U.S. patent application number 17/386883 was filed with the patent office on 2021-11-18 for polyphenolic additives in sequencing-by-synthesis.
The applicant listed for this patent is IsoPlexis Corporation. Invention is credited to Jerzy OLEJNIK, Michel Georges PERBOST.
Application Number | 20210355529 17/386883 |
Document ID | / |
Family ID | 1000005740307 |
Filed Date | 2021-11-18 |
United States Patent
Application |
20210355529 |
Kind Code |
A1 |
OLEJNIK; Jerzy ; et
al. |
November 18, 2021 |
POLYPHENOLIC ADDITIVES IN SEQUENCING-BY-SYNTHESIS
Abstract
The invention relates to methods, compositions, devices, systems
and kits as described including, without limitation, reagents and
mixtures for determining the identity of nucleic acids in
nucleotide sequences using, for example, sequencing by synthesis
methods. In particular, the present invention contemplates the use
of polyphenolic compounds, known as antioxidant additives, to
improve the efficiency of Sequencing-By-Synthesis reactions. For
example, gallic acid (GA) is shown herein to be one of many
exemplary SBS polyphenolic additives.
Inventors: |
OLEJNIK; Jerzy; (Brookline,
MA) ; PERBOST; Michel Georges; (Belmont, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
IsoPlexis Corporation |
Branford |
CT |
US |
|
|
Family ID: |
1000005740307 |
Appl. No.: |
17/386883 |
Filed: |
July 28, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16422056 |
May 24, 2019 |
11078526 |
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17386883 |
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15427664 |
Feb 8, 2017 |
10337050 |
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16422056 |
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62419685 |
Nov 9, 2016 |
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62293969 |
Feb 11, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6869 20130101;
C12Q 1/6823 20130101 |
International
Class: |
C12Q 1/6823 20060101
C12Q001/6823; C12Q 1/6869 20060101 C12Q001/6869 |
Claims
1. A method of incorporating labeled nucleotides, comprising: a)
providing i) a plurality of nucleic acid primers and template
molecules, ii) a polymerase, iii) a cleave reagent comprising a
reducing agent and a polyphenolic compound, and iv) a plurality of
nucleotide analogues wherein at least a portion of said nucleotide
analogues is labeled with a label attached through a cleavable
disulfide linker to the base; b) hybridizing at least a portion of
said primers to at least a portion of said template molecules so as
to create hybridized primers; c) incorporating a first labeled
nucleotide analogue with said polymerase into at least a portion of
said hybridized primers so as to create extended primers comprising
an incorporated labeled nucleotide analogue; d) detecting said
incorporated labeled nucleotide analogue; and e) cleaving the
cleavable linker of said incorporated nucleotide analogues with
said cleave reagent.
2. The method of claim 1, wherein said polyphenolic compound is
selected from the group consisting of gallic acid, gentisic acid,
pryocatechol, pyrogallol, hydroquinone and resorcinol.
3. The method of claim 1, wherein said reducing agent of said
cleave reagent comprises TCEP (tris(2-carboxyethyl)phosphine).
4. The method of claim 1, wherein said incorporated nucleotide
analogues of step c) further comprise a removable chemical moiety
capping the 3'-OH group.
5. The method of claim 3, wherein the cleaving of step e) removes
the removable chemical moiety capping the 3'-OH group.
6. The method of claim 5, wherein the method further comprises: f)
incorporating a second nucleotide analogue with said polymerase
into at least a portion of said extended primers.
7. The method of claim 1, wherein said label is fluorescent.
8. A cleave reagent comprising i) a reducing agent, and ii) a
polyphenolic compound.
9. The cleave reagent of claim 8, wherein said polyphenolic
compound is selected from the group consisting of gallic acid,
gentisic acid, pryocatechol, pyrogallol, hydroquinone, and/or
resorcinol.
10. The cleave reagent of claim 8, wherein said reducing agent is
TCEP Tris(2-carboxyethyl)phosphine).
11. A kit, comprising i) the cleave reagent of claim 8 and ii) a
plurality of nucleotide analogues wherein at least a portion of
said nucleotide analogues is labeled with a label attached through
a cleavable disulfide linker to the base.
12. A system comprising primers hybridized to template in solution,
said solution comprising the cleave reagent of claim 8.
13. The system of claim 11, wherein said hybridized primers and
template are immobilized.
14. The system of claim 12, wherein said hybridized primers and
template are in a flow cell.
Description
CROSS-REFEENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 16/422,056, filed May 24, 20919, which is a continuation of
U.S. application Ser. No. 15/427,664, filed Feb. 08, 2017, (now
U.S. Pat. No. 10,337,050), which claims priority to, and the
benefit of, U.S. provisional application Nos. 62/293,969, filed
Feb. 11, 2016 and 62/419,685 filed Nov. 09, 2016, under 35 USC
.sctn. 119(e). The contents of each of these applications are
hereby incorporated by reference in their entireties.
FIELD OF THE INVENTION
[0002] The invention relates to methods, compositions, devices,
systems and kits as described including, without limitation,
reagents and mixtures for determining the identity of nucleic acids
in nucleotide sequences using, for example, sequencing by synthesis
methods. In particular, the present invention contemplates the use
of polyphenolic compounds, known as antioxidant additives, to
improve the efficiency of Sequencing-By-Synthesis reactions. For
example, gallic acid (GA) is shown herein to be one of many
exemplary SBS polyphenolic additives.
BACKGROUND
[0003] Over the past 25 years, the amount of DNA sequence
information that has been generated and deposited into Genbank has
grown exponentially. Traditional sequencing methods (e.g., for
example Sanger sequencing) are being replaced by next-generation
sequencing technologies that use a form of sequencing by synthesis
(SBS), wherein specially designed nucleotides and DNA polymerases
are used to read the sequence of chip-bound, single-stranded DNA
templates in a controlled manner. To attain high throughput, many
millions of such template spots are arrayed across a sequencing
chip and their sequence is independently read out and recorded.
[0004] Systems for using arrays for DNA sequencing are known (e.g.,
Ju et al., U.S. Pat. No. 6,664,079). However, there is a continued
need for methods and compositions for increasing the accuracy
and/or efficiency of sequencing nucleic acid sequences and
increasing the read lengths available for automated sequencing.
