U.S. patent application number 15/092063 was filed with the patent office on 2016-10-06 for dna templates for rolling circle amplification.
This patent application is currently assigned to McMaster University. The applicant listed for this patent is McMaster University. Invention is credited to Yingfu Li.
Application Number | 20160289741 15/092063 |
Document ID | / |
Family ID | 57015139 |
Filed Date | 2016-10-06 |
United States Patent
Application |
20160289741 |
Kind Code |
A1 |
Li; Yingfu |
October 6, 2016 |
DNA TEMPLATES FOR ROLLING CIRCLE AMPLIFICATION
Abstract
The present application provides circular templates that are
high in adenine and cytosine and methods and uses thereof for
rolling circle amplification (RCA). These templates are
particularly suitable for biosensing applications involving
RCA.
Inventors: |
Li; Yingfu; (Dundas,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
McMaster University |
Hamilton |
|
CA |
|
|
Assignee: |
McMaster University
Hamilton
CA
|
Family ID: |
57015139 |
Appl. No.: |
15/092063 |
Filed: |
April 6, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62143256 |
Apr 6, 2015 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6823 20130101;
C12Q 2531/125 20130101; C12Q 1/682 20130101; C12Q 1/6806 20130101;
C12Q 1/6844 20130101; C12Q 1/6844 20130101; G01N 33/5308
20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method of amplification comprising: a) annealing a primer to a
circular template; wherein the circular template comprises a region
complementary to the primer and an AC rich nucleotide region; b)
amplifying the circular template under rolling circle amplification
conditions; and c) optionally, detecting the product of the rolling
circle amplification.
2. The method of claim 1, further comprising subjecting the product
of the amplification to restriction digestion.
3. The method of claim 1, wherein the primer is linked to a
recognition moiety that detects the presence of an analyte.
4. The method of claim 3, wherein the recognition moiety is an
aptamer that changes conformation in the presence of the
analyte.
5. The method of claim 3, wherein the recognition moiety is an
antibody or nucleic acid probe that binds the analyte.
6. The method of claim 3, wherein the analyte is a nucleic acid,
protein or small molecule.
7. The method of claim 1, wherein the AC rich nucleotide region is
at least 70% AC rich, at least 80% AC rich, or at least 85% AC
rich.
8. The method of claim 1, wherein the AC rich nucleotide region
comprises one of the sequence as shown in SEQ ID NOs: 1-10 or a
variant thereof.
9. The method of claim 1 wherein the AC rich nucleotide region
comprises a scrambled sequence that contains the nucleotide content
of one of the sequences shown in SEQ ID NOs: 1-10 or a variant
thereof.
10. The method of claim 9, wherein the AC rich nucleotide region
comprises one of the sequences shown in SEQ ID NOs:36-40 or a
variant thereof.
11. The method of claim 1, wherein conditions that allow for
rolling circle amplification comprise the presence of phi29 DNA
polymerase.
12. The method of claim 1, wherein prior to a), the method
comprises increasing the AC content of the circular template.
13. A kit comprising a circular template, wherein the circular
template comprises a region complementary to a primer and an AC
rich nucleotide region; and one or more reagents for carrying out
rolling circle amplification.
14. The kit of claim 13, wherein the one or more reagents for
carrying out rolling circle amplification is a DNA polymerase,
dNTPs, the primer, labelled probes and/or reaction buffer.
15. The kit of claim 13, wherein the AC rich nucleotide region is
at least 70% AC rich, at least 80% AC rich, or at least 85% AC
rich.
16. The kit of claim 13, wherein the AC rich nucleotide region
comprises the sequence as shown in SEQ ID NOs: 1-10 or a variant
thereof.
17. The kit of claim 13, wherein the AC rich nucleotide region
comprises a scrambled sequence that contains the nucleotide content
of the sequences shown in SEQ ID NOs: 1-10 or a variant
thereof.
18. The kit of claim 17, wherein the AC rich nucleotide region
comprises one of the sequences as shown in SEQ ID NOs:36-40 or a
variant thereof.
19. The kit of claim 13, wherein the DNA polymerase is phi29 DNA
polymerase.
20. A nucleic acid molecule comprising one of the sequences as
shown in SEQ ID NOs:1-10 and 36-40 or a variant thereof.
Description
RELATED APPLICATIONS
[0001] The present application claims the benefit of provisional
patent application No. 62/143,256, filed Apr. 6, 2015, the contents
of which are herein incorporated by reference.
INCORPORATION OF SEQUENCE LISTING
[0002] A computer readable form of the Sequence Listing
"3244-P48282US01_SequenceListing.txt" (8,192 bytes), submitted via
EFS-WEB and created on Apr. 6, 2016, is herein incorporated by
reference.
FIELD
[0003] The application relates to DNA templates for rolling circle
amplification and methods and uses thereof.
BACKGROUND
[0004] Rolling circle amplification (RCA) involves growing a long
DNA chain with a repetitive sequence by continuously adding
nucleotides to a primer annealed to a circular DNA
template..sup.[1,2] The DNA polymerases used for this reaction,
such as phi29 DNA polymerase, are special because they possess both
strand displacement ability and high processivity..sup.[3,4] These
properties empower these enzymes to make cyclic copying of the same
circular template, producing extremely long DNA molecules with
thousands of sequence repeats.
[0005] RCA has emerged as a popular DNA amplification technique
because it offers some key advantages that cannot be matched by
polymerase chain reaction (PCR). One advantage is that it does not
require equipment: while PCR needs temperature cycling, RCA is an
isothermal process. No need for special equipment makes RCA better
suited for point-of-care (POC) and field applications. Another
advantage is the compatibility with most molecular recognition
elements (MREs). Unlike PCR that requires a high-temperature
(>90.degree. C.) step that deactivates most MREs, RCA can be
conducted at temperatures that are more suited for optimal MRE
functions. This particular benefit facilitates the use of RCA for
the detection of not only nucleic acid targets (both DNA and
RNA),.sup.[5-8] but also other analytes (small molecules, proteins
and even cells).sup.[9-11] when combined with functional nucleic
acid probes (e.g. aptamers and DNAzymes)..sup.[12-16] The key
element in nearly all reported biosensing strategies involving RCA
is linking a molecular recognition event into the formation of a
primer-template complex from which the DNA polymerase synthesizes
long-chain DNA amplicons. Such coupling delivers high detection
sensitivity, which is crucial for diagnostic and biosensing
applications.
[0006] Cyclic copying of the same circular DNA template, however,
comes with an inherent disadvantage: when the target concentration
is very low (thus the formation of a limited amount of
primer-template complex), the RCA process needs a long time to
produce enough amplicons to be detected. This drawback makes RCA
less desirable for undertakings that require time-sensitive
detection of a trace amount of target, a hallmark of POC and field
applications. Therefore, strategies that can significantly improve
the efficiency of the RCA process are highly desirable. Branched
RCA where an additional primer is used to further amplify the RCA
product represents an excellent strategy..sup.[17]
SUMMARY
[0007] It has been found that increasing the adenine and cytosine
(AC) content of a circular template increases the efficiency of
rolling circle amplification. Accordingly, in some embodiments, the
present application includes optimized circular templates, which
may be used in a rolling circle amplification, such as for
biosensing applications involving RCA, for example which link a
molecular recognition event to the formation of a primer-template
complex from which the DNA polymerase synthesizes long-chain DNA
amplicons (or concatemers).
[0008] Accordingly, herein provided is a method of amplification
comprising: [0009] a) annealing a primer to a circular template;
wherein the circular template comprises a region complementary to
the primer and an AC rich nucleotide region; and [0010] b)
amplifying the circular template under rolling circle amplification
conditions.
[0011] In an embodiment, the method further comprises subjecting
the product of the amplification to restriction digestion.
[0012] In another embodiment, prior to a), the method comprises
increasing the AC content of the circular template.
[0013] In another embodiment, the method further comprises c)
detecting the product of the rolling circle amplification.
[0014] In an embodiment, the primer is linked to a recognition
moiety. Any recognition moiety that is able to detect the presence
of an analyte may be used, for example, an aptamer that changes
conformation in the presence of the analyte, a DNAzyme that cleaves
RNA in the presence of the analyte, or an antibody or nucleic acid
probe that binds the analyte.
[0015] In one embodiment, the analyte is a nucleic acid, protein or
small molecule.
[0016] In an embodiment, the AC rich nucleotide region is at least
70% AC rich, at least 80% AC rich, or at least 85% AC rich.
[0017] In a particular embodiment, the AC rich nucleotide region
comprises one of the sequences as shown in SEQ ID NOs: 1-10 or a
variant thereof. In another embodiment, the AC rich nucleotide
region comprises a scrambled sequence that contains the nucleotide
content of one of the sequences shown in SEQ ID NOs: 1-10 or a
variant thereof, such as the CTA5 mutants shown in SEQ ID NOs:36-40
or variants thereof. In an embodiment, the variant maintains
substantially the same amount of adenine and cytosine as the
reference sequence.
