a device. Such transfer of sample or reagent may be
accomplished without providing a continuous fluid pathway from
cartridge to device. Such transfer of sample or reagent may be
accomplished without providing a continuous fluid pathway within a
device. In embodiments, such transfer of sample or reagent may be
accomplished by a sample handling system (e.g., a pipette); for
example, a sample, reagent, or aliquot thereof may be aspirated
into an open-tipped transfer component, such as a pipette tip,
which may be operably connected to a sample handling system which
transfers the tip, with the sample, reagent, or aliquot thereof
contained within the tip, to a location on or within the sample
processing device. The sample, reagent, or aliquot thereof can be
deposited at a location on or within the sample processing device.
Sample and reagent, or multiple reagents, may be mixed using a
sample handling system in a similar manner. One or more components
of the cartridge may be transferred in an automated fashion to
other portions of the sample processing device, and vice versa.
[0132] A device, such as a sample processing device, may have a
fluid handling system. A fluid handling system may perform, or may
aid in performing, transport, dilution, extraction, aliquotting,
mixing, and other actions with a fluid, such as a sample. In some
embodiments, a fluid handling system may be contained within a
device housing. A fluid handling system may permit the collection,
delivery, processing and/or transport of a fluid, dissolution of
dry reagents, mixing of liquid and/or dry reagents with a liquid,
as well as collection, delivery, processing and/or transport of
non-fluidic components, samples, or materials. The fluid may be a
sample, a reagent, diluent, wash, dye, or any other fluid that may
be used by the device, and may include, but not limited to,
homogenous fluids, different liquids, emulsions, suspensions, and
other fluids. A fluid handling system, including without limitation
a pipette, may also be used to transport vessels (with or without
fluid contained therein) around the device. The fluid handling
system may dispense or aspirate a fluid. The sample may include one
or more particulate or solid matter floating within a fluid.
[0133] In embodiments, a fluid handling system may comprise a
pipette, pipette tip, syringe, capillary, or other component. The
fluid handling system may have portion with an interior surface and
an exterior surface and an open end. The fluid handling system may
comprise a pipette, which may include a pipette body and a pipette
nozzle, and may comprise a pipette tip. A pipette tip may or may
not be removable from a pipette nozzle. In embodiments, a fluid
handling system may use a pipette mated with a pipette tip; a
pipette tip may be disposable. A tip may form a fluid-tight seal
when mated with a pipette. A pipette tip may be used once, twice,
or more times. In embodiments, a fluid handling system may use a
pipette or similar device, with or without a pipette tip, to
aspirate, dispense, mix, transport, or otherwise handle the fluid.
The fluid may be dispensed from the fluid handling system when
desired. The fluid may be contained within a pipette tip prior to
being dispensed, e.g., from an orifice in the pipette tip. In
embodiments, or instances during use, all of the fluid may be
dispensed; in other embodiments, or instances during use, a portion
of the fluid within a tip may be dispensed. A pipette may
selectively aspirate a fluid. The pipette may aspirate a selected
amount of fluid. The pipette may be capable of actuating stirring
mechanisms to mix the fluid within the tip or within a vessel. The
pipette may incorporate tips or vessels creating continuous flow
loops for mixing, including of materials or reagents that are in
non-liquid form. A pipette tip may also facilitate mixture by
metered delivery of multiple fluids simultaneously or in sequence,
such as in 2-part substrate reactions.
[0134] The fluid handling system may include one or more
fluidically isolated or hydraulically independent units. For
example, the fluid handling system may include one, two, or more
pipette tips. The pipette tips may be configured to accept and
confine a fluid. The tips may be fluidically isolated from or
hydraulically independent of one another. The fluid contained
within each tip may be fluidically isolated or hydraulically
independent from one fluids in other tips and from other fluids
within the device. The fluidically isolated or hydraulically
independent units may be movable relative to other portions of the
device and/or one another. The fluidically isolated or
hydraulically independent units may be individually movable. A
fluid handling system may comprise one or more base or support. A
base or support may support one or more pipette or pipette units. A
base or support may connect one or more pipettes of the fluid
handling system to one another.
[0135] A sample processing device may be configured to perform
processing steps or actions on a sample obtained from a subject.
Sample processing may include sample preparation, including, e.g.,
sample dilution, division of a sample into aliquots, extraction,
contact with a reagent, filtration, separation, centrifugation, or
other preparatory or processing action or step. A sample processing
device may be configured to perform one or more sample preparation
action or step on the sample. Optionally, a sample may be prepared
for a chemical reaction and/or physical processing step. A sample
preparation action or step may include one or more of the
following: centrifugation, separation, filtration, dilution,
enriching, purification, precipitation, incubation, pipetting,
transport, chromatography, cell lysis, cytometry, pulverization,
grinding, activation, ultrasonication, micro column processing,
processing with magnetic beads, processing with nanoparticles, or
other sample preparation action or steps. For example, sample
preparation may include one or more step to separate blood into
serum and/or particulate fractions, or to separate any other sample
into various components. Sample preparation may include one or more
step to dilute and/or concentrate a sample, such as a blood sample,
or other biological samples. Sample preparation may include adding
an anti-coagulant or other ingredients to a sample. Sample
preparation may also include purification of a sample. In
embodiments, all sample processing, preparation, or assay actions
or steps are performed by a single device. In embodiments, all
sample processing, preparation, or assay actions or steps are
performed within a housing of a single device. In embodiments, most
sample processing, preparation, or assay actions or steps are
performed by a single device, and may be performed within a housing
of a single device. In embodiments, many sample processing,
preparation, or assay actions or steps are performed by a single
device, and may be performed within a housing of a single device.