SUMMARY OF THE INVENTION
[0005] The invention relates to methods, compositions, devices,
systems and kits as described including, without limitation,
reagents and mixtures for determining the identity of nucleic acids
in nucleotide sequences using, for example, sequencing by synthesis
methods. In particular, the present invention contemplates the use
of polyphenolic compounds, known as antioxidant additives, to
improve the efficiency of Sequencing-By-Synthesis reactions. For
example, gallic acid (GA) is shown herein to be one of many
exemplary SBS polyphenolic additives.
[0006] In one embodiment, the present invention contemplates the
use polyphenolic compounds as antioxidant additives to the cleave
reagent during the cleave step in sequencing by synthesis (SBS).
Such method of application is leads to significant improvement of
the sequencing performance (raw error rate thus supporting longer
read length). Such effect may be due to enhanced efficacy of the
cleave reaction via scavenging of radical by-products and
deactivation of excess cleave reagent. Such radical by-products may
build up in the flow cell leading to carry over into the subsequent
extension step thus causing premature de-protection of the 3'-OH
moiety and impairing single base incorporation rate.
[0007] The present invention contemplates, in one embodiment, a
method of incorporating labeled nucleotides, comprising: a)
providing i) a plurality of nucleic acid primers and template
molecules, ii) a polymerase, iii) a cleave reagent comprising a
reducing agent and a polyphenolic compound, and iv) a plurality of
nucleotide analogues wherein at least a portion of said nucleotide
analogues is labeled with a label attached through a cleavable
linker (e.g. a disulfide linker) to the base; b) hybridizing at
least a portion of said primers to at least a portion of said
template molecules so as to create hybridized primers; c)
incorporating a first labeled nucleotide analogue with said
polymerase into at least a portion of said hybridized primers so as
to create extended primers comprising an incorporated labeled
nucleotide analogue; d) detecting said incorporated labeled
nucleotide analogue; and e) cleaving the cleavable linker of said
incorporated nucleotide analogues with said cleave reagent. In one
embodiment, the polyphenolic compound is selected from the group
consisting of gallic acid, gentisic acid, pryocatechol, pyrogallol,
hydroquinone, and/or resorcinol (or combinations thereof). In one
embodiment, said reducing agent of said cleave reagent comprises
TCEP (tris(2-carboxyethyl)phosphine). In one embodiment, said
incorporated nucleotide analogues of step c) further comprise a
removable chemical moiety capping the 3'-OH group. In one
embodiment, the cleaving of step e) removes the removable chemical
moiety capping the 3'-OH group. In one embodiment, the method
further comprises f) incorporating a second nucleotide analogue
with said polymerase into at least a portion of said extended
primers.
[0008] It is not intended that the present invention be limited to
the type of label. A variety of labels are contemplated. In a
preferred embodiment, said label is fluorescent.
[0009] The present invention also contemplates compositions and
reagents. In one embodiment, the present invention contemplates a
cleave reagent comprising i) a reducing agent, and ii) a
polyphenolic compound. In one embodiment, the polyphenolic compound
includes, but is not limited to, gallic acid, gentisic acid,
pryocatechol, pyrogallol, hydroquinone, and/or resorcinol. In one
embodiment, said reducing agent is TCEP
Tris(2-carboxyethyl)phosphine).
[0010] The present invention also contemplates kits, where reagents
are supplied with instructions for their use. In one embodiment,
the present invention contemplates a kit, comprising: i) the cleave
reagent and ii) a plurality of nucleotide analogues wherein at
least a portion of said nucleotide analogues is labeled with a
label attached through a cleavable linker (e.g. a disulfide linker)
to the base. In one embodiment, the cleave reagent comprises i) a
reducing agent, and ii) a polyphenolic compound. In one embodiment,
the polyphenolic compound includes, but is not limited to, gallic
acid, gentisic acid, pryocatechol, pyrogallol, hydroquinone, and/or
resorcinol. In one embodiment, said reducing agent is TCEP
Tris(2-carboxyethyl)phosphine).
[0011] The present invention also contemplates systems, such as
systems with flow cells where the flow cells are linked to sources
of reagents. See e.g. U.S. Pat. No. 9,145,589, herein incorporated
by reference. In one embodiment, the present invention contemplates
a system comprising primers hybridized to template in solution,
said solution comprising a cleave reagent, the cleave reagent
comprising i) a reducing agent, and ii) a polyphenolic compound. In
one embodiment, the polyphenolic compound includes, but is not
limited to, gallic acid, gentisic acid, pryocatechol, pyrogallol,
hydroquinone, and/or resorcinol. In one embodiment, said reducing
agent is TCEP Tris(2-carboxyethyl)phosphine). In one embodiment,
said hybridized primers and template are immobilized. In one
embodiment, said hybridized primers and template are in a flow
cell.
Definitions
[0012] To facilitate the understanding of this invention, a number
of terms are defined below. Terms defined herein have meanings as
commonly understood by a person of ordinary skill in the areas
relevant to the present invention. Terms such as "a", "an" and
"the" are not intended to refer to only a singular entity but also
plural entities and also includes the general class of which a
specific example may be used for illustration. The terminology
herein is used to describe specific embodiments of the invention,
but their usage does not delimit the invention, except as outlined
in the claims.
[0013] The term "about" as used herein, in the context of any of
any assay measurements refers to +/- 5% of a given measurement.
[0014] The term "linker" as used herein, refers to any molecule (or
collection of molecules) capable of attaching a label and/or
chemical moiety that is susceptible to cleavage. In one embodiment,
cleavage of the linker may produce toxic radical products. For
example, a linker may include, but is not limited to, a disulfide
linker and/or an azide linker.
[0015] The term "attached" as used herein, refers to any
interaction between a first molecule (e.g., for example, a nucleic
acid) and a second molecule (e.g., for example, a label molecule).
Attachment may be reversible or irreversible. Such attachment
includes, but is not limited to, covalent bonding, ionic bonding,
Van der Waals forces or friction, and the like.
[0016] "Nucleic acid sequence" and "nucleotide sequence" as used
herein refer to an oligonucleotide or polynucleotide, and fragments
or portions thereof, and to DNA or RNA of genomic or synthetic
origin which may be single- or double-stranded, and represent the
sense or antisense strand. Such nucleic acids may include, but are
not limited to, cDNA, mRNA or other nucleic acid sequences.