[0018] In an embodiment, the rolling circle amplification
conditions comprise the presence of phi29-, Bst- or Vent exo-DNA
polymerase. In an embodiment, the rolling circle amplification
conditions comprise the presence of phi29-DNA polymerase.
[0019] Also provided herein are nucleic acid molecules comprising
the AC rich nucleotide region as well as circular template
molecules disclosed herein and a use of a circular template
disclosed herein for rolling circle amplification.
[0020] Even further provided is a method of optimizing rolling
circle amplification comprising increasing the AC content of a
circular template before amplification.
[0021] Yet further provided is a kit comprising a circular template
disclosed herein and one or more reagents necessary for carrying
out rolling circle amplification, such as a suitable DNA
polymerase, NTPs, a primer complementary to the circular template,
labelled probes, reaction buffer, and instructions for use. In one
embodiment, the DNA polymerase is phi29 DNA polymerase.
[0022] Other features and advantages of the present application
will become apparent from the following detailed description. It
should be understood, however, that the detailed description and
the specific examples, while indicating embodiments of the
application, are given by way of illustration only and the scope of
the claims should not be limited by these embodiments, but should
be given the broadest interpretation consistent with the
description as a whole.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] The embodiments of the application will now be described in
greater detail with reference to the attached drawings in
which:
[0024] FIG. 1 shows an in vitro selection of optimal DNA templates
for RCA in one embodiment of the application. (a) In vitro
selection scheme. (b) The top 10 CTA sequences (SEQ ID NOs:1-10
respectively). Only the random-sequence domain is shown. (c) The
sequence abundance (SA) and nucleotide distributions of the top 10
CTAs, and their matching CTBs.
[0025] FIG. 2 shows determination of RCA efficiency (RE) of
selective CTAs and CTBs in an embodiment of the application. (a)
dPAGE analysis of digested RCA products obtained from varying time
of RCA with CTA1, CTB1, and LB. The top band: digested RCA monomer
(60 nt); the bottom band: DNA loading control (51 nt). ARU: average
repeating units of the RCA product from a given circular template.
(b) ARU vs. RCA time for CTA1, CTB1 and LB. (c) RE values of 5
CTA/CTB pairs. ARU values that were used to derive RE values are
provided in Table 2. SD: standard deviation. (d) Percent ACGT in
CTA5, CTA109 and CTA1548.
[0026] FIG. 3 shows the RCA efficiency comparison of CTA5 and CTA5
mutants (SEQ ID NOs:5, 36, 37, 38, 39 and 40, respectively) in an
exemplary embodiment of the application. ARU values that were used
to derive RE values are provided in Table 2.
[0027] FIG. 4 is a comparison of time-dependent amplicon production
using CTA5 (grey line) and LB (black line) as the circular
templates in an exemplary embodiment of the application. RPC:
relative production of RCA product at a given template
concentration. CT: circular template. RPC=100.times.C.sub.M,
t/C.sub.M, 320 where CM, t is the concentration of digested RCA
product of CTA5 or LB at time t and CM, 320 is the concentration
produced from CTA5 at 320 min.
[0028] FIG. 5 shows thrombin detection using CTA5-assisted RCA in
an exemplary embodiment of the application. (a) Detection strategy.
(b) The sequences of circular templates (SEQ ID NO:16 and 30,
respectively) and the aptamer probe (SEQ ID NO:31). (c) Comparison
of time-dependent amplicon formation from CTA5 and CDT1. (d)
Fluorescence spectra of SYBR Gold-RCA product mixtures obtained
with CTA5 and CDT1. (e) Relative fluorescence (RF) vs. thrombin
concentration. RF=F/Fc, where F is the fluorescence of a given
mixture and Fc is the fluorescence of the no RCA control. The RCA
time for d and e is 60 min.
[0029] FIG. 6 shows time-dependent digestion of RCA products made
from CTA1 using EcoRV in an exemplary embodiment of the
application. The RCA reaction was performed at 30.degree. C. for 20
min in 50 .mu.L of 1.times.RCA buffer containing 0.4 nM CTA1, 2
.mu.M DT1, 1 mM each of dGTP, dATP, dTTP, dCTP, and 5 U phi29 DNA
polymerase. The digestion reaction was performed at 37.degree. C.
for 0.5, 1, 2, 4, 8 and 16 h in 10 .mu.L made of 5 .mu.L of the
above RCA reaction mixture, 2 .mu.L of 50 .mu.M DT2, and 1 .mu.L of
10.times. Fast Digestion Buffer and 2 .mu.L of FastDigest EcoRV.
DT2 was added to make the EcoRV recognition site into double
stranded DNA to facilitate the restriction digestion. The reaction
mixture was then subjected to dPAGE analysis.
DETAILED DESCRIPTION
I. Definitions
[0030] Unless otherwise indicated, the definitions and embodiments
described in this and other sections are intended to be applicable
to all embodiments and aspects of the present application herein
described for which they are suitable as would be understood by a
person skilled in the art.
[0031] The term "analyte" as used herein means any agent,
including, but not limited to, nucleic acids, small inorganic and
organic molecules, metal ions, hormonal growth factors,
biomolecules, toxins, biopolymers (such as carbohydrates, lipids,
peptides and proteins), cells, tissues and microorganisms
(including bacteria and viruses), for which one would like to sense
or detect. In an embodiment, the analyte is either isolated from a
natural source or is synthetic. The term analyte also includes
mixtures of compounds or agents such as, but not limited to,
combinatorial libraries and samples from an organism or a natural
environment.
[0032] The term "AC rich nucleotide region" as used herein refers
to a nucleic acid sequence that has at least 65%, at least 70%, at
least 75%, at least 80% or at least 85% content made of adenine (A)
and/or cytosine (C) residues. In the Examples section, the AC rich
nucleotide region corresponds to the 35 nt random nucleotide region
when it is enriched for adenine and cytosine.
[0033] The term "nucleic acid" refers to polynucleotides such as
deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
[0034] The term "recognition moiety" as used herein refers to an
agent that is able to recognize the presence of an analyte.
Recognition moieties, include without limitation, aptamers,
structure-switching aptamers, reporter aptamers, DNAzymes,
antibodies, and nucleic acid probes.
[0035] The term "aptamer" as used herein refers to short,
chemically synthesized, single stranded (ss) RNA or DNA
oligonucleotides which fold into specific three-dimensional (3D)
structures that bind to a specific analyte with dissociation
constants, for example, in the pico- to nano-molar range.
[0036] The term "structure-switching nucleic acid aptamers" or
"reporter nucleic acid aptamers" as used herein refers to
aptamer-based reporters that function by switching structures from
a DNA/DNA or RNA/RNA complex to a DNA/analyte or RNA/analyte
complex.
[0037] The term "concatemeric nucleic acid molecules" or
"concatemer" as used herein refers to a long continuous DNA or RNA
molecule that contains multiple copies of the same DNA or RNA
sequences linked in a tandem series.
[0038] The term "rolling circle amplification" as used herein
refers to a unidirectional nucleic acid replication that can
rapidly synthesize multiple copies of circular molecules of DNA or
RNA. In an embodiment, rolling circle amplification is an
isothermal enzymatic process where a short DNA or RNA primer is
amplified to form a long single stranded DNA or RNA using a
circular DNA template and an appropriate DNA or RNA polymerase. The
product of this process is a concatemer containing ten to thousands
of tandem repeats that are complementary to the circular
template.
[0039] The phrase "detecting the product of the rolling circle
amplification" as used herein refers to detection of concatemers,
for example, by colorimetric, electrochemical and/or spectroscopic
methods. For example, the recognition moiety may be an aptamer that
changes conformation in the presence of an analyte allowing the
primer to anneal to the circular template to allow for rolling
circle amplification and the concatemer produced is detected using
a labelled probe that is complementary to a portion of the product.
Other biosensing strategies that utilize rolling circle
amplification are known in the art and are encompassed herein.
[0040] The term "primer" as used herein refers to a nucleic acid
sequence, whether occurring naturally as in a purified restriction
digest or produced synthetically, which is capable of acting as a
point of synthesis when placed under conditions in which synthesis
of a primer extension product, which is complementary to a nucleic
acid strand is induced (e.g. in the presence of nucleotides and an
inducing agent such as DNA polymerase and at a suitable temperature
and pH). The primer is sufficiently long to prime the synthesis of
the desired extension product in the presence of the inducing
agent. The exact length of the primer will depend upon factors,
including temperature, sequences of the primer and the methods
used. A primer typically contains 15-25 or more nucleotides,
although it can contain less. The factors involved in determining
the appropriate length of primer are readily known to one of
ordinary skill in the art. It can be a DNA, an RNA, or a chimeric
DNA/RNA sequence.