In embodiments, sample processing, preparation, or assay actions or
steps may be performed by more than one device.
[0136] A sample processing device may be configured to run one or
more assays on a sample, and to obtain data from the sample. A
sample processing device may perform methods provided herein, as
well as additional assays. An assay may include one or more
physical or chemical treatments, and may include running one or
more chemical or physical reactions. A sample processing device may
be configured to perform one, two or more assays on a small sample
of bodily fluid. One or more chemical reaction may take place on a
sample having a volume, as described elsewhere herein. For example
one or more chemical reaction may take place in a pill having less
than femtoliter volumes. In an instance, the sample collection unit
is configured to receive a volume of the bodily fluid sample
equivalent to a single drop or less of blood or interstitial fluid.
In embodiments, the volume of a sample may be a small volume, where
a small volume may be a volume that is less than about 1000 .mu.L,
or less than about 500 .mu.L, or less than about 250 .mu.L, or less
than about 150 .mu.L, or less than about 100 .mu.L, or less than
about 75 .mu.L, or less than about 50 .mu.L, or less than about 40
.mu.L, or less than about 20 .mu.L, or less than about 10 .mu.L,
less than about 5 .mu.L, less than about 1 .mu.L, less than about
0.5 .mu.L, less than about 0.1 .mu.L, or other small volume. In
embodiments, all sample assay actions or steps are performed on a
single sample. In embodiments, all sample assay actions or steps
are performed by a single device. In embodiments, all sample assay
actions or steps are performed within a housing of a single device.
In embodiments, most sample assay actions or steps are performed by
a single device, and may be performed within a housing of a single
device. In embodiments, many sample assay actions or steps are
performed by a single device, and may be performed within a housing
of a single device. In embodiments, sample processing, preparation,
or assay actions or steps may be performed by more than one
device.
[0137] A sample processing device may be configured to perform a
plurality of assays on a sample. In some embodiments, a sample
processing device may be configured to perform a method provided
herein and one, two, or more additional assays. In embodiments, a
sample processing device may be configured to perform a plurality
of assays on a single sample. In embodiments, a sample processing
device may be configured to perform a plurality of assays on a
single sample, where the sample is a small sample. For example, a
small sample may have a sample volume that is a small volume of
less than about 1000 .mu.L, or less than about 500 .mu.L, or less
than about 250 .mu.L, or less than about 150 .mu.L, or less than
about 100 .mu.L, or less than about 75 .mu.L, or less than about 50
.mu.L, or less than about 40 .mu.L, or less than about 20 .mu.L, or
less than about 10 .mu.L, less than about 5 .mu.L, less than about
1 .mu.L, less than about 0.5 .mu.L, less than about 0.1 .mu.L, or
other small volume. A sample processing device may be capable of
performing multiplexed assays on a single sample. A plurality of
assays may be run simultaneously; may be run sequentially; or some
assays may be run simultaneously while others are run sequentially.
One or more control assays and/or calibrators (e.g., including a
configuration with a control of a calibrator for the assay/tests)
can also be incorporated into the device; control assays and assay
on calibrators may be performed simultaneously with assays
performed on a sample, or may be performed before or after assays
performed on a sample, or any combination thereof. In embodiments,
all sample assay actions or steps are performed by a single device.
In embodiments, all of a plurality of assay actions or steps are
performed within a housing of a single device. In embodiments, most
sample assay actions or steps, of a plurality of assays, are
performed by a single device, and may be performed within a housing
of a single device. In embodiments, many sample assay actions or
steps, of a plurality of assays, are performed by a single device,
and may be performed within a housing of a single device. In
embodiments, sample processing, preparation, or assay actions or
steps may be performed by more than one device.
[0138] In embodiments, all of a plurality of assays may be
performed in a short time period. In embodiments, such a short time
period comprises less than about three hours, or less than about
two hours, or less than about one hour, or less than about 40
minutes, or less than about 30 minutes, or less than about 25
minutes, or less than about 20 minutes, or less than about 15
minutes, or less than about 10 minutes, or less than about 5
minutes, or less than about 4 minutes, or less than about 3
minutes, or less than about 2 minutes, or less than about 1 minute,
or other short time period.
[0139] A sample processing device may be configured to detect one
or more signals relating to the sample. A sample processing device
may be configured to identify one or more properties of the sample.
For instance, the sample processing device may be configured to
detect the presence or concentration of one analyte (e.g. a target
nucleic acid) or a plurality of analytes or a disease condition in
the sample (e.g., in or through a bodily fluid, secretion, tissue,
or other sample). Alternatively, the sample processing device may
be configured to detect a signal or signals that may be analyzed to
detect the presence or concentration of one or more analytes (which
may be indicative of a disease condition) or a disease condition in
the sample. The signals may be analyzed on board the device, or at
another location. Running a clinical test may or may not include
any analysis or comparison of data collected.