[0017] The term "an isolated nucleic acid", as used herein, refers
to any nucleic acid molecule that has been removed from its natural
state (e.g., removed from a cell and is, in a preferred embodiment,
free of other genomic nucleic acid).
[0018] In some embodiments, the present invention contemplates
hybridizing nucleic acid together. This requires some degree of
complementarity. As used herein, the terms "complementary" or
"complementarity" are used in reference to "polynucleotides" and
"oligonucleotides" (which are interchangeable terms that refer to a
sequence of nucleotides) related by the base-pairing rules. For
example, the sequence "C-A-G-T," is complementary to the sequence
"G-T-C-A." Complementarity can be "partial" or "total." "Partial"
complementarity is where one or more nucleic acid bases is not
matched according to the base pairing rules. "Total" or "complete"
complementarity between nucleic acids is where each and every
nucleic acid base is matched with another base under the base
pairing rules. The degree of complementarity between nucleic acid
strands has significant effects on the efficiency and strength of
hybridization between nucleic acid strands. This is of particular
importance in amplification reactions, as well as detection methods
which depend upon binding between nucleic acids.
[0019] The terms "homology" and "homologous" as used herein in
reference to nucleotide sequences refer to a degree of
complementarity with other nucleotide sequences. There may be
partial homology or complete homology (i.e., identity). A
nucleotide sequence which is partially complementary, i.e.,
"substantially homologous," to a nucleic acid sequence is one that
at least partially inhibits a completely complementary sequence
from hybridizing to a target nucleic acid sequence. The inhibition
of hybridization of the completely complementary sequence to the
target sequence may be examined using a hybridization assay
(Southern or Northern blot, solution hybridization and the like)
under conditions of low stringency. A substantially homologous
sequence or probe will compete for and inhibit the binding (i.e.,
the hybridization) of a completely homologous sequence to a target
sequence under conditions of low stringency. This is not to say
that conditions of low stringency are such that non-specific
binding is permitted; low stringency conditions require that the
binding of two sequences to one another be a specific (i.e.,
selective) interaction. The absence of non-specific binding may be
tested by the use of a second target sequence which lacks even a
partial degree of complementarity (e.g., less than about 30%
identity); in the absence of non-specific binding the probe will
not hybridize to the second non-complementary target.
[0020] Low stringency conditions comprise conditions equivalent to
binding or hybridization at 42.degree. C. in a solution consisting
of 5 x SSPE (43.8 g/l NaCl, 6.9 g/lNaH2PO4H2O and 1.85 g/l EDTA, pH
adjusted to 7.4 with NaOH), 0.1% SDS, 5x Denhardt's reagent {50x
Denhardt's contains per 500 ml: 5 g Ficoll (Type 400, Pharmacia), 5
g BSA (Fraction V; Sigma)} and 100 .mu.g/ml denatured salmon sperm
DNA followed by washing in a solution comprising 5x SSPE, 0.1% SDS
at 42.degree. C. when a probe of about 500 nucleotides in length.
is employed. Numerous equivalent conditions may also be employed to
comprise low stringency conditions; factors such as the length and
nature (DNA, RNA, base composition) of the probe and nature of the
target (DNA, RNA, base composition, present in solution or
immobilized, etc.) and the concentration of the salts and other
components (e.g., the presence or absence of formamide, dextran
sulfate, polyethylene glycol), as well as components of the
hybridization solution may be varied to generate conditions of low
stringency hybridization different from, but equivalent to, the
above listed conditions. In addition, conditions which promote
hybridization under conditions of high stringency (e.g., increasing
the temperature of the hybridization and/or wash steps, the use of
formamide in the hybridization solution, etc.) may also be
used.
[0021] As used herein, the term "hybridization" is used in
reference to the pairing of complementary nucleic acids using any
process by which a strand of nucleic acid joins with a
complementary strand through base pairing to form a hybridization
complex. Hybridization and the strength of hybridization (i.e., the
strength of the association between the nucleic acids) is impacted
by such factors as the degree of complementarity between the
nucleic acids, stringency of the conditions involved, the Tm of the
formed hybrid, and the G:C ratio within the nucleic acids.
[0022] As used herein the term "hybridization complex" refers to a
complex formed between two nucleic acid sequences by virtue of the
formation of hydrogen bounds between complementary G and C bases
and between complementary A and T bases; these hydrogen bonds may
be further stabilized by base stacking interactions. The two
complementary nucleic acid sequences hydrogen bond in an
antiparallel configuration. A hybridization complex may be formed
in solution (e.g., C0 t or R0 t analysis) or between one nucleic
acid sequence present in solution and another nucleic acid sequence
immobilized to a solid support (e.g., a nylon membrane or a
nitrocellulose filter as employed in Southern and Northern
blotting, dot blotting or a glass slide as employed in in situ
hybridization, including FISH (fluorescent in situ
hybridization)).
[0023] As used herein, the term "Tm " is used in reference to the
"melting temperature." The melting temperature is the temperature
at which a population of double-stranded nucleic acid molecules
becomes half dissociated into single strands. As indicated by
standard references, a simple estimate of the Tm value may be
calculated by the equation: Tm=81.5+0.41 (% G+C), when a nucleic
acid is in aqueous solution at 1M NaCl. Anderson et al.,
"Quantitative Filter Hybridization" In: Nucleic Acid Hybridization
(1985). More sophisticated computations take structural, as well as
sequence characteristics, into account for the calculation of
Tm.
[0024] As used herein the term "stringency" is used in reference to
the conditions of temperature, ionic strength, and the presence of
other compounds such as organic solvents, under which nucleic acid
hybridizations are conducted. "Stringency" typically occurs in a
range from about Tm to about 20.degree. C. to 25.degree. C. below
Tm. A "stringent hybridization" can be used to identify or detect
identical polynucleotide sequences or to identify or detect similar
or related polynucleotide sequences. For example, when fragments
are employed in hybridization reactions under stringent conditions
the hybridization of fragments which contain unique sequences
(i.e., regions which are either non-homologous to or which contain
less than about 50% homology or complementarity) are favored.
Alternatively, when conditions of "weak" or "low" stringency are
used hybridization may occur with nucleic acids that are derived
from organisms that are genetically diverse (i.e., for example, the
frequency of complementary sequences is usually low between such
organisms).