[0041] The term "probe" refers to a nucleic acid sequence that will
hybridize to a nucleic acid target sequence. In one example, the
probe hybridizes to the circular template or its complement. The
length of probe depends on the hybridization conditions and the
sequences of the probe and nucleic acid target sequence. In one
embodiment, the probe is 8-100, 8-200 or 8-500 nucleotides in
length, such as 8-10, 11-15, 16-20, 21-25, 26-50, 51-75, 76-100,
101-150 or 151-200 nucleotides in length or at least 200, 250, 400,
500 or more nucleotides in length. In other embodiments, 10, 15, 20
or 25 nucleotides provide a lower end for the aforementioned
nucleotide ranges.
[0042] The term "circular template" as used herein refers to a
nucleic acid sequence of at least 20 nucleotides that is ligated to
form a circular nucleic acid molecule that can serve as a template
for rolling circle amplification.
[0043] The term "sequence identity" as used herein refers to the
percentage of sequence identity between two polypeptide sequences
or two nucleic acid sequences. To determine the percent identity of
two amino acid sequences or of two nucleic acid sequences, the
sequences are aligned for optimal comparison purposes (e.g., gaps
can be introduced in the sequence of a first amino acid or nucleic
acid sequence for optimal alignment with a second amino acid or
nucleic acid sequence). The amino acid residues or nucleotides at
corresponding amino acid positions or nucleotide positions are then
compared. When a position in the first sequence is occupied by the
same amino acid residue or nucleotide as the corresponding position
in the second sequence, then the molecules are identical at that
position. The percent identity between the two sequences is a
function of the number of identical positions shared by the
sequences (i.e., % identity=number of identical overlapping
positions/total number of positions.times.100%). In one embodiment,
the two sequences are the same length. The determination of percent
identity between two sequences can also be accomplished using a
mathematical algorithm. One non-limiting example of a mathematical
algorithm utilized for the comparison of two sequences is the
algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci.
U.S.A. 87:2264-2268, modified as in Karlin and Altschul, 1993,
Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is
incorporated into the NBLAST and XBLAST programs of Altschul et
al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be
performed with the NBLAST nucleotide program parameters set, e.g.,
for score=100, wordlength=12 to obtain nucleotide sequences
homologous to a nucleic acid molecules of the present application.
BLAST protein searches can be performed with the XBLAST program
parameters set, e.g., to score-50, wordlength=3 to obtain amino
acid sequences homologous to a protein molecule of the present
invention. To obtain gapped alignments for comparison purposes,
Gapped BLAST can be utilized as described in Altschul et al., 1997,
Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-BLAST can be
used to perform an iterated search which detects distant
relationships between molecules. When utilizing BLAST, Gapped
BLAST, and PSI-Blast programs, the default parameters of the
respective programs (e.g., of XBLAST and NBLAST) can be used (see,
e.g., the NCBI website). Another non-limiting example of a
mathematical algorithm utilized for the comparison of sequences is
the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an
algorithm is incorporated in the ALIGN program (version 2.0) which
is part of the GCG sequence alignment software package. When
utilizing the ALIGN program for comparing amino acid sequences, a
PAM120 weight residue table, a gap length penalty of 12, and a gap
penalty of 4 can be used. The percent identity between two
sequences can be determined using techniques similar to those
described above, with or without allowing gaps. In calculating
percent identity, typically only exact matches are counted.
[0044] By "at least moderately stringent hybridization conditions"
it is meant that conditions are selected which promote selective
hybridization between two complementary nucleic acid molecules in
solution. Hybridization may occur to all or a portion of a nucleic
acid sequence molecule. The hybridizing portion is typically at
least 15 (e.g. 20, 25, 30, 40 or 50) nucleotides in length. Those
skilled in the art will recognize that the stability of a nucleic
acid duplex, or hybrids, is determined by the Tm, which in sodium
containing buffers is a function of the sodium ion concentration
and temperature (Tm=81.5.degree. C.-16.6 (Log 10
[Na+])+0.41(%(G+C)-600/l), or similar equation). Accordingly, the
parameters in the wash conditions that determine hybrid stability
are sodium ion concentration and temperature. In order to identify
molecules that are similar, but not identical, to a known nucleic
acid molecule a 1% mismatch may be assumed to result in about a
1.degree. C. decrease in Tm, for example if nucleic acid molecules
are sought that have a >95% identity, the final wash temperature
will be reduced by about 5.degree. C. Based on these considerations
those skilled in the art will be able to readily select appropriate
hybridization conditions. In some embodiments, stringent
hybridization conditions are selected. By way of example the
following conditions may be employed to achieve stringent
hybridization: hybridization at 5.times. sodium chloride/sodium
citrate (SSC)/5.times.Denhardt's solution/1.0% SDS at Tm-5.degree.
C. based on the above equation, followed by a wash of
0.2.times.SSC/0.1% SDS at 60.degree. C. Moderately stringent
hybridization conditions include a washing step in 3.times.SSC at
42.degree. C. It is understood, however, that equivalent
stringencies may be achieved using alternative buffers, salts and
temperatures. Additional guidance regarding hybridization
conditions may be found in: Current Protocols in Molecular Biology,
John Wiley & Sons, N.Y., 2002, and in: Sambrook et al.,
Molecular Cloning: a Laboratory Manual, Cold Spring Harbor
Laboratory Press, 2001.
[0045] As used in this specification and the appended claims, the
singular forms "a", "an" and "the" include plural references unless
the content clearly dictates otherwise.
[0046] As used in this application and claim(s), the words
"comprising" (and any form of comprising, such as "comprise" and
"comprises"), "having" (and any form of having, such as "have" and
"has"), "including" (and any form of including, such as "include"
and "includes") or "containing" (and any form of containing, such
as "contain" and "contains"), are inclusive or open-ended and do
not exclude additional, unrecited elements or process steps.
[0047] As used in this application and claim(s), the word
"consisting" and its derivatives, are intended to be close ended
terms that specify the presence of stated features, elements,
components, groups, integers, and/or steps, and also exclude the
presence of other unstated features, elements, components, groups,
integers and/or steps.
[0048] The term "consisting essentially of", as used herein, is
intended to specify the presence of the stated features, elements,
components, groups, integers, and/or steps as well as those that do
not materially affect the basic and novel characteristic(s) of
these features, elements, components, groups, integers, and/or
steps.
[0049] The terms "about", "substantially" and "approximately" as
used herein mean a reasonable amount of deviation of the modified
term such that the end result is not significantly changed. These
terms of degree should be construed as including a deviation of at
least .+-.5% of the modified term if this deviation would not
negate the meaning of the word it modifies.
[0050] The term "and/or" as used herein means that the listed items
are present, or used, individually or in combination. In effect,
this term means that "at least one of" or "one or more" of the
listed items is used or present.
[0051] The term "suitable" as used herein means that the selection
of the particular compound or conditions would depend on the
specific synthetic manipulation to be performed, and the identity
of the molecule(s) to be transformed, but the selection would be
well within the skill of a person trained in the art. All
process/method steps described herein are to be conducted under
conditions sufficient to provide the product shown. A person
skilled in the art would understand that all reaction conditions,
including, for example, reaction solvent, reaction time, reaction
temperature, reaction pressure, reactant ratio and whether or not
the reaction should be performed under an anhydrous or inert
atmosphere, can be varied to optimize the yield of the desired
product and it is within their skill to do so.
II. Methods and Uses
[0052] A new approach that can make the rolling circle
amplification (RCA) process more effective at low primer-template
concentrations has been developed. In particular, the present
inventor has found optimal DNA templates that exist that allow for
the production of more DNA amplicons within an allocated time, and
has shown that these molecules can be isolated from a
random-sequence DNA pool using "in vitro selection"..sup.[18]
[0053] Herein provided is a method of amplification comprising:
[0054] a) annealing a primer to a circular template; wherein the
circular template comprises a region complementary to the primer
and an AC rich nucleotide region; and [0055] b) amplifying the
circular template under conditions that allow rolling circle
amplification.
[0056] In an embodiment, the method further comprises subjecting
the product of the amplification to restriction digestion. In such
an embodiment, the circular template may be designed to include a
restriction enzyme site at the position of ligation of the linear
sequence such that upon restriction enzyme digestion of the
product, the concatemer is separated into individual monomer
amplicons, which can then optionally be detected or quantified. A
person skilled in the art would understand that any restriction
recognition sites would be compatible.
[0057] In another embodiment, the method further comprises c)
detecting the product of the rolling circle amplification. Methods
for detection of products of rolling circle amplification are known
in the art. For example, a nucleotide probe that is complementary
to a portion of the concatemer can be labeled and incubated with
the product under stringent conditions to allow for hybridization
and subsequent detection of the label. Detection includes
qualitative and quantitative detection.
[0058] In an embodiment, the method further comprises selecting a
linear sequence comprising a region complementary to the primer and
an AC rich nucleotide region, followed by circularizing the
sequence to form the circular template prior to a). In an
embodiment, the circularization is performed using DNA ligase, such
as T4 DNA ligase.