[0140] A chemical reaction or other processing step may be
performed, with or without the sample. Examples of steps, tests, or
assays that may be prepared or run by the device may include, but
are not limited to immunoassay, nucleic acid assay (e.g. methods
provided herein), receptor-based assay, cytometric assay,
colorimetric assay, enzymatic assay, electrophoretic assay,
electrochemical assay, spectroscopic assay, chromatographic assay,
microscopic assay, topographic assay, calorimetric assay,
turbidimetric assay, agglutination assay, radioisotope assay,
viscometric assay, coagulation assay, clotting time assay, protein
synthesis assay, histological assay, culture assay, osmolarity
assay, and/or other types of assays, centrifugation, separation,
filtration, dilution, enriching, purification, precipitation,
pulverization, incubation, pipetting, transport, cell lysis, or
other sample preparation action or steps, or combinations thereof.
Steps, tests, or assays that may be prepared or run by the device
may include imaging, including microscopy, cytometry, and other
techniques preparing or utilizing images. Steps, tests, or assays
that may be prepared or run by the device may further include an
assessment of histology, morphology, kinematics, dynamics, and/or
state of a sample, which may include such assessment for cells.
[0141] A device may be capable of performing all on-board steps
(e.g., steps or actions performed by a single device) in a short
amount of time. A device may be capable of performing all on-board
steps on a single sample in a short amount of time. For example,
from sample collection from a subject to transmitting data and/or
to analysis may take about 3 hours or less, 2 hours or less, 1 hour
or less, 50 minutes or less, 45 minutes or less, 40 minutes or
less, 30 minutes or less, 20 minutes or less, 15 minutes or less,
10 minutes or less, 5 minutes or less, 4 minutes or less, 3 minutes
or less, 2 minutes or less, or 1 minute or less. The amount of time
from accepting a sample within the device to transmitting data
and/or to analysis from the device regarding such a sample may
depend on the type or number of steps, tests, or assays performed
on the sample. The amount of time from accepting a sample within
the device to transmitting data and/or to analysis from the device
regarding such a sample may take about 3 hours or less, 2 hours or
less, 1 hour or less, 50 minutes or less, 45 minutes or less, 40
minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes
or less, 10 minutes or less, 5 minutes or less, 4 minutes or less,
3 minutes or less, 2 minutes or less, or 1 minute or less.
[0142] A device may be configured to prepare a sample for disposal,
or to dispose of a sample, such as a biological sample, following
processing or assaying of a sample.
[0143] In embodiments, a sample processing device may be configured
to transmit data obtained from a sample. In embodiments, a sample
processing device may be configured to communicate over a network.
A sample processing device may include a communication module that
may interface with the network. A sample processing device may be
connected to the network via a wired connection or wirelessly. The
network may be a local area network (LAN) or a wide area network
(WAN) such as the Internet. In some embodiments, the network may be
a personal area network. The network may include the cloud. The
sample processing device may be connected to the network without
requiring an intermediary device, or an intermediary device may be
required to connect a sample processing device to a network. A
sample processing device may communicate over a network with
another device, which may be any type of networked device,
including but not limited to a personal computer, server computer,
or laptop computer; personal digital assistants (PDAs) such as a
Windows CE device; phones such as cellular phones, smartphones
(e.g., iPhone, Android, Blackberry, etc.), or location-aware
portable phones (such as GPS); a roaming device, such as a
network-connected roaming device; a wireless device such as a
wireless email device or other device capable of communicating
wireless with a computer network; or any other type of network
device that may communicate possibly over a network and handle
electronic transactions. Such communication may include providing
data to a cloud computing infrastructure or any other type of data
storage infrastructure which may be accessed by other devices.
[0144] A sample processing device may provide data regarding a
sample to, e.g., a health care professional, a health care
professional location, such as a laboratory, or an affiliate
thereof. One or more of a laboratory, health care professional, or
subject may have a network device able to receive or access data
provided by the sample processing device. A sample processing
device may be configured to provide data regarding a sample to a
database. A sample processing device may be configured to provide
data regarding a sample to an electronic medical records system, to
a laboratory information system, to a laboratory automation system,
or other system or software. A sample processing device may provide
data in the form of a report.
[0145] A laboratory, device, or other entity or software may
perform analysis on data regarding a sample in real-time. A
software system may perform chemical analysis and/or pathological
analysis, or these could be distributed amongst combinations of
lab, clinical, and specialty or expert personnel. Analysis may
include qualitative and/or quantitative evaluation of a sample.
Data analysis may include a subsequent qualitative and/or
quantitative evaluation of a sample. Optionally, a report may be
generated based on raw data, pre-processed data, or analyzed data.
Such a report may be prepared so as to maintain confidentiality of
the data obtained from the sample, the identity and other
information regarding the subject from whom a sample was obtained,
analysis of the data, and other confidential information. The
report and/or the data may be transmitted to a health care
professional. Data obtained by a sample processing device, or
analysis of such data, or reports, may be provided to a database,
an electronic medical records system, to a laboratory information
system, to a laboratory automation system, or other system or
software.