[0025] As used herein, the term "amplifiable nucleic acid" is used
in reference to nucleic acids which may be amplified by any
amplification method. It is contemplated that "amplifiable nucleic
acid" will usually comprise "sample template."
[0026] As used herein, the term "sample template" or (more simply)
"template" refers to nucleic acid originating from a sample which
is analyzed for the presence of a target sequence of interest. In
contrast, "background template" is used in reference to nucleic
acid other than sample template which may or may not be present in
a sample. Background template is most often inadvertent. It may be
the result of carryover, or it may be due to the presence of
nucleic acid contaminants sought to be purified away from the
sample. For example, nucleic acids from organisms other than those
to be detected may be present as background in a test sample.
[0027] "Amplification" is defined as the production of additional
copies of a nucleic acid sequence and is generally carried out
using polymerase chain reaction. Dieffenbach C. W. and G. S.
Dveksler (1995) In: PCR Primer, a Laboratory Manual, Cold Spring
Harbor Press, Plainview, N.Y.
[0028] As used herein, the term "polymerase chain reaction" ("PCR")
refers to the method of K. B. Mullis U.S. Pat. Nos. 4,683,195 and
4,683,202, herein incorporated by reference, which describe a
method for increasing the concentration of a segment of a target
sequence in a mixture of genomic DNA without cloning or
purification. The length of the amplified segment of the desired
target sequence is determined by the relative positions of two
oligonucleotide primers with respect to each other, and therefore,
this length is a controllable parameter. By virtue of the repeating
aspect of the process, the method is referred to as the "polymerase
chain reaction" (hereinafter "PCR"). Because the desired amplified
segments of the target sequence become the predominant sequences
(in terms of concentration) in the mixture, they are said to be
"PCR amplified". With PCR, it is possible to amplify a single copy
of a specific target sequence in genomic DNA to a level detectable
by several different methodologies (e.g., hybridization with a
labeled probe; incorporation of biotinylated primers followed by
avidin-enzyme conjugate detection; incorporation of 32P-labeled
deoxynucleotide triphosphates, such as dCTP or dATP, into the
amplified segment). In addition to genomic DNA, any oligonucleotide
sequence can be amplified with the appropriate set of primer
molecules. In particular, the amplified segments created by the PCR
process itself are, themselves, efficient templates for subsequent
PCR amplifications.
[0029] As used herein, the term "primer" refers to an
oligonucleotide, whether occurring naturally as in a purified
restriction digest or produced synthetically, which is capable of
acting as a point of initiation of synthesis when placed under
conditions in which synthesis of a primer extension product which
is complementary to a nucleic acid strand is induced, (i.e., in the
presence of nucleotides and an inducing agent such as DNA
polymerase and at a suitable temperature and pH). The primer is
preferably single stranded for maximum efficiency in amplification,
but may alternatively be double stranded. If double stranded, the
primer is first treated to separate its strands before being used
to prepare extension products. Preferably, the primer is an
oligodeoxy-ribonucleotide. The primer must be sufficiently long to
prime the synthesis of extension products in the presence of the
inducing agent. The exact lengths of the primers will depend on
many factors, including temperature, source of primer and the use
of the method.
[0030] As used herein, the term "probe" refers; to an
oligonucleotide (i.e., a sequence of nucleotides), whether
occurring naturally as in a purified restriction digest or produced
synthetically, recombinantly or by PCR amplification, which is
capable of hybridizing to another oligonucleotide of interest. A
probe may be single-stranded or double-stranded. Probes are useful
in the detection, identification and isolation of particular gene
sequences. It is contemplated that any probe used in the present
invention will be labeled with any "reporter molecule," so that is
detectable in any detection system, including, but not limited to
enzyme (e.g., ELISA, as well as enzyme-based histochemical assays),
fluorescent, radioactive, and luminescent systems. It is not
intended that the present invention be limited to any particular
detection system or label.
[0031] The term "label" or "detectable label" are used herein, to
refer to any composition detectable by spectroscopic,
photochemical, biochemical, immunochemical, electrical, optical or
chemical means. Such labels include biotin for staining with
labeled streptavidin conjugate, magnetic beads (e.g.,
Dynabeads.RTM.), fluorescent dyes (e.g., fluorescein, texas red,
rhodamine, green fluorescent protein, and the like), radiolabels
(e.g., 3H, 125I, 35S, 14C, or 32P), enzymes (e.g., horse radish
peroxidase, alkaline phosphatase and others commonly used in an
ELISA), and calorimetric labels such as colloidal gold or colored
glass or plastic (e.g., polystyrene, polypropylene, latex, etc.)
beads. Patents teaching the use of such labels include, but are not
limited to, U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;
3,996,345; 4,277,437; 4,275,149; and 4,366,241 (all herein
incorporated by reference).
[0032] In a preferred embodiment, the label is typically
fluorescent and is linked to the base of the nucleotide. For
cytosine and thymine, the attachment is usually to the 5-position.
For the other bases, a deaza derivative is created and the label is
linked to a 7-position of deaza-adenine or deaza-guanine.
[0033] The labels contemplated in the present invention may be
detected by many methods. For example, radiolabels may be detected
using photographic film or scintillation counters, fluorescent
markers may be detected using a photodetector to detect emitted
light. Enzymatic labels are typically detected by providing the
enzyme with a substrate and detecting, the reaction product
produced by the action of the enzyme on the substrate, and
calorimetric labels are detected by simply visualizing the colored
label.
[0034] The term "luminescence" and/or "fluorescence", as used
herein, refers to any process of emitting electromagnetic radiation
(light) from an object, chemical and/or compound. Luminescence
and/or fluorescence results from a system which is "relaxing" from
an excited state to a lower state with a corresponding release of
energy in the form of a photon. These states can be electronic,
vibronic, rotational, or any combination of the three. The
transition responsible for luminescence can be stimulated through
the release of energy stored in the system chemically or added to
the system from an external source. The external source of energy
can be of a variety of types including, but not limited to,
chemical, thermal, electrical, magnetic, electromagnetic, physical
or any other type capable of causing a system to be excited into a
state higher than the ground state. For example, a system can be
excited by absorbing a photon of light, by being placed in an
electrical field, or through a chemical oxidation-reduction
reaction. The energy of the photons emitted during luminescence can
be in a range from low-energy microwave radiation to high-energy
x-ray radiation. Typically, luminescence refers to photons in the
range from UV to IR radiation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0036] FIG. 1 presents exemplary data showing a comparison of
sequencing performance for runs containing gallic acid (addG) in
Cleave solution vs Baseline runs. Sequencing parameters include
average error rate, average percentage of perfect (error free)
reads, phasing parameters (lead and lag).