[0059] A nucleotide probe may be labelled with a detectable marker
such as a radioactive label which provides for an adequate signal
and has sufficient half-life such as .sup.32P, .sup.3H, .sup.14C or
the like. Other detectable markers which may be used include
antigens that are recognized by a specific labelled antibody,
fluorescent compounds, enzymes, antibodies specific for a labelled
antigen, and chemiluminescent compounds. An appropriate label may
be selected having regard to the rate of hybridization and binding
of the probe to the nucleotide to be detected and the amount of
nucleotide available for hybridization.
[0060] Alternatively, the primer may be labelled with detectable
markers which allow for detection of the amplified product.
Suitable detectable markers are radioactive markers such as
.sup.32P, .sup.35S, .sup.125I, and .sup.3H, luminescent markers
such as chemiluminescent markers, such as luminol, and fluorescent
markers, such as dansyl chloride, fluorcein-5-isothiocyanate, and
4-fluor-7-nitrobenz-2-axa-1,3 diazole, enzyme markers such as
horseradish peroxidase, alkaline phosphatase, .beta.-galactosidase,
acetylcholinesterase, or biotin.
[0061] It will be appreciated that the primer may contain
non-complementary sequences provided that a sufficient amount of
the primer contains a sequence which is complementary to a region
of the circular template disclosed herein, which is to be
amplified, to allow hybridization of the primer to the circular
template.
[0062] In an embodiment, the primer is linked to a recognition
moiety that is able to detect the presence of an analyte. In one
embodiment, the recognition moiety is a functional nucleic acid
probe, such as an aptamer that changes conformation in the presence
of the analyte or a DNAzyme that cleaves an RNA linkage in the
presence of the analyte.
[0063] In other embodiments, the recognition moiety is an antibody
or a nucleic acid probe specific for the analyte.
[0064] In some embodiments, detection by the recognition moiety of
the analyte permits the primer to anneal to the circular template
whereas in the absence of analyte, the primer is unable to anneal
to the circular template. These include, without limitation,
structure-switching aptamers and RNA-cleaving DNAzymes.
[0065] In alternative embodiments, the analyte is immobilized on a
solid support and the recognition moiety then binds to the analyte
on the solid support allowing the rolling circle amplification
product to be immobilized on the solid support. In such an
embodiment, detection of the rolling circle amplification product
occurs after the solid support is washed. The solid support can be
nanoparticles, metal surfaces, inorganic surfaces, organic
surfaces, paper or modified paper surfaces.
[0066] In an embodiment, the analyte is a nucleic acid, protein or
small molecule.
[0067] In one embodiment, the AC rich nucleotide region is at least
70% AC rich, at least 80% AC rich, or at least 85% AC rich.
[0068] In a particular embodiment, the AC rich nucleotide region
comprises one of the sequences as shown in SEQ ID NOs: 1-10 or a
variant thereof. In another embodiment, the AC rich nucleotide
region comprises a scrambled sequence that contains the nucleotide
content of one of the sequences shown in SEQ ID NOs: 1-10 or a
variant thereof. For example, in an embodiment, the AC rich
nucleotide region comprises CTA5 (SEQ ID NO:5) scrambled sequences:
CTA5M1, CTA5M2, CTA5M3, CTA5M4 and CTA5M5 (SEQ ID NOs:36-40) or
variants thereof. A person skilled in the art could readily create
similar scrambled sequences of CTA1-4 (SEQ ID NOs:1-4) and CTA6-10
(SEQ ID NOs:6-10) as well as other CTA5 (SEQ ID NO:5) scrambled
sequences.
[0069] In an embodiment, the variant sequence has at least 50%, or
at least 60%, or at least 70%, or at least 80%, or at least 90%, or
at least 95% sequence identity to the sequences disclosed herein.
In an embodiment, the variant has substantially the same AC content
as the reference sequence.
[0070] In another embodiment, the variants provided herein include
nucleotide sequences that hybridize to the nucleic acid sequences
under at least moderately stringent hybridization conditions.
[0071] Rolling circle amplification conditions are known in the
art. For example, rolling circle amplification occurs in the
presence of a polymerase that possesses both strand displacement
ability and high processivity in the presence of template, primer
and nucleotides. In an embodiment, rolling circle amplification
conditions comprise temperatures of from about 25.degree. C. to
about 35.degree. C., or about 30.degree. C., a reaction time
sufficient for the generation of detectable amounts of amplicon and
performing the reaction in a buffer. In an embodiment, the rolling
circle amplification conditions comprise the presence of phi29-,
Bst-, or Vent exo-DNA polymerase. In an embodiment, the rolling
circle amplification conditions comprise the presence of phi29-DNA
polymerase.
[0072] Even further provided is a method of optimizing rolling
circle amplification comprising increasing the AC content of a
circular template before amplification. In an embodiment, the AC
content is increased by at least 5%, 10%, 15% 20% or more. In one
embodiment, optimizing means increasing the product by 3-fold,
5-fold, 7-fold or more compared to the circular template before the
AC content was increased. In another embodiment, optimizing means
producing more DNA amplicons within an allocated time, such as 10%,
20%, 30% or more DNA amplicons within an allocated time.
[0073] Even further provided is use of a circular template
disclosed herein for rolling circle amplification. In certain
embodiment, the use of the circular template is for biosensing
applications that use rolling circle amplification as known in the
art.
III. Nucleic Acids and Kits
[0074] Also provided herein is a circular template disclosed
herein. Accordingly, in one embodiment, there is provided a
circular template comprising a region complementary to a primer and
an AC rich nucleotide region. In one embodiment, the AC rich
nucleotide region comprises one of the sequences as shown in SEQ ID
NOs: 1-10 or a variant thereof.
[0075] In another embodiment, the AC rich nucleotide region
comprises a scrambled sequence that contains the nucleotide content
of one of the sequences shown in SEQ ID NOs: 1-10 or a variant
thereof. In a particular embodiment, the AC rich nucleotide region
comprises one of the sequences shown in SEQ ID NOs:36-40 (CTA5
scrambled sequences). A person skilled in the art could readily
create similar scrambled sequences of CTA1-4 (SEQ ID NOs:1-4) and
CTA6-10 (SEQ ID NOs:6-10) as well as other CTA5 (SEQ ID NO:5)
scrambled sequences.
[0076] Also provided herein is a nucleic acid molecule comprising
one of the sequences as shown in SEQ ID NOs:1-10 and 36-40 or a
variant thereof or a scrambled sequence thereof.
[0077] In an embodiment, the variant sequence has at least 50%, or
at least 60%, or at least 70%, or at least 80%, or at least 90%, or
at least 95% sequence identity to the sequences disclosed herein.
In an embodiment, the variant sequence contains substantially the
same amount of AC content as the reference sequence.
[0078] Also provided herein is a kit comprising a circular template
disclosed herein and one or more reagents necessary for carrying
out rolling circle amplification, such as a suitable DNA
polymerase, NTPs, a primer complementary to the circular template,
labelled probes, reaction buffer, and instructions for use. In one
embodiment, the DNA polymerase is phi29 DNA polymerase.
[0079] The above disclosure generally describes the present
application. A more complete understanding can be obtained by
reference to the following specific examples. These examples are
described solely for the purpose of illustration and are not
intended to limit the scope of the disclosure. Changes in form and
substitution of equivalents are contemplated as circumstances might
suggest or render expedient. Although specific terms have been
employed herein, such terms are intended in a descriptive sense and
not for purposes of limitation.
[0080] The following non-limiting examples are illustrative of the
present application:
Examples
Results
[0081] The present inventor developed a selection strategy
illustrated in FIG. 1a. The method features three enzymatic
reactions: templated DNA circularization catalyzed by T4 DNA
ligase, RCA by phi29 DNA polymerase, and restriction digestion by
EcoRV (FIG. 1a).
[0082] The original linear DNA pool (made of .about.10.sup.14 60-nt
DNA molecules with a 35-nt random region; nt: nucleotide) was first
end-ligated into circular template (denoted CTA; step 1). Following
RCA and restriction conversion of long RCA products into monomers
(step 2), end-ligation was again performed to produce a new
circular template, CTB (step 3). The cycle was completed with
another RCA and restriction digestion (step 4). seven iterations of
steps 1-4 were performed. To derive the best templates, the amount
of CTA and CTB was reduced from 100 pmol in round 1 (R1) to 10 pmol
in R2, 1 pmol in R3-R5, and 0.1 pmol in R6 and R7.
[0083] The monomeric DNA pool following the step 2 of R7 was
subjected to deep sequencing to acquire individual CTA sequences.
296,430 sequence reads were obtained and they can be classified
into 235,315 distinct classes. FIG. 1b lists the sequences of the
random nucleotide portion of the top 10 CTAs. Consistent with the
observed sequence diversity, the top ranked sequence, CTA1, only
had a sequence abundance (SA, percentage of a given sequence in the
sequenced pool) of 0.16%. Likewise, the accumulative SA of the top
10 sequences was only 0.58%. These observations are not entirely
surprising considering DNA polymerases are evolved by nature to
copy diverse DNA templates.