[0146] Description and disclosure of examples of reagents, assays,
methods, kits, devices, and systems which may use, or be used with,
methods, compositions, or other reagents disclosed herein may be
found, for example, in U.S. Pat. No. 8,088,593; U.S. Pat. No.
8,380,541; U.S. patent application Ser. No. 13/769,798, filed Feb.
18, 2013; U.S. patent application Ser. No. 13/769,779, filed Feb.
18, 2013; U.S. patent application Ser. No. 13/244,947 filed Sep.
26, 2011; PCT/US2012/57155, filed Sep. 25, 2012; U.S. application
Ser. No. 13/244,946, filed Sep. 26, 2011; U.S. patent application
Ser. No. 13/244,949, filed Sep. 26, 2011; and U.S. Application Ser.
No. 61/673,245, filed Sep. 26, 2011, the disclosures of which
patents and patent applications are all hereby incorporated by
reference in their entireties.
EXAMPLES
[0147] The following examples are offered for illustrative purposes
only, and are not intended to limit the present disclosure in any
way.
Example 1
[0148] A method as provided herein was used to amplify a target
nucleic acid. Reactions were prepared to assay for a target nucleic
acid from T102A1, which is a DNA molecule from Staphylococcus
aureus, subspecies SSCmec. T102A1 has the sequence 5'
CAACTAATGAAACAGAAAGTCGTAACTATCCTCTAGAAAAAGCGACTTCACATCTAT
TAGGTTATGTTGGTCCCATTAACTCTGAAGAATTAAAACAAAAAGAATATAAAGGCT
ATAAAGATGATGCAGTTATTGGTAAAAAGGGACTCGAAAAACTTTACGATAAAAAG
CTCCAACATGAAGATGGCTATCGTGTCACAATCGTTGACGATAATAGCAATACAATC
GCACATACATTAATAGAGAAAAAGAAAAAAGATGGCAAAGATATTCAACTAACTAT
TGATGCTAAAGTTCAAAAGAGTATTTATAACAACATGAAAAATGATTATGGCTCAG
GTACTGCTATCCACCCTCAAACAGGTGAATTATTAGCACTTGTAAGCACACCTTCAT
ATGACGTCTATCCATTTATGTATGCATGAGTAACGAAGAATATAATAAATTAACCGA
AGATAAAAAAGAACCTCTGCTCAACAAGTTCCAGATTACAACTTCACCAGGTTCAAC
TCAAAAAATATTAACAGCA 3'.
[0149] First primer "RLX0513" (nucleotide sequence: 5'
GGCTCAGGTACTGCTATCCACCC 3') and second primer "RLX0514" (nucleotide
sequence: 5' TTTTGAGTTGAACCTGGTGAAGTTG 3') were used to amplify a
target sequence from T102A1.
[0150] 25 microliter reaction mixtures were prepared, each
containing: 50 mM potassium acetate, 20 mM Tris-acetate, pH 7.9, 10
mM magnesium acetate, 1 mM DTT, 0.2 .mu.g/.mu.l bovine serum
albumin (BSA), 0.8 M betaine, 1.4 mM each of dATP, dTTP, dGTP, and
dCTP, 1 mM rATP, 1.times.SYTO.RTM. 59 (Life Technologies), 1.2
units/.mu.l Bst DNA polymerase (New England BioLabs), 20
units/.mu.l T4 DNA ligase (New England BioLabs), 0.8 .mu.M of first
primer RLX0513, 0.8 .mu.M of second primer RLX0514, and 0, 100,
1,000, 10,000, 100,000, or 1,000,000 copies T102A1 template per
microliter. The template was preheated at 85 C for 5 minutes and
cooled on ice for 5 minutes before addition to the reaction
mixture. Upon addition of the template, reaction mixtures were
incubated at 42 C for 15 minutes, followed by at 56 C for 90
minutes in a CFX 96 Touch instrument (Bio-Rad). Under these
reaction conditions, at 42 C, ligation reactions are relatively
favored; at 56 C, polymerase reactions are relatively favored. The
inflection points for the assays were determined using a
single-threshold method with CFX Manager software (Bio-Rad), and
are shown in FIG. 2. The X-axis provides the concentration of
template molecules/microliter, and the Y-axis provides the
inflection time (in minutes) of the assay. As shown in FIG. 2, each
of the reactions, including a few as 100 copies template/microliter
have inflection times significantly faster than the no template
control (NTC) reaction. No template control reactions sometimes
show an inflection time; this is due to background non-specific
products that are formed over time. The specific inflection times
for the reactions are provided below in Table 1. "Cq" refers to the
quantification cycle value (time) of the inflection point.