[0037] FIG. 2 presents exemplary data showing a comparison of
sequencing performance for runs containing gallic acid (addG) in
Cleave solution vs Baseline runs. Sequencing included assessment of
called variants (true positives--TP, false positives--FP).
[0038] FIG. 3 presents exemplary data showing a comparison of
sequencing performance for runs containing gallic acid (addG) in
Cleave solution vs Baseline runs. Sequencing included assessment of
average error rate per cycle.
[0039] FIG. 4 presents exemplary data showing a comparison of
sequencing performance for runs containing gallic acid (addG) in
Cleave solution vs Baseline runs. Sequencing included assessment of
the following performance indicators: average error rate per cycle,
average percentage of perfect reads, percent of signal retention,
false positive rate and lead/lag. Values higher than 0.95 on the
third bar indicate statistical significance, first bar corresponds
to baseline, second to experiment with relative improvement
factor.
[0040] FIG. 5 presents exemplary data showing an LC-MS analysis of
a cleaved spacer arm terminating with free SH group and exposure to
Cleave without additives. Formation of alkene moiety detected as a
result of side reactions during cleavage step.
[0041] FIG. 6 presents exemplary data showing a comparison of
sequencing performance for runs containing gallic acid (addG) in
Cleave solution vs solutions with gallic acid and Tween detergent.
Sequencing included assessment of the following performance
indicators: average error rate per cycle, average percentage of
perfect reads, percent of signal retention, false positive rate and
lead/lag. Values higher than 0.95 on the third bar indicate
statistical significance, first bar corresponds to baseline, second
to experiment with relative improvement factor
[0042] FIG. 7 presents exemplary data showing a comparison of
sequencing performance for runs containing addG alternatives in
Cleave solution (pyrogallol, pyrocatechol, gentisic acid) vs
baseline runs without any additive. Results are provided for two
sample types (Clones and Gene Panel). Improvements are noted for
sequencing KPIs such as error rate, percent perfect and lead/lag
when addG alternatives such as pyrogallol, pyrocatechol, gentisic
acid are added to Cleave.
[0043] FIG. 8 presents representative electropherograms of
sequencing products after sequencing under baseline conditions (A)
and with gallic acid additive in Cleave (B). Higher yield of full
length product observed in case with additive (B).
[0044] FIG. 9 presents exemplary RP-HPLC cleavage studies of
labeled dCTP nucleotide in the absence (baseline) and presence of
additive (examples of gallic and gentisic acid). Byproducts are
clearly visible in baseline and absent in chromatograms with
additives.
[0045] FIG. 10 presents exemplary data showing a comparison of
sequencing performance for runs containing baseline imaging buffer
vs imaging buffer containing additives: GR 5.10: Replace baseline
Imaging buffer with a single HEPES pH 7.5 buffer with Gentisic acid
at final concentration of 25 mM; GR 5.4: Add Gentisic acid (25 mM)
to Image B; GR 5.5: Image B buffer with Trolox removed and Gentisic
acid added (25 mM).
[0046] FIG. 11 presents exemplary data showing the relative bead
loss in flow cells during runs comparing indole-3-propionic acid
(IPA: AddC_15FC) and gallic acid (GA: AddG_7FC).
[0047] FIG. 12 presents exemplary data comparing the raw error rate
for ascorbic acid (Add AA), additive C (Add C), additive G (light)
along with an additive-free control (No add) where the results were
generated in a sequencing run using the NA12878/101X gene panel as
template. The raw error rate for ascorbic acid is significantly
better than i) the no additive run, and ii) the run with additive C
(while performing comparably to additive G).
DETAILED DESCRIPTION OF THE INVENTION
[0048] The invention relates to methods, compositions, devices,
systems and kits as described including, without limitation,
reagents and mixtures for determining the identity of nucleic acids
in nucleotide sequences using, for example, sequencing by synthesis
methods. In particular, the present invention contemplates the use
of polyphenolic compounds, known as antioxidant additives, to
improve the efficiency of Sequencing-By-Synthesis reactions. For
example, gallic acid (GA) is shown herein to be one of many
exemplary SBS polyphenolic additives.
1. Sequencing-By-Synthesis (SBS)
[0049] One step in the sequencing-by-synthesis workflow is the
removal of the fluorescent label which is covalently attached via a
cleavable linker molecule to the ring-position of the heterocyclic
base of the nucleotide (reversible terminator) involved in the
incorporation step. The efficacy of the cleave step is reflected
not only in the efficiency of the fluorescent label cleavage but
also in the mitigation of reaction by-products that could
accumulate in the flow cell and interfere with subsequent base
incorporation step. Examples of such compounds are radical
by-products that may form due to radical pathways involved in the
omolytic scission of the linker molecule to release the fluorescent
label and excess cleave reagent (i. e.,
tris(2-carboxyethyl)phosphine or TCEP). These may build up in the
flow cell and carry over into the subsequent base extension step
thus causing premature de-protection of the 3'-OH moiety and
causing more than one base to incorporate. An effective cleave step
is important for single nucleotide incorporation throughout the
sequencing reaction, as well as a prerequisite for low error rate
and long read length. To improve the efficacy of the cleave step,
molecules that quench radical pathways and oxidize excess TCEP are
contemplated, such as ascorbic acid, so as to enhance the efficacy
of this reactive step.
[0050] In one embodiment, the present invention contemplates a
series of method steps performed by an automated sequencing by
synthesis instrument. See U.S. Pat. No. 9,145,589, hereby
incorporated by reference. In one embodiment, the instrument is
comprised of numerous reagent reservoirs. Each reagent reservoir
has a specific reactivity reagent dispensed within the reservoir to
support the SBS process, for example:
[0051] One reactive step in a method for sequencing by synthesis
using cleavable fluorescent nucleotide reversible terminators
comprises cleaving a fluorescent label from a nucleotide analogue
molecule. It is not intended that the present invention be limited
by the nature of the cleaving agent.