[0084] The top 10 sequences were found to be highly rich in A and C
(85.4%; top 10 average) and poor in G and T (14.6%), especially in
G (only 2.3%). This feature also applied to the top 100, 1,000 and
10,000 sequences: these respective groups exhibited an average AC
content of 83.2, 81.7 and 80.2% (the corresponding SA values are
1.64, 4.78 and 16.06%). In fact, CTA sequences as a whole showed a
significant AC bias (76.1%). As a control experiment, the original
DNA pool was also sequenced and only a small bias toward AC (58.1%)
was found.
[0085] Next, the DNA pool was sequenced following the step 4 of R7
to gather sequence information on CTBs. 580,491 sequence reads were
obtained, which can be classified into 433,639 distinct classes. As
expected, CTB sequences exhibit an overall bias towards G and T:
the average GT content of the top 10, 100, 1,000 and 10,000
sequences is 85.7, 84.9, 83.8 and 82.2%, respectively, reflecting
the fact that CTBs are the complements of CTAs. The top 3 CTBs
match the top 3 CTAs in the correct order (FIG. 1c). However, the
CTB counterparts of CTA4-10, though most of which fall within the
top 10, have a different ranking order (FIG. 1c).
[0086] Taken together, the results discussed above are indicative
of a successful sequence enrichment experiment. However, the great
sequence diversity called for experimental confirmation that these
sequences were enriched due to better RCA efficiencies.
[0087] Because it is difficult to directly quantify long-chain RCA
products, a strategy was developed that first converts RCA products
into monomeric amplicons via digestion with EcoRV, followed by
denaturing polyacrylamide gel electrophoresis (dPAGE) and DNA
staining with SYBR Gold, a fluorescent DNA binding dye. FIG. 6
shows the result of digesting the RCA product made with CTA1. A
full digestion was achieved after 4 hours; a 16-hour digestion was
chosen for the remainder of the study to ensure complete
monomerization. Because the fully digested RCA product is 60-nt
long, a 51-nt DNA molecule with a defined concentration was
included as an internal control. By determining the fluorescence
ratio (FR) of the two DNA bands in each lane, the concentration of
the digested monomer (CM) could be calculated, which was used to
estimate the average repeat unit (ARU) of the RCA product based on
the input concentration of the circular template.
[0088] The method was applied to compare the RCA efficiency (RE) of
CTA1 and CTB1, with the inclusion of the original random-sequence
DNA library (LB) as the control. FIG. 2a lists FR and ARU values
determined for time-dependent RCA of the three templates and FIG.
2b plots ARU values vs. RCA time. The RE of each template was
measured as the slope of line.
[0089] The above analysis revealed that CTA1 performed
.about.7-fold better than the control (39.0 vs. 6.1 in RE). It also
showed that, although there were two RCA reactions in each
selection cycle (steps 1 and 3, FIG. 1a), the template for the
first RCA was the driver of the selection (the RE of CTB1 is less
than 2-fold higher than that of LB). This observation was
consistent with the fact that the first RCA reaction was directly
linked to the initial DNA pool.
[0090] The RCA efficiency of a few more CTA/CTB pairs was also
analyzed and their RE values are provided in FIG. 2c. CTA5 and
CTA10 were selected as additional top 10 sequences because their AC
content and ACGT distribution (both measured in percentage) closely
match the top 10 average values (see FIG. 1c). CTA109 was included
because it has an AC content (91.4%) higher than the top 10 average
(85.4%). Finally, CTA1548 was chosen because, although lowly
ranked, it has top 10-like AC content (85.7%). For comparison, all
CTB counterparts of the chosen CTAs were included for the RE
analysis.
[0091] Like CTA1, both CTA5 and CTA10 were highly efficient RCA
templates (RE=43.4 and 36.5, respectively). In comparison, both
CTA109 and CTA1548 were less effective as templates (RE=25.9 and
26.4, respectively). As expected, all CTBs had smaller RE values
(RE between 7-12). The RE results presented above indicate: (1) the
top 10 CTAs were enriched because they can function as better RCA
templates; (2) CTAs have an competitive edge over CTBs.
[0092] The data in FIG. 2c also suggests other factors beyond high
AC content may contribute to the RE of a DNA template. These may
include the requirement of a defined sequence and an optimal ACGT
composition. To probe into these possibilities, mutagenesis studies
of CTA5, the best performing template, were carried out. Five CTA5
mutants were prepared that have the same ACGT composition of CTA5
but vary in sequence arrangements (FIG. 3). The first four mutants
differed from their parent in two relocated 5-nt elements and the
final mutant was highly scrambled. All five mutants performed
similarly as the RCA templates and this observation indicated that
the high RCA efficiency of CTA5 was sequence-independent. All
considered, the top ranked CTAs have been selected for both its
high AC content and optimal ACGT composition, but not for their
precise sequences.
[0093] To further demonstrate the competitive advantage of
high-performing templates at low primer-template concentrations,
comparative RCA reactions with CTA5 and LB were carried out at
varying template concentrations (1, 10 and 100 pM) and RCA times
(5-320 min). The concentration of monomeric amplicon (CM) following
RCA-digestion-dPAGE steps was then calculated and provided in Table
2. To simplify the comparison, the relative production of RCA
product at a given concentration (RPC) was determined by setting
the concentration of digested monomeric amplicon produced from CTA5
at 320 minutes to be 100. As depicted in FIG. 4, at relatively high
template concentration (10 and 100 pM), the control DNA template
(LB) still produced 15% and 35% of the amplicons made from CTA5. At
very low template concentration (1 pM), CTA5 produced detectable
amplicon in 80 minutes whereas no RCA product was observed even at
320 minutes with LB as the template.
[0094] To demonstrate the analytical utility of the selected DNA
templates, a biosensing experiment was conducted where CTA5,
reduced graphene oxide (rGO) and a DNA aptamer were used to achieve
protein detection using a method reported previously..sup.[19] As
illustrated in FIG. 5a, this method features a rGO-adsorbed DNA
probe that contains an aptamer sequence at its 5' end and a primer
for RCA at its 3' end. In the presence of the cognate target, the
DNA probe is released from the rGO surface, which is captured by
the circular template to enable the RCA reaction for signal
amplification. The well-known model thrombin-binding DNA
aptamer.sup.[20] was chosen for this demonstration. The sequences
of thrombin-binding probe (TP1) and circular templates (CTA5 and
CDT1, a template used in the previous study that is not AC-rich)
are given in FIG. 5b.
[0095] RCA reactions using 40 pM CTA5 or CDT1 was first conducted
to capture TP1 that was released in the presence of 1 nM thrombin.
The concentration of monomer equivalent (CME) in the RCA product
produced from both CTA5 and CDT1 was determined using the
digestion-dPAGE method (the data is provided in Table 4). FIG. 5c
(CME vs. RCA time) clearly showed that CTA5 was more effective than
CDT1.
[0096] The production of RCA product was next monitored by
measuring the solution fluorescence upon addition of SYBR Gold. As
expected, a higher level of fluorescence was observed with the
amplicons produced with CTA5 than with CDT1. For example,
.about.10-fold fluorescence was observed for the RCA reaction
conducted with CTA5 than with CDT1 at 1 nM thrombin (FIG. 5d).
Fluorescence intensities of RCA products were also measured in
response to thrombin concentrations that varied by 6 orders of
magnitude (0.1-10,000 pM; FIG. 5e). The use of CTA5 can lead to the
detection of 1 pM thrombin, which is .about.10-fold better than the
detection limit observed with CDT1 (see the insert in FIG. 5e). To
the present inventor's knowledge, the 1 pM limit of detection
represents the best sensitivity ever achieved with the
thrombin-binding DNA aptamer.
[0097] In summary, the present inventor has developed an in vitro
selection method to search for DNA sequences that can function as
highly effective templates for RCA. Diverse sequences were selected
with AC-richness as the common feature. The top 10 sequences, in
particular, were highly rich in A and C as their average AC content
surpassed 85%. To the best of the present inventor's knowledge,
this finding represents a novel observation as no prior literature
evidence exists for the high AC preference by phi29 DNA polymerase
or other DNA polymerases. The genome of phi29 bacteriophage has a
well-balanced ACGT distribution (30.1% A, 19.7% C, 20.3% G and
29.9% T) and the host bacterium, Bacillus subtilis, has a similar
ACGT allocation (28.2% A, 21.8% C, 21.7% G and 28.3% T), and
therefore, the observed AC-preference does not seem to have a
biological relevance.
[0098] Without wishing to be bound by theory, the observed
AC-richness may reflect phi29 DNA polymerase's propensity in
handling AC-rich DNA templates (template selectivity) or utilizing
dTTP and dGTP better than dATP and dCTP (nucleotide selectivity).
Although the polymerase may only have a very subtle template or
nucleotide selectivity for each nucleotide addition, the repetitive
copying of the same template for thousands of times can
significantly amplify this selectivity.