TABLE-US-00002 TABLE 1 RLX513/514 T102A1 Cq st. dev 10{circumflex
over ( )}6 44.29 3.53 10{circumflex over ( )}5 50.27 4.83
10{circumflex over ( )}4 59.11 1.07 10{circumflex over ( )}3 62.19
6.74 10{circumflex over ( )}2 73.36 1.41 NTC 92.75 8.14
Example 2
[0151] A method as provided herein was used to amplify a target
nucleic acid. Reactions were prepared to assay for a target nucleic
acid from TH1SC, which is a mRNA molecule from hemagglutinin gene,
from influenza A virus, H1N1. TH1SC mRNA has the sequence: 5'
CGCCGGAUGGCUCUUGGGAAACCGAAGACAGCCACAACGGGAAACUAUGUAAAU
UAAAAGGAAUAGCCCCACUACAAUUGGGGAAAUGUAACAUCGCCGGAUGGCUCU
UGGGAAACCCAGAAUGCGACUCACUGCUUCCAGCGAAAUCAUGGUCCUACAUUGU
AGAAACACCAAACUCUGAGAAUGGAGCAUGUUAUCCAGGAGAUUUCAUCGACUA
UGAGGAACUGAAGGAGCAAUUGAGCUCAGUAUCAUCAUUAGAAAGAUUCGAAAU
AUUUCCCAAGGAAAGUUCAUGGCCCAACCACAACACACUCAAAGGAGUAACAGCA
GCAUGCUCCCAUAGGGGAAAAAGCAGUUUUUACAGAAAUUUGCUAUGGCUGACG
AAAACGGGGGACUCAUACCCAAAGCUGAACAAUUCCUAUGUGAACAAUAAAGGG AAAGAAGUC
3.
[0152] First primer "RLX0585" (nucleotide sequence: 5'
CGCCGGATGGCTCTTGGGAAACC 3') and second primer "RLX0586" (nucleotide
sequence: 5' TCGCTGGAAGCAGTGAGTCGCATTC 3') were used to amplify a
target sequence from TH1SC.
[0153] 25 microliter reaction mixtures were prepared, each
containing: 50 mM potassium acetate, 20 mM Tris-acetate, pH 7.9, 10
mM magnesium acetate, 1 mM DTT, 0.2 .mu.g/.mu.l bovine serum
albumin (BSA), 0.8 M betaine, 1.4 mM each of dATP, dTTP, dGTP, and
dCTP, 1 mM rATP, 1.times.SYTO.RTM. 59 (Life Technologies), 1.2
units/.mu.l Bst DNA polymerase (New England BioLabs), 20
units/.mu.l T4 DNA ligase (New England BioLabs), 0.016 units/.mu.l
AMV reverse trancriptase enzyme (New England Biolabs), 1 unit/.mu.l
murine RNase inhibitor (New England Biolabs), 0.8 .mu.M of first
primer RLX0585, 0.8 .mu.M of second primer RLX0586, and 0, 100,
1,000, or 10,000 copies TH1SC template per microliter. The reaction
mixtures were prepared in triplicate for the different quantities
of TH 1 SC template. The template was preheated at 85 C for 5
minutes and cooled on ice for 5 minutes before addition to the
reaction mixture. Upon addition of the template, reaction mixtures
were incubated at 42 C for 15 minutes, followed by at 65 C for 90
minutes in a CFX 96 Touch instrument (Bio-Rad). Under these
reaction conditions, at 42 C, ligation reactions are relatively
favored; at 65 C, polymerase reactions are relatively favored. The
inflection points for the assays were determined using a
single-threshold method with CFX Manager software (Bio-Rad), and
are shown in FIG. 3. The X-axis provides the concentration of
template molecules/microliter, and the Y-axis provides the
inflection time (in minutes) of the assay. As shown in FIG. 3, each
of the template-containing reactions has an inflection time under
40 minutes.
Example 3
[0154] A method as provided herein was used to amplify a target
nucleic acid. Reactions were prepared to assay for a target nucleic
acid from T129, which is a mRNA molecule from hemagglutinin gene,
HA1 domain (segment 4), from influenza B virus. T129 mRNA has the
sequence: 5' CUUCUUGAAUUUGAUGUCUAAGAGUAAUUUGCCAACGUGAGGCCAUCAGAAAGU
AUGGUGCGCAAGUGGCAGGAGCAAGGUAAUAAAAGGGUCCUUGCCUUUAAUUGG
UGAAGCAGAUUGCCUCCACGAAAAAUACGGUGGAUUAAACAAAAGCAAGCCUUA
CUACACAGGAGAACAUGCAAAAGCCAUAGGAAAUUGCCCAAUAUGGGUGAAAAC
ACCCUUGAAGCUGGCCAAUGGAACCAAAUAUAGACCGCCUGCAAAACUAUUAAAG
GAAAGAGGUUUCUUCGGAAGCGACAGACAGUAACACUCAACUCUCGACCAUCUGG
UGUAACAACCUCG 3'.
[0155] First primer "RLX0479" (nucleotide sequence: 5'
CGGTGGATTAAACAAAAGCAAGCC 3') and second primer "RLX0480"
(nucleotide sequence: 5' ATTGGCCAGCTTCAAGGGTG 3') were used to
amplify a target sequence from T129.