[0052] In one embodiment, the SBS method comprises doing different
steps at different stations. By way of example, each station is
associated with a particular step. While not limited to particular
formulations, some examples for these steps and the associated
reagents are shown below: [0053] 1) Extend A Reagent: Comprises
reversibly terminated labeled nucleotides and polymerase.
[0054] The composition of Extend A is as follows:
TABLE-US-00001 Component Conc PNSE (% wt/vol) 0.005% Tris x HCl (pH
8.8), mM 50 NaCl (mM) 50 EDTA (mM) 1 MgSO4 (mM) 10 Cystamine (mM) 1
Glycerol (% wt/vol) 0.01% Therminator IX* (U/ml) 10 N3-dCTP (.mu.M)
3.83 N3-dTTP (.mu.M) 3.61 N3-dATP (.mu.M) 4.03 N3-dGTP (.mu.M) 0.4
Alexa488-dCTP (nM) 550 R6G-dUTP (nM) 35 ROX-dATP (nM) 221 Cy5-dGTP
(nM) 66 *with Alkylated free Cysteine
[0055] 2) Extend B Reagent: Comprises reversibly terminated
unlabeled nucleotides and polymerase, but lacks labeled nucleotide
analogues. The composition of Extend B is as follows:
TABLE-US-00002 [0055] Component Conc PNSE (% wt/vol) 0.005% Tris x
HCl (pH 8.8), mM 50 NaCl (mM) 50 EDTA (mM) 1 MgSO4 (mM) 10 Glycerol
(% wt/vol) 0.01% Therminator IX* (U/ml) 10 N3-dCTP (.mu.M) 21
N3-dTTP (.mu.M) 17 N3-dATP (.mu.M) 21 N3-dGTP (.mu.M) 2 *Alkylated
free Cysteine
[0056] 3) Wash solution 1 with a detergent (e.g., polysorbate 20)
citrate buffer (e.g., saline) [0057] 4) Cleave Reagent: A cleaving
solution composition is as follows:
TABLE-US-00003 [0057] Component Conc NaOH (mM) 237.5 TrisHCl (pH
8.0) (mM) 237.5 TCEP (mM) 50
[0058] 5) Wash solution 2 with a detergent (e.g., polysorbate 20) a
tris(hydroxymethyl)-aminomethane (Tris) buffer.
II. Polyphenolic Sequencing Additives
[0059] In one embodiment, the present invention contemplates
compositions and compounds that are polyphenolic compounds as
antioxidant additives which improve methods of sequencing by
synthesis. In one embodiment, the polyphenolic compound includes,
but is not limited to, gallic acid, gentisic acid, pryocatechol,
pyrogallol, hydroquinone, and/or resorcinol.
[0060] One embodiment of the invention includes addition of
polyphenolic additives in sequencing reactions of Cleavage solution
to improve lifetime of solution, to reduce undesirable free radical
driven side reactions, allow premixing, and as a result improve
sequencing performance. Another embodiment includes addition of
polyphenolic compounds to Imaging solution to improve lifetime of
solution and to reduce undesirable free radical driven side
reactions.
[0061] Yet another embodiment of the invention is addition of
polyphenolic compounds to Extend solution to improve lifetime of
solution and to reduce undesirable free radical driven side
reactions. In one embodiment of the invention the polyphenolic
compounds are antioxidants. In yet another embodiment the
polyphenolic compounds have free radical scavenging properties.
[0062] Current sequencing processes includes a Cleave solution with
buffered phosphine to deprotect 3'-OH groups and disulfide dye
linker. This solution has limited activity window due to oxygen
absorption from the air and open Cleave container on GeneReader
instrument. Literature reports indicate that phosphines such as
TCEP (Tris(carboxyethyl)phosphine) can lead to by-products with
thiol-based compounds. One example is conversion of cysteine to
dehydroalanine residues in peptides. The process is thought to
involve a free radical path. Zhouxi et al., Rapid Commun Mass
Spectrom. (2010) 24(3):267-275. Analysis of SBS nucleotides
cleavage reactions in solution by means of LC-MS indicates
formation of additional species in addition to expected products.
Analysis of sequencing products by means of denaturing capillary
electrophoresis indicates presence of non-full length products.
[0063] One Imaging solution currently used on GeneReader uses an
active oxygen scavenging system and radical/triplet state
scavenger. Extend A/B solutions do not contain reducing agents due
to compatibility with sequencing chemistry (disufide bridges). In
another embodiment, polyphenolic anti-oxidant compounds are
identified that actively scavenge dissolved oxygen out of a Cleave
solution and prolong useful life time of a Cleave solution and
increase its efficiency. In another embodiment, improved
performance of a Cleave step and reduction of side reactions is
disclosed.
[0064] Preliminary data included tests with polyphenolic additives
to a Cleave reagent to assess improvements SBS performance. The
results suggested that several polyphenolic compounds had a high
antioxidant potential. For example, two promising polyphenolic
compounds were chosen for further studies: gallic acid and gentisic
acid. In addition to reducing available oxygen and having positive
impact on the lifetime of a Cleave solution (reducing agent) these
two polyphenolic compounds had additional positive impact on
sequencing performance possibly due to reducing side reactions.
[0065] Sequencing SBS chemistry performance was assessed using
standard baseline SBS conditions (50 mM TCEP at pH=8.5) versus runs
with additives at the same pH. To this effect, the following
conclusions were made based on experimental data and described
further in detail: [0066] 1. Analysis of cleavage reactions at
nucleotide level by means of analytical HPLC and LC-MS (labeled and
terminating nucleotide) in the absence and presence of additives
was performed. These analyses revealed that cleavage reactions
containing antioxidant compounds (50 mM) including, but not limited
to, gallic acid, gentisic acid, pyrocatechol or pyrogallol revealed
fewer side products. [0067] 2. Sequencing runs containing Cleave
solution with phosphine only or containing gallic acid, gentisic
acid, pyrocatechol, pyrogallol were conducted at varying
concentrations. Analysis of sequencing KPIs indicates better
performance as indicated by lower error rate, higher signal margin
and lower lead values as well as lower false positive rate for
variants effectively extending usable read length by 25-50%.