[0099] Through the study of a few AC-rich sequences with
wide-ranging rankings as well as several mutants of the best
performing template, strong evidence was uncovered signifying that
the superior RCA efficiency of high-ranking sequences is not the
property of a precise sequence but a trait reflecting their high AC
content and optimal distribution of component nucleotides.
[0100] The most significant advantage offered by the selected
AC-rich RCA templates is the production of more DNA amplicons at
low primer/template concentrations. This benefit may allow for
ultrasensitive detection involving RCA because the amplification
under this scenario has to be carried out with limited amounts of
the primer/template complex. Therefore the use of AC-rich templates
may significantly shorten the detection time and increase the
detection sensitivity. These optimal template sequences may be
useful in RCA as a versatile signal amplification tool for
diagnostic, biosensing and related applications.
Materials and Methods
[0101] Enzymes, Chemicals and Other Materials.
[0102] T4 DNA ligase, phi29 DNA polymerase, T4 polynucleotide
kinase (PNK), EcoRV, adenosine 5'-triphosphates (ATP) and
deoxynucleoside 5'-triphosphates (dNTPs) were purchased from Thermo
Scientific (Ottawa, ON, Canada). SYBR Gold (10,000.times.
concentrated stock in DMSO) was purchased from Life Technologies
(Burlington, ON, Canada). [a-.sup.32P]-dGTP was acquired from
Perkin Elmer (Woodbridge, ON, Canada). Water was purified with a
Milli-Q Synthesis A10 water purification system. 10.times.PBS (pH
7.4) was purchased from BioShop Canada (Burlington, ON. Canada),
which contains 80 g/L sodium chloride, 2 g/L potassium chloride,
14.2 g/L sodium phosphate, and 2.4 g/L potassium phosphate. All
other materials were purchased from Sigma-Aldrich (Oakville, ON,
Canada). 2.times. denaturing gel loading buffer (2.times.GLB) was
made in house with the following recipe (for 100 mL): 20 g sucrose,
10 mL of 10.times.TBE, 1 mL of 10% w/v SDS, 25 mg bromophenol blue,
25 mg xylene cyanole FF, and 110 g urea. The recipe for
10.times.TBE (1 L): 108 g Tris-base, 55 g boric acid, 20 mL of 0.5
M EDTA (pH 8.0).
[0103] Synthesis and Purification of Oligonucleotides.
[0104] DNA oligonucleotides were prepared by automated DNA
synthesis using standard phosphoramidite chemistry (Integrated DNA
Technologies, Coralville, Iowa, USA). The random DNA library for in
vitro selection was synthesized using an equimolar mixture of the
four standard phosphoramidites. All DNA oligonucleotides were
purified by 10% denaturing (8 M urea) polyacrylamide gel
electrophoresis (dPAGE), and their concentrations were determined
spectroscopically. The DNA sequences are provided in Table 1.
[0105] Preparation of Circular DNA Templates.
[0106] Circular DNA templates were prepared from 5'-phosphorylated
linear DNA oligonucleotides through template-assisted ligation with
T4 DNA ligase. Each linear DNA oligonucleotide was phosphorylated
as follows: a reaction mixture (50 .mu.L) was made to contain 1 nM
linear oligonucleotide, 20 U PNK (U: unit), 1.times.PNK buffer A
(50 mM Tris-HCl, pH 7.6 at 25.degree. C., 10 mM MgCl.sub.2, 5 mM
DTT, 0.1 mM spermidine), and 2 mM ATP. The mixture was incubated at
37.degree. C. for 30 min, followed by heating at 90.degree. C. for
5 min. The circularization reaction was conducted in a volume of
400 .mu.L, produced by adding 306 .mu.L of H.sub.2O and 2 .mu.L of
a DNA template (DT1 or DT2, 500 .mu.M) to the phosphorylation
reaction mixture above. After heating at 90.degree. C. for 3 min
and cooling down at room temperature (RT) for 10 min, 40 .mu.L of
10.times.T4 DNA ligase buffer (400 mM Tris-HCl, 100 mM MgCl.sub.2,
100 mM DTT, 5 mM ATP, pH 7.8 at 25.degree. C.) and 2 .mu.L of T4
DNA ligase (5 U/.mu.L) were added. This mixture was incubated at RT
for 2 h before heating at 90.degree. C. for 5 min to deactivate the
ligase. The ligated circular DNA molecules were concentrated by
standard ethanol precipitation and purified by 10% dPAGE. The
concentration of the circular DNA template was determined
spectroscopically.
[0107] In Vitro Selection Protocol.
[0108] The DNA library, denoted DL1, contained 60 nucleotides (nt)
distributed into a central random-sequence domain of 35 nt and two
constant regions, 15 nt at the 5' end and 10 nt at 3' ends. Its
sequence is provided in Table 1. After circularization in the
presence of DT1 using the protocol described above, the circular
template, denoted CTA, was purified by 10% dPAGE. Note that CTA
contains a recognition site for the restriction enzyme EcoRV (shown
in italic-bold in Table 1). The RCA for the first round of
selection was performed in 50 .mu.L of 1.times.RCA buffer (made
from 10.times. stock, which is made of 330 mM Tris-acetate, pH 7.9
at 37.degree. C., 100 mM magnesium acetate, 660 mM potassium
acetate, 1% (v/v) Tween 20, 10 mM DTT) containing 2 .mu.M CTA (100
pmol), 2 .mu.M DT1 (100 pmol), 1 mCi [a-.sup.32P]-dGTP, 0.7 mM dGTP
(35 nmol), 1 mM dATP, dTTP and dCTP (50 nmol each). After heating
at 90.degree. C. for 3 min, the solution was cooled to room
temperature for 10 min. Subsequently, 0.5 .mu.L of phi29 DNA
polymerase (10 U/.mu.L) was added, followed by incubation at
30.degree. C. for 20 min. Finally, the mixture was heated to
65.degree. C. for 10 min to deactivate the polymerase.
[0109] To the RCA reaction mixture above, 2 .mu.L of 500 .mu.M DT2
(1 nmol) was introduced. The mixture was heated at 90.degree. C.
for 3 min and cooled at RT for 10 min, followed by the addition of
10 .mu.L of 10.times. Fast Digestion Buffer (100 mM Tris-HCl, pH
8.0, 50 mM MgCl.sub.2, 1 M NaCl, 1 mg/mL BSA) and 5 .mu.L of
FastDigest EcoRV (unit size 400 reactions; the total volume is 400
.mu.L). The total reaction volume was increased to 100 .mu.L. The
reaction mixture was then incubated at 37.degree. C. for 16 h. The
restriction enzyme was inactivated at 65.degree. C. for 10 min. The
monomerized RCA products were concentrated by standard ethanol
precipitation and purified by dPAGE. The DNA was then eluted and
circularized into circular DNA template B (CTB), which was used for
the second RCA reaction. The reaction condition was identical to
the first RCA except for the replacement of DT1 with DT2. For the
restriction digestion after RCA, DT2 was replaced DT1.
[0110] Seven rounds of selection were conducted while the amount of
the circular template was reduced from 100 pmol (round 1) to 10
pmol (round 2), 1 pmol (rounds 3-5), and 0.1 pmol (rounds 6 and 7),
which was used as a strategy to favor the selection of highly
efficient DNA templates. DNA pool from the 7.sup.th round was used
for deep sequencing as described next.
[0111] Sequencing Protocol.
[0112] CTA, CTB in round 7 and LB, was digested into linear DNA
sequences as previously described. 2 .mu.L of 0.05 .mu.M linear
CTA, CTB and LB were amplified by PCR. There were two PCR steps. In
PCR1, a reaction mixture (50 .mu.L) was prepared to contain the DNA
above, 0.4 .mu.M each of forward primer (FP) and reverse primer
(RP; their sequences are provided in Table 1), 200 .mu.M each of
dNTPs (dATP, dCTP, dGTP and dTTP), 1.times.PCR buffer (75 mM
Tris-HCl, pH 9.0, 2 mM MgCl.sub.2, 50 mM KCl, 20 mM
(NH4).sub.2SO.sub.4) and 1.5 U Taq DNA polymerase. The DNA was
amplified using the following thermocycling steps: 94.degree. C.
for 3 min; 15 circles of 94.degree. C. (30 s), 42.degree. C. (45 s)
and 72.degree. C. (45 s), 72.degree. C. for 1 min. 1 .mu.L of the
PCR1 product was diluted with H.sub.2O to 100 .mu.L, 2 .mu.L of
which was used as the template for PCR2 using deep sequencing
primers DF and DR (their sequences are provided in Table 1) while
following the same protocol above for PCR1 except that the
annealing temperature increased to 48.degree. C. Note that the
numbers of amplification cycles between CTA, CTB and LB were
adjusted, typically between 12 and 15 cycles. The DNA product
generated in PCR2 was analyzed by 2% agarose gel electrophoresis
and sent out for deep sequencing. Paired-end next generation
sequencing (NGS) was done using an Illumina Miseq system at the
Famcombe Metagenomics Facility, McMaster University. Forward and
reverse reads were sorted by tag and exported as FASTQ files using
the Illumina Basespace platform. Primer domains were removed and
paired-end reads were merged using PANDAseq 2.6, only sequences
possessing perfect complementarity between paired-end reads were
output in FASTA format for further analysis (Ref. 1). Sequences
were dereplicated and tagged with copy number using USEARCH
v7.0.1090_i86linux32 sequence analysis package (Ref. 2). USEARCH
was also used for clustering of dereplicated populations using
the--cluster_smallmem command at 0.9 identity threshold. PANDAseq
and USEARCH software packages were run on Ubuntu Linux 12.04 LTS.