[0156] 25 microliter reaction mixtures were prepared, each
containing: 50 mM potassium acetate, 20 mM Tris-acetate, pH 7.9, 10
mM magnesium acetate, 1 mM DTT, 0.2 .mu.g/.mu.l bovine serum
albumin (BSA), 0.8 M betaine, 1.4 mM each of dATP, dTTP, dGTP, and
dCTP, 1 mM rATP, 1.times.SYTO.RTM. 59 (Life Technologies), 1.2
units/.mu.l Bst DNA polymerase (New England BioLabs), 20
units/.mu.l T4 DNA ligase (New England BioLabs), 0.016 units/.mu.l
AMV reverse trancriptase enzyme (New England Biolabs), 1 unit/.mu.l
murine RNase inhibitor (New England Biolabs), 0.8 .mu.M of first
primer RLX0479, 0.8 .mu.M of second primer RLX0480, and 0, 100,
1,000, 10,000, 100,000, or 1,000,000 copies T129 template per
microliter. Six replicates of the reaction mixture was prepared for
each of the different quantities of T129 template. The template was
preheated at 85 C for 5 minutes and cooled on ice for 5 minutes
before addition to the reaction mixture. Upon addition of the
template, reaction mixtures were incubated at 42 C for 15 minutes,
followed by at 65 C for 90 minutes in a CFX 96 Touch instrument
(Bio-Rad). Under these reaction conditions, at 42 C, ligation
reactions are relatively favored; at 65 C, polymerase reactions are
relatively favored. The inflection points for the assays were
determined using a single-threshold method with CFX Manager
software (Bio-Rad), and are shown in FIG. 4. The X-axis provides
the concentration of template molecules/microliter, and the Y-axis
provides the inflection time (in minutes) of the assay. As shown in
FIG. 4, each of the template-containing reactions has an inflection
time under 40 minutes and significantly faster than the no template
control (NTC) reaction.
[0157] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. Any feature, whether preferred or not,
may be combined with any other feature, whether preferred or not.
It should also be understood that while the invention provided
herein has been described herein using a limited number of terms
and phrases for purposes of expediency, the invention could also be
described using other terms and phrases not provided herein which
also accurately describe the invention. The appended claims are not
to be interpreted as including means-plus-function limitations,
unless such a limitation is explicitly recited in a given claim
using the phrase "means for." It should be understood that as used
in the description herein and throughout the claims that follow,
the meaning of "a," "an," and "the" includes plural reference
unless the context clearly dictates otherwise. For example, a
reference to "an assay" may refer to a single assay or multiple
assays. Also, as used in the description herein and throughout the
claims that follow, the meaning of "in" includes "in" and "on"
unless the context clearly dictates otherwise. As used in the
description herein and through the claims that follow, a first
object described as containing "at least a portion" of a second
object may contain the full amount of/the complete second object.
As used in the description herein and throughout the claims that
follow, the terms "comprise", "include", and "contain" and related
tenses are inclusive and open-ended, and do not exclude additional,
unrecited elements or method steps. Finally, as used in the
description herein and throughout the claims that follow, the
meaning of "or" includes both the conjunctive and disjunctive
unless the context expressly dictates otherwise. Thus, the term
"or" includes "and/or" unless the context expressly dictates
otherwise.
[0158] This document contains material subject to copyright
protection. The copyright owner (Applicant herein) has no objection
to facsimile reproduction by anyone of the patent documents or the