Analysis by CE reveals higher yield of full length sequencing
products. Flowcell data homegeneity was also improved indicating
possibly beneficial impact on Cleave solution clearance from the
flowcell. [0068] 3. Sequencing runs containing gallic acid,
gentisic acid, pyrocatechol, or pyrogallol in Imaging solution
indicated better performance as shown by lower error rate, higher
signal margin and lower lead values as well as lower false positive
rate for variants. Analysis by CE reveals higher yield of full
length sequencing products. [0069] 4. Identification of additional
compounds with similar properties were identified. Additional
compounds evalauted as Cleave additives showed similar benefits as
demonstrated in 1-3 above. These compounds contain poly-phenolic
groups or have antioxidant properties. Compounds tested include
pyrogallol, pyrocatechol, hydroquinone, resorcinol, but it is not
intended that the invention is limited to this set of
compounds.
[0070] A. Gallic Acid
[0071] Gallic acid (GA) has been shown to improve sequencing
performance and allow the system to provide a filtered trimmed
sequence output of 1 Gb.
[0072] Gallic acid is found in a number of land plants, such as the
parasitic plant, Cynomorium coccineum, the aquatic plant,
Myriophyllum spicatum, and the blue-green alga, Microcystis
aeruginosa. Zucca et al., "Evaluation of Antioxidant Potential of
"Maltese Mushroom" (Cynomorium coccineum) by Means of Multiple
Chemical and Biological Assays" Nutrients 5(1):149-161; and Nakai,
S (2000). "Myriophyllum spicatum-released allelopathic polyphenols
inhibiting growth of blue-green algae Microcystis aeruginosa" Water
Research 34(11):3026-3032. Gallic acid is a trihydroxybenzoic acid,
a type of phenolic acid, a type of organic acid, also known as
3,4,5-trihydroxybenzoic acid, found in gallnuts, sumac, witch
hazel, tea leaves, oak bark, and other plants. The chemical formula
is C.sub.6H.sub.2(OH).sub.3COOH, having the following
structure:
##STR00001##
[0073] Gallic acid is found both free and as part of hydrolyzable
tannins. The gallic acid groups are usually bonded to form dimers
such as ellagic acid. Hydrolysable tannins break down on hydrolysis
to give gallic acid and glucose or ellagic acid and glucose, known
as gallotannins and ellagitannins respectively. Gallic acid may
also form intermolecular esters (depsides) such as digallic and
trigallic acid, and cyclic ether-esters (depsidones) and is
commonly used in the pharmaceutical industry. Fiuza et al.,
"Phenolic acid derivatives with potential anticancer properties--a
structure--activity relationship study. Part 1: Methyl, propyl and
octyl esters of caffeic and gallic acids". Bioorganic &
Medicinal Chemistry (Elsevier) 12 (13): 3581-3589. Gallic acid is
easily freed from gallotannins by acidic or alkaline hydrolysis.
When gallic acid is heated with concentrated sulfuric acid,
rufigallol is produced by condensation. Oxidation with arsenic
acid, permanganate, persulfate, or iodine yields ellagic acid, as
does reaction of methyl gallate with iron(III) chloride.
[0074] Gallic acid is formed from 3-dehydroshikimate by the action
of the enzyme shikimate dehydrogenase to produce
3,5-didehydroshikimate. This latter compound tautomerizes to form
the redox equivalent gallic acid, where the equilibrium lies
essentially entirely toward gallic acid because of the
coincidentally occurring aromatization. Dewick et al., (1969)
"Phenol biosynthesis in higher plants. Gallic acid". Biochemical
Journal 113 (3): 537-542. Gallate dioxygenase and gallate
decarboxylase are enzymes responsible for the degradation of gallic
acid.
[0075] The data presented herein demonstrate that SBS runs with
gallic acid showed no bead loss as shown by its comparison to SBS
runs using indole-3-propionic acid (IPA). See, FIG. 11. These data
show that gallic acid, like IPA, does not undergo any bead loss
during SBS, contradicting previous reports. Although it is not
necessary to understand the mechanism of an invention, it is
believed that when a certain pH value is reached, Gallic acid
undergoes an irreversible transition to a new chemical entity. It
is believed that this chemical transition generates an active
gallic acid derivative that is responsible for previously observed
bead loss. In one embodiment, the present invention contemplates an
SBS reagent (e.g., for example, a Cleave 1 buffer, where gallic
acid is not mixed in the absence of TCEP. When gallic acid and TCEP
are both present in the SBS reagent, no bead loss is observed.
[0076] This lack of bead loss is reflected in data showing improved
error rates when SBS runs were compared between the presence of
gallic acid, IPA and ascorbic acid. All three additives improved
SBS error rates when compared to no additive. See, FIG. 12. The
data show that gallic acid (AddG) results in a raw error rate that
is significantly better than no additive and IPA (AddC).
[0077] B. Gentisic Acid
[0078] Gentisic acid is a dihydroxybenzoic acid. It is a derivative
of benzoic acid and a minor (1%) product of the metabolic break
down of aspirin. It is also found in the African tree Alchornea
cordifolia and in wine.
[0079] Gentisic acid may be produced by carboxylation of
hydroquinone:
C.sub.6H.sub.4(OH).sub.2+CO.sub.2.fwdarw.C.sub.6H.sub.3(CO.sub.2H)(OH).s-
ub.2
This conversion is an example of a Kolbe-Schmitt reaction and
results in the following structure:
##STR00002##
Alternatively the compound can be synthesized from Salicylic acid
via Elbs persulfate oxidation (50% yield). Schock Jr. et al.,
(1951) "The Persulfate Oxidation of Salicylic Acid.
2,3,5-Trihydroxybenzoic Acid" The Journal of Organic Chemistry
16(11):1772-1775. As a hydroquinone, gentisic acid is readily
oxidized and is used as an antioxidant excipient in some
pharmaceutical preparations. In the laboratory, it is used as a
sample matrix in matrix-assisted laser desorption/ionization
(MALDI) mass spectrometry, and has been shown to conveniently
detect peptides incorporating the boronic acid moiety by MALDI.
Strupat et al., (1991) "2,5-Dihidroxybenzoic acid: a new matrix for
laser desorption-ionization mass spectrometry" Int. J. Mass
Spectrom. Ion Processes 72(111):89-102; and Crumpton et al., (2011)
"Facile Analysis and Sequencing of Linear and Branched Peptide
Boronic Acids by MALDI Mass Spectrometry" Analytical Chemistry
83(9):3548-3554.