Analysis of sequence populations, rankings and base composition
were done using Microsoft Excel 2010 running on a Windows 8 PC.
Experimental Details for FIGS. 2 and 3:
[0113] RCA Reaction.
[0114] The RCA reaction was performed in 50 .mu.L. 2 .mu.L of a
relevant, 0.01 .mu.M circular DNA template (the final
concentration=0.4 nM) was mixed with 2 .mu.L of 50 .mu.M DT1 (used
for CTAs and LB) or DT2 (used for CTBs; the final DT1 or DT2
concentration=2 .mu.M), 5 .mu.L of 10 mM each of dGTP, dATP, dTTP
and dCTP (the final concentration=1 mM each), 5 .mu.L 10.times.RCA
buffer and 35.5 .mu.L of H.sub.2O. After heating at 90.degree. C.
for 3 min, the solution was cooled to room temperature for 10 min.
0.5 .mu.L of 10 U/.mu.L phi29 DNA polymerase was then added,
followed by incubation at 30.degree. C. for 5, 10, 20, 30 and 60
min. This mixture was heated to 65.degree. C. for 10 min to
deactivate the polymerase.
[0115] Restriction Digestion.
[0116] The digestion reaction was performed in 10 .mu.L. A 5-.mu.L
aliquot of the above RCA reaction mixture was combined with 2 .mu.L
of 50 .mu.M DT2 (used for CTAs and LB) or CT1 (used for CTBs; the
final DT1 or DT2 concentration=10 .mu.M), heated at 90.degree. C.
for 3 min and cooled at RT for 10 min. This was followed by the
addition of 1 .mu.L of 10.times. Fast Digestion Buffer and 2 .mu.L
of FastDigest EcoRV. The reaction mixture was then incubated at
37.degree. C. for 16 h.
[0117] Analysis of Monomeric RCA Products.
[0118] The above digestion mixture was combined with 10 .mu.L of
2.times. denaturing gel loading buffer (2.times.GLB), and 4 .mu.L
of 200 nM DLC (in 1.times.GLB, the final concentration=33.3 nM; its
sequence is provided in Table 1). The final volume of this DNA
mixture was 24 .mu.L. A 5-.mu.L aliquot was then run on a 10% dPAGE
gel. After electrophoresis, the gel was stained with 1.times.SYBR
Gold (diluted from the 10,000.times. concentrated stock solution).
A fluorescent image of the stained gel was obtained using Typhoon
9200 and analyzed using Image Quant software (Molecular
Dynamics).
[0119] Calculation of FR, C.sub.M, C.sub.ME, and ARU. The
fluorescence intensity of the 60-nt monomeric DNA band (F.sub.60nt)
and the 51-nt DLC band (F.sub.51nt) from each digestion mixture was
calculated and used to derive an FR (fluorescence ratio) value
using Equation 1:
FR=F.sub.60nt/F.sub.51nt Eq. 1
[0120] The FR value is used to calculate the concentration of the
monomer, C.sub.M, of the digestion mixture using equation 2:
C.sub.M=FR.times.33.3 nM.times.2.4 Eq. 2
[0121] Note that 2.4 is the volume correction factor, which is
calculated from 24/10 (the final volume of the digested monomer-DLC
mixture was 24 .mu.L whereas the volume of the digestion reaction
mixture was 10 .mu.L). The C.sub.M values are further used to
calculate the concentration of monomer equivalent (C.sub.ME) in the
RCA product in the RCA reaction mixture using Equation 3:
C.sub.ME=C.sub.M.times.2 Eq. 3
[0122] Note that 2 is the volume correction factor, which is
calculated from 10/5 (5 .mu.L of the RCA reaction mixture were used
to produce 10 .mu.L of the digestion reaction mixture). C.sub.ME
can also be calculated from FR using Equation 4:
C.sub.ME=FR.times.33.3 nM.times.2.4.times.2=FR.times.159.8 nM Eq.
4
[0123] Since the concentration of each circular template, [CT] (in
nM), was the limiting factor of the RCA reaction, the average
repeating units (ARU) of the RCA product can be estimated using
Equation 5:
ARU=FR/[CT].times.159.8 Eq. 5
[0124] The calculated ARU values for all the RCA reactions
(performed twice) featured in FIGS. 2 and 3 are summarized in Table
2.
Experimental Details for FIG. 4.
[0125] RCA Reaction.
[0126] The RCA reactions were carried in the same way as described
in the experimental details for FIGS. 2 and 3, with the following
exceptions: (1) two circular templates, CTA5 and LB, were examined
for RCA at the template concentration of 0.001, 0.01, and 0.1 nM,
(2) each RCA reaction was carried out for 5, 10, 20, 40, 80, 160
and 320 min.
[0127] Restriction Digestion and Analysis of Monomeric RCA
Products.
[0128] Both were carried out identically as described in the
experimental details for FIGS. 2 and 4 except for the following: a
4-.mu.L aliquot of the RCA reaction mixture (instead of 5 .mu.L)
was used to set up the digestion reaction.
[0129] Calculation of FR, C.sub.M, RPC.
[0130] FR and C.sub.M were calculated using Equations 1 and 2.
Table 3 lists C.sub.M values for each reaction time at each
template concentration, which were used to calculate the relative
production of RCA product at a given concentration (RPC) using
Equation 6.
RPC=100.times.C.sub.M,t/C.sub.M,320M Eq. 6
[0131] C.sub.M, t is the concentration of digested RCA product of
CTA5 or LB at time t and C.sub.M, 320M is the concentration
produced from CTA5 at 320 min.
Experimental details for FIG. 5.
[0132] Preparation of Reduced Graphene Oxide.
[0133] Graphene oxide (GO) was prepared according to a previously
reported modified Hummers method (Ref. 3). To produce reduced
graphene oxide (rGO), an aqueous solution containing 1 mL of 0.1
mg/mL GO, 10 .mu.L of 10 mg/mL L-ascorbic acid and 2 .mu.L of
ammonia solution was heated at 90.degree. C. for 5 min. After that,
the mixture was cooled to room temperature and the stably dispersed
rGO solution was obtained.
[0134] DNA Probe Adsorption by rGO.
[0135] 450 .mu.L of target binding buffer (TBB, 20 mM PBS, 150 mM
NaCl, 20 mM KCl, 5 mM MgCl.sub.2, pH 7.5), 10 .mu.L of 15 .mu.M
thrombin-binding DNA probe (TP1), and 40 .mu.L of 100 .mu.g/mL rGO
solution were incubated at 30.degree. C. for 30 min. The final TP1
concentration was 300 nM whereas the final rGO concentration was 8
.mu.g/mL. Under this condition, the DNA probe is completely
adsorbed by rGO (Ref. 4).
[0136] DNA Probe Release by Thrombin.
[0137] 48 .mu.L of the TP1-rGO mixture was transferred into a
1.5-mL microcentrifuge tube, and combined with 2 .mu.L of a
thrombin stock solution with a defined concentration of thrombin.
The reaction mixture was incubated at 30.degree. C. for 30 min,
then centrifuged for 10 min at 15,000 g to remove the rGO. The
supernatant was used for RCA reactions as described below.
Experimental Details for FIG. 5c.
[0138] The RCA reactions were carried out in the same way as
described in the experimental procedure for FIGS. 2 and 4, with the
following exceptions: (1) two circular templates, CTA5 and CDT1,
were examined for RCA at the template concentration of 0.04 nM and
5 .mu.L of TP1 solution released by 1 nM thrombin; (2) each RCA
reaction was carried out for 5, 10, 20, 40, 80, 160 and 320 min.
Restriction digestion and analysis of monomeric RCA products were
carried out identically as described for FIGS. 2 and 4 except for
the following: a 4-.mu.L aliquot of the RCA reaction mixture
(instead of 5 .mu.L) was used to set up the digestion reaction. FR
and C.sub.M were calculated using Equations 1 and 2; however
C.sub.ME values were calculated using Equation 7, owing to the use
of 4 .mu.L (rather than 5 .mu.L) of the RCA reaction mixture to set
up the 10-.mu.L digestion reaction (the volume correction factor
thus becomes 2.5).
C.sub.ME=FR.times.33.3 nM.times.2.4.times.2.5=FR.times.199.8 nM Eq.
7
[0139] The calculated C.sub.ME values for all the RCA reactions
featured in FIG. 5c are summarized in Table 4.
[0140] Experimental Details for FIG. 5d.