patent disclosure, as they appear in the US Patent and Trademark
Office patent file or records, but otherwise reserves all copyright
rights whatsoever. The following notice shall apply: Copyright 2013
Theranos, Inc.
Sequence CWU 1
1
1111038PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 1Met Gly His His His His His His His His His
His Ser Ser Gly His 1 5 10 15 Ile Glu Gly Arg Ala Ser Ala Asp Gly
Pro Tyr Leu Gln Ile Leu Glu 20 25 30 Gln Pro Lys Gln Arg Gly Phe
Arg Phe Arg Tyr Val Cys Glu Gly Pro 35 40 45 Ser His Gly Gly Leu
Pro Gly Ala Ser Ser Glu Lys Asn Lys Lys Ser 50 55 60 Tyr Pro Gln
Val Lys Ile Cys Asn Tyr Val Gly Pro Ala Lys Val Ile 65 70 75 80 Val
Gln Leu Val Thr Asn Gly Lys Asn Ile His Leu His Ala His Ser 85 90
95 Leu Val Gly Lys His Cys Glu Asp Gly Ile Cys Thr Val Thr Ala Gly
100 105 110 Pro Lys Asp Met Val Val Gly Phe Ala Asn Leu Gly Ile Leu
His Val 115 120 125 Thr Lys Lys Lys Val Phe Glu Thr Leu Glu Ala Arg
Met Thr Glu Ala 130 135 140 Cys Ile Arg Gly Tyr Asn Pro Gly Leu Leu
Val His Pro Asp Leu Ala 145 150 155 160 Tyr Leu Gln Ala Glu Gly Gly
Gly Asp Arg Gln Leu Gly Asp Arg Glu 165 170 175 Lys Glu Leu Ile Arg
Gln Ala Ala Leu Gln Gln Thr Lys Glu Met Asp 180 185 190 Leu Ser Val
Val Arg Leu Met Phe Thr Ala Phe Leu Pro Asp Ser Thr 195 200 205 Gly
Ser Phe Thr Arg Arg Leu Glu Pro Val Val Ser Asp Ala Ile Tyr 210 215
220 Asp Ser Lys Ala Pro Asn Ala Ser Asn Leu Lys Ile Val Arg Met Asp
225 230 235 240 Arg Thr Ala Gly Cys Val Thr Gly Gly Glu Glu Ile Tyr
Leu Leu Cys 245 250 255 Asp Lys Val Gln Lys Asp Asp Ile Gln Ile Arg
Phe Tyr Glu Glu Glu 260 265 270 Glu Asn Gly Gly Val Trp Glu Gly Phe
Gly Asp Phe Ser Pro Thr Asp 275 280 285 Val His Arg Gln Phe Ala Ile
Val Phe Lys Thr Pro Lys Tyr Lys Asp 290 295 300 Ile Asn Ile Thr Lys
Pro Ala Ser Val Phe Val Gln Leu Arg Arg Lys 305 310 315 320 Ser Asp
Leu Glu Thr Ser Glu Pro Lys Pro Phe Leu Tyr Tyr Pro Glu 325 330 335
Ile Lys Asp Lys Glu Glu Val Gln Arg Lys Arg Gln Lys Gly Ser Ser 340
345 350 Gly Thr Ser Gly Gly Gly Ser Gly Gly Gly Met Thr Leu Glu Glu
Ala 355 360 365 Arg Lys Arg Val Asn Glu Leu Arg Asp Leu Ile Arg Tyr
His Asn Tyr 370 375 380 Arg Tyr Tyr Val Leu Ala Asp Pro Glu Ile Ser
Asp Ala Glu Tyr Asp 385 390 395 400 Arg Leu Leu Arg Glu Leu Lys Glu
Leu Glu Glu Arg Phe Pro Glu Leu 405 410 415 Lys Ser Pro Asp Ser Pro
Thr Leu Gln Val Gly Ala Arg Pro Leu Glu 420 425 430 Ala Thr Phe Arg
Pro Val Arg His Pro Thr Arg Met Tyr Ser Leu Asp 435 440 445 Asn Ala
Phe Asn Leu Asp Glu Leu Lys Ala Phe Glu Glu Arg Ile Glu 450 455 460
Arg Ala Leu Gly Arg Lys Gly Pro Phe Ala Tyr Thr Val Glu His Lys 465
470 475 480 Val Asp Gly Leu Ser Val Asn Leu Tyr Tyr Glu Glu Gly Val
Leu Val 485 490 495 Tyr Gly Ala Thr Arg Gly Asp Gly Glu Val Gly Glu
Glu Val Thr Gln 500 505 510 Asn Leu Leu Thr Ile Pro Thr Ile Pro Arg
Arg Leu Lys Gly Val Pro 515 520 525 Glu Arg Leu Glu Val Arg Gly Glu
Val Tyr Met Pro Ile Glu Ala Phe 530 535 540 Leu Arg Leu Asn Glu Glu
Leu Glu Glu Arg Gly Glu Arg Ile Phe Lys 545 550 555 560 Asn Pro Arg
Asn Ala Ala Ala Gly Ser Leu Arg Gln Lys Asp Pro Arg 565 570 575 Ile
Thr Ala Lys Arg Gly Leu Arg Ala Thr Phe Tyr Ala Leu Gly Leu 580 585
590 Gly Leu Glu Glu Val Glu Arg Glu Gly Val Ala Thr Gln Phe Ala Leu
595 600 605 Leu His Trp Leu Lys Glu Lys Gly Phe Pro Val Glu His Gly
Tyr Ala 610 615 620 Arg Ala Val Gly Ala Glu Gly Val Glu Ala Val Tyr
Gln Asp Trp Leu 625 630 635 640 Lys Lys Arg Arg Ala Leu Pro Phe Glu
Ala Asp Gly Val Val Val Lys 645 650 655 Leu Asp Glu Leu Ala Leu Trp
Arg Glu Leu Gly Tyr Thr Ala Arg Ala 660 665 670 Pro Arg Phe Ala Ile
Ala Tyr Lys Phe Pro Ala Glu Glu Lys Glu Thr 675 680 685 Arg Leu Leu
Asp Val Val Phe Gln Val Gly Arg Thr Gly Arg Val Thr 690 695 700 Pro
Val Gly Ile Leu Glu Pro Val Phe Leu Glu Gly Ser Glu Val Ser 705 710
715 720 Arg Val Thr Leu His Asn Glu Ser Tyr Ile Glu Glu Leu Asp Ile