[0080] C. Pryocatechol
[0081] Pyrocatechol, also known as catechol or
1,2-dihydroxybenzene, is an organic compound with the molecular
formula C.sub.6H.sub.4(OH)2. It is the ortho isomer of the three
isomeric benzenediols. This colorless compound occurs naturally in
trace amounts. It was first discovered by destructive distillation
of the plant extract catechin. About 20 million kg are now
synthetically produced annually as a commodity organic chemical,
mainly as a precursor to pesticides, flavors, and fragrances.
[0082] Catechol is produced industrially by the hydroxylation of
phenol using hydrogen peroxide:
C.sub.6H.sub.5OH+H.sub.2O.sub.2.fwdarw.C.sub.6H.sub.4(OH).sub.2+H.sub.2O
and results in the following structure:
##STR00003##
Previously, it was produced by hydrolysis of 2-substituted phenols,
especially 2-chlorophenol, with hot aqueous solutions containing
alkali metal hydroxides. Its methyl ether derivative, guaiacol,
converts to catechol via hydrolysis of the CH3-O bond as promoted
by hydriodic acid.
[0083] D. Pyrogallol
[0084] Pyrogallol is an organic compound with the formula
C.sub.6H.sub.3(OH).sub.3 having the following chemical
structure:
##STR00004##
It is a white solid although because of its sensitivity toward
oxygen, samples are typically brownish. It is one of three isomeric
benzenetriols. It is produced by heating gallic acid that results
in decarboxylation. An alternate preparation involves treating
para-chlorophenoldisulphonic acid with potassium hydroxide.
[0085] E. Hydroquinone
[0086] Hydroquinone, also benzene-1,4-diol or quinol, is an
aromatic organic compound that is a type of phenol, a derivative of
benzene, having the chemical formula C6H4(OH)2, having the
following structure:
##STR00005##
Its chemical structure features two hydroxyl groups bonded to a
benzene ring in a para position. It is a white granular solid.
Substituted derivatives of this parent compound are also referred
to as hydroquinones.
[0087] The reactivity of hydroquinone's O--H groups resembles other
phenols, being weakly acidic. The resulting conjugate base
undergoes easy O-alkylation to give mono- and diethers. Similarly,
hydroquinone is highly susceptible to ring substitution by
Friedel-Crafts reactions such as alkylation. This reaction is
exploited en route to popular antioxidants such as
2-tert-butyl-4-methoxyphenol ("BHA"). The useful dye quinizarin is
produced by diacylation of hydroquinone with phthalic anhydride.
Hydroquinone undergoes oxidation under mild conditions to give
benzoquinone. This process can be reversed. Some naturally
occurring hydroquinone derivatives exhibit this sort of reactivity,
one example being coenzyme Q. Industrially this reaction is
exploited both with hydroquinone itself but more often with its
derivatives where one OH has been replaced by an amine.
[0088] There are various other uses associated with its reducing
power. As a polymerization inhibitor, hydroquinone prevents
polymerization of acrylic acid, methyl methacrylate, cyanoacrylate,
and other monomers that are susceptible to radical-initiated
polymerization. This application exploits the antioxidant
properties of hydroquinone.
[0089] Hydroquinone can undergo mild oxidation to convert to the
compound parabenzoquinone, C6H4O2, often called p-quinone or simply
quinone. Reduction of quinone reverses this reaction back to
hydroquinone. Some biochemical compounds in nature have this sort
of hydroquinone or quinone section in their structures, such as
Coenzyme Q, and can undergo similar redox interconversions.
[0090] Hydroquinone can lose an H+ from both to form a diphenolate
ion. The disodium diphenolate salt of hydroquinone is used as an
alternating comonomer unit in the production of the polymer
PEEK.
[0091] F. Resorcinol
[0092] Resorcinal is the 1,3-isomer (or meta-isomer) of benzenediol
with the formula C.sub.6H.sub.4(OH).sub.2, having the following
structure:
##STR00006##
Resorcinol crystallizes from benzene as colorless needles that are
readily soluble in water, alcohol, and ether, but insoluble in
chloroform and carbon disulfide. Sodium amalgam reduces it to
dihydroresorcin, which when heated to 150 to 160.degree. C. with
concentrated barium hydroxide solution gives .gamma.-acetylbutyric
acid and when fused with potassium hydroxide, resorcinol yields
phloroglucin, pyrocatechol, and diresorcin.
Experimental
EXAMPLE 1
[0093] In one embodiment, the present invention contemplates a SBS
method comprising the steps shown in Table 1. See Olejink et al.,
"Methods And Compositions For Inhibiting Undesired Cleaving Of
Labels" U.S. Pat. No. 8,623,598 (herein incorporated by reference
in its entirety).
TABLE-US-00004 TABLE 1 An Exemplary SBS Workflow Fluid Movements
Volume Speed Station Temp Time Step Reagent mL mL/s Number .degree.
C. [s] 1. Dispense Reagent Reagent 1 100 67 3 65 7 2. Incubate
Reagent Reagent 1 n/a n/a 3 65 210 3. Dispense Reagent Reagent 2
100 67 4 65 7 4. Incubate Reagent Reagent 2 n/a n/a 4 65 210 5.
Dispense Reagent Reagent 3 330 27 5 Ambient 12 6. Dispense Reagent
Reagent 200 27 5 Ambient 15 4 + 5 7. Image n/a n/a n/a 11 Ambient
210 8. Dispense Reagent Reagent 3 330 27 20 65 12 9. Dispense
Reagent Reagent 6 100 67 1 65 7 10. Incubate Reagent Reagent 6 n/a
n/a 1 65 210 11. Incubate Reagent Reagent 6 n/a n/a 2 65 210 12.
Dispense Reagent Reagent 7 990 27 2 65 37 13. Go to Step 1 Reagent
1 = Extend A; Reagent 2 = Extend B; Reagent 3 = Wash; Reagent 4 =
Image A; Reagent 5 = Image B; Reagent 6 = Cleave; and Reagent 7 =
Wash 11
[0094] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described methods and system of
the invention will be apparent to those skilled in the art without
departing from the scope and spirit of the invention. Although the
invention has been described in connection with specific
embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. Indeed,
various modifications of the described modes for carrying out the
invention which are obvious to those skilled in the art and in
fields related thereto are intended to be within the scope of the
following claims.
* * * * *