Arg 725 730 735 Ile Gly Asp Trp Val Leu Val His Lys Ala Gly Gly Val
Ile Pro Glu 740 745 750 Val Leu Arg Val Leu Lys Glu Arg Arg Thr Gly
Glu Glu Arg Pro Ile 755 760 765 Arg Trp Pro Glu Thr Cys Pro Glu Cys
Gly His Arg Leu Leu Lys Glu 770 775 780 Gly Lys Val His Arg Cys Pro
Asn Pro Leu Cys Pro Ala Lys Arg Phe 785 790 795 800 Glu Ala Ile Arg
His Phe Ala Ser Arg Lys Ala Met Asp Ile Gln Gly 805 810 815 Leu Gly
Glu Lys Leu Ile Glu Arg Leu Leu Glu Lys Gly Leu Val Lys 820 825 830
Asp Val Ala Asp Leu Tyr Arg Leu Arg Lys Glu Asp Leu Val Gly Leu 835
840 845 Glu Arg Met Gly Glu Lys Ser Ala Gln Asn Leu Leu Arg Gln Ile
Glu 850 855 860 Glu Ser Lys Lys Arg Gly Leu Glu Arg Leu Leu Tyr Ala
Leu Gly Leu 865 870 875 880 Pro Gly Val Gly Glu Val Leu Ala Arg Asn
Leu Ala Ala Arg Phe Gly 885 890 895 Asn Met Asp Arg Leu Leu Glu Ala
Ser Leu Glu Glu Leu Leu Glu Val 900 905 910 Glu Glu Val Gly Glu Leu
Thr Ala Arg Ala Ile Leu Glu Thr Leu Lys 915 920 925 Asp Pro Ala Phe
Arg Asp Leu Val Arg Arg Leu Lys Glu Ala Gly Val 930 935 940 Glu Met
Glu Ala Lys Glu Lys Gly Gly Glu Ala Leu Lys Gly Leu Thr 945 950 955
960 Phe Val Ile Thr Gly Glu Leu Ser Arg Pro Arg Glu Glu Val Lys Ala
965 970 975 Leu Leu Arg Arg Leu Gly Ala Lys Val Thr Asp Ser Val Ser
Arg Lys 980 985 990 Thr Ser Tyr Leu Val Val Gly Glu Asn Pro Gly Ser
Lys Leu Glu Lys 995 1000 1005 Ala Arg Ala Leu Gly Val Pro Thr Leu
Thr Glu Glu Glu Leu Tyr 1010 1015 1020 Arg Leu Leu Glu Ala Arg Thr
Gly Lys Lys Ala Glu Glu Leu Val 1025 1030 1035
2529DNAStaphylococcus aureusSubspecies SSCmec 2caactaatga
aacagaaagt cgtaactatc ctctagaaaa agcgacttca catctattag 60gttatgttgg
tcccattaac tctgaagaat taaaacaaaa agaatataaa ggctataaag
120atgatgcagt tattggtaaa aagggactcg aaaaacttta cgataaaaag
ctccaacatg 180aagatggcta tcgtgtcaca atcgttgacg ataatagcaa
tacaatcgca catacattaa 240tagagaaaaa gaaaaaagat ggcaaagata
ttcaactaac tattgatgct aaagttcaaa 300agagtattta taacaacatg
aaaaatgatt atggctcagg tactgctatc caccctcaaa 360caggtgaatt
attagcactt gtaagcacac cttcatatga cgtctatcca tttatgtatg
420catgagtaac gaagaatata ataaattaac cgaagataaa aaagaacctc
tgctcaacaa 480gttccagatt acaacttcac caggttcaac tcaaaaaata ttaacagca
529323DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 3ggctcaggta ctgctatcca ccc 23425DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
4ttttgagttg aacctggtga agttg 255443RNAInfluenza A virus 5cgccggaugg
cucuugggaa accgaagaca gccacaacgg gaaacuaugu aaauuaaaag 60gaauagcccc
acuacaauug gggaaaugua acaucgccgg auggcucuug ggaaacccag
120aaugcgacuc acugcuucca gcgaaaucau gguccuacau uguagaaaca
ccaaacucug 180agaauggagc auguuaucca ggagauuuca ucgacuauga
ggaacugaag gagcaauuga 240gcucaguauc aucauuagaa agauucgaaa
uauuucccaa ggaaaguuca uggcccaacc 300acaacacacu caaaggagua
acagcagcau gcucccauag gggaaaaagc aguuuuuaca 360gaaauuugcu
auggcugacg aaaacggggg acucauaccc aaagcugaac aauuccuaug
420ugaacaauaa agggaaagaa guc 443623DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
6cgccggatgg ctcttgggaa acc 23725DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 7tcgctggaag cagtgagtcg
cattc 258339RNAInfluenza B virus 8cuucuugaau uugaugucua agaguaauuu
gccaacguga ggccaucaga aaguauggug 60cgcaaguggc aggagcaagg uaauaaaagg
guccuugccu uuaauuggug aagcagauug 120ccuccacgaa aaauacggug
gauuaaacaa aagcaagccu uacuacacag gagaacaugc 180aaaagccaua
ggaaauugcc caauaugggu gaaaacaccc uugaagcugg ccaauggaac
240caaauauaga ccgccugcaa aacuauuaaa ggaaagaggu uucuucggaa
gcgacagaca 300guaacacuca acucucgacc aucuggugua acaaccucg
339924DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 9cggtggatta aacaaaagca agcc 241020DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
10attggccagc ttcaagggtg 201110PRTArtificial SequenceDescription of
Artificial Sequence Synthetic 10xHis tag 11His His His His His His
His His His His 1 5 10
* * * * *