[0141] The RCA reactions were carried in the same way as described
in the experimental procedure for FIGS. 2 and 4, with the following
exceptions: (1) two circular templates, CTA5 and CDT1, were
examined for RCA at the template concentration of 0.4 nM and 5
.mu.L of TP1 solution released by 1,000 pM thrombin; (2) each RCA
reaction was carried out for 60 min; (3) two no-RCA control
reactions were also carried out for both CTA5 and CDT1 and these
reactions contained the same RCA components except phi29 DNA
polymerase. Following the RCA reaction, the fluorescence
measurement was carried out as follows: 7 .mu.L of the RCA reaction
mixture was mixed with 6 .mu.L of 10.times.SYBR Gold (diluted from
the 10,000.times. concentrated stock), 6 .mu.L of 10.times.TBE and
41 .mu.L of H.sub.2O. Note that the reaction tube was wrapped with
aluminum foil to prevent photo-bleaching. The mixture was incubated
at RT for 5 min and the fluorescence spectrum (.lamda.em=500-700
nm) was obtained using a Cary Eclipse fluorescence
spectrophotometer (Varian) with an excitation wavelength
(.lamda.ex) at 495 nm. The bandpasses for excitation and emission
were both set at 5 nm.
Experimental Details for FIG. 5e.
[0142] The experimental procedure for this panel was identical to
that used for panel 5d except that several more RCA reactions were
performed using the TP1 solution released by 0.1, 1, 10, 100, 1,000
and 10,000 pM thrombin. The fluorescence intensity at the maximal
emission wavelength (A=545 nm) was obtained for each test solution
and used to calculate the relative fluorescence using Equation
8:
RF=(F.sub.T-F.sub.C)/F.sub.C Eq. 8
[0143] F.sub.T: fluorescence reading of a test RCA-SYBR Gold
mixture; F.sub.C: [0144] fluorescence reading of the control
RCA-SYBR Gold mixture. RF vs. the original thrombin concentration
is plotted as FIG. 5e.
[0145] While the present application has been described with
reference to examples, it is to be understood that the scope of the
claims should not be limited by the embodiments set forth in the
examples, but should be given the broadest interpretation
consistent with the description as a whole.
[0146] All publications, patents and patent applications are herein
incorporated by reference in their entirety to the same extent as
if each individual publication, patent or patent application was
specifically and individually indicated to be incorporated by
reference in its entirety. Where a term in the present application
is found to be defined differently in a document incorporated
herein by reference, the definition provided herein is to serve as
the definition for the term.
TABLE-US-00001 TABLE 1 DNA oligonucleotides used Name Sequence
(5'-3') DL1 (LB) TCGACTA GTCAG-N.sub.35- TGTCTCG (SEQ ID NO: 11)
DT1 TAGTCGA CGAGACA (SEQ ID NO: 12) DT2 TGTCTCG TCGACTA (SEQ ID NO:
13) DLC TTCGGAAGAG ATGGCGACGC CGAACTATCT CTCGAGCTGA TCCTGATGGA A
(SEQ ID NO: 14) CTA1 TCGACTA GTCAGAACAA TACCCATCAA GCACCAACCA
CATTTCACAC TGTCTCG (SEQ ID NO: 15) CTA5 TCGACTA GTCAGCACAC
ATCAAAGCCC ATACTACAAC AACTACAACA TGTCTCG (SEQ ID NO: 16) CTA10
TCGACTA GTCAGAAGAA CAAACACTTC CCCATACCAC ACAACATCAA TGTCTCG (SEQ ID
NO: 17) CTA109 TCGACTA GTCAGCACAT ACAACACACA AATCACCAAC AACACAACAT
TGTCTCG (SEQ ID NO: 18) CTA1548 TCGACTA GTCAGAATAA TCAAACAACA
CCACAACTAT CAAAATACCA TGTCTCG (SEQ ID NO: 19) CTB1 CGAGACA
GTGTGAAATG TGGTTGGTGC TTGATGGGTA TTGTTCTGAC TAGTCGA (SEQ ID NO: 20)
CTB10 CGAGACA TGTTGTAGTT GTTGTAGTAT GGGCTTTGAT GTGTGCTGAC TAGTCGA
(SEQ ID NO: 21) CTB13 CGAGACA TTGATGTTGT GTGGTATGGG GAAGTGTTTG
TTCTTCTGAC TAGTCGA (SEQ ID NO: 22) CTB62 CGAGACA ATGTTGTGTT
GTTGGTGATT TGTGTGTTGT ATGTGCTGAC TAGTCGA (SEQ ID NO: 23) CTB965
CGAGACA TGGTATTTTG ATAGTTGTGG TGTTGTTTGA TTATTCTGAC TAGTCGA (SEQ ID
NO: 24) CTA5M1 TCGACTA GTCAGACAAC ATCAAAGCCC ATACTCACAC AACTACAACA
TGTCTCG (SEQ ID NO: 25) CTA5M2 TCGACTA GTCAGCACAC AACTAAGCCC
ATACTACAAC ATCAACAACA TGTCTCG (SEQ ID NO: 26) CTA5M3 TCGACTA
GTCAGCACAC ATCAACAACA ATACTACAAC AACTAAGCCC TGTCTCG (SEQ ID NO: 27)
CTA5M4 TCGACTA GTCAGATACT ATCAAAGCCC CACACACAAC AACTACAACA TGTCTCG
(SEQ ID NO: 28) CTA5M5 TCGACTA GTCAGCAAAA ACATGTCAAC CCAACAAACC
ATCCACTCAA TGTCTCG (SEQ ID NO: 29) CDT1 TCGACTA GTCAGGTTTC
CTTTCCTTGA AACTTCTTCC TTTCCTTTAC TGTCTCG (SEQ ID NO: 30) TP1
GGTTGGTGTG GTTGGAATAG TCGAGATATC CGAGACA (SEQ ID NO: 31) FP
GCCTCAACTT ATCCGAGACA (SEQ ID NO: 32) RP ATCTCGACTA GTCAGGCACT (SEQ
ID NO: 33) DF AATGATACGG CGACCACCGA GATCTACACT CTTTCCCTAC
ACGACGCTCT TCCGATCTGC CTCAACTTAT CCGAGACA (SEQ ID NO: 34) DR
CAAGCAGAAG ACGGCATACG AGATTTCTTG GTGACTGGAG TTCAGACGTG TGCTCTTCCG
ATCTATCTCG ACTAGTCAGG CACT (SEQ ID NO: 35)
TABLE-US-00002 TABLE 2 ARU values for FIGS. 2 and 3 Name Repeat
RCA5M RCA10M RCA20M RCA30M RCA60M CTA1 1 227.7 411.5 711.1 1082.6
2285.1 2 239.7 455.4 970.8 1250.4 2452.9 CTA5 1 254.8 456.8 791.9
1204.1 2540.4 2 267.3 508.5 1080.8 1390.8 2725.3 CTA10 1 174.1
377.6 735.4 1167.8 2132.9 2 164.8 340.0 671.2 1089.6 2212.5 CTA109
1 247.7 379.8 731.8 859.0 1648.7 2 196.0 383.9 705.4 994.7 1539.2
CTA1548 1 210.2 361.2 622.3 996.8 1749.2 2 202.0 352.3 669.1 906.5
1459.4 CTB1 1 59.9 115.9 255.7 415.5 715.1 2 67.9 127.8 251.7 431.5
695.1 CTB10 1 49.8 93.9 195.0 388.4 752.0 2 40.8 82.5 169.0 336.6
651.7 CTB13 1 73.5 128.8 259.8 313.5 627.2 2 71.2 106.9 221.1 294.4
542.4 CTB62 1 54.5 87.7 203.0 325.6 631.7 2 50.5 80.9 210.0 368.2
648.0 CTB965 1 32.9 64.0 117.4 178.6 373.9 2 43.4 65.2 140.2 217.2
429.1 CTA5M1 1 175.7 309.7 564.5 1055.7 2086.1 2 182.5 339.3 707.3
1298.3 2181.2 CTA5M2 1 173.2 303.4 544.2 979.5 1872.9 2 177.8 359.1
594.1 1075.7 2119.1 CTA5M3 1 218.8 359.4 763.9 1264.5 2279.5 2
290.1 542.4 1097.5 1432.5 2468.3 CTA5M4 1 264.2 477.8 964.8 1500.7
2520.6 2 197.4 360.3 704.1 1200.7 2374.6 CTA5M5 1 231.2 480.4 850.0
1298.4 2398.1 2 164.3 404.7 985.1 1278.8 2444.4 LB 1 24.0 55.9
123.8 203.7 379.5 2 24.0 43.9 103.9 171.8 343.6
TABLE-US-00003 TABLE 3 C.sub.M value for each reaction time at each
template concentration Name Repeat C.sub.M, 5M C.sub.M, 10M
C.sub.M, 20M C.sub.M40M C.sub.M, 80M C.sub.M, 160M C.sub.M, 320M
CTA5 - 0.001 nM 1 0 0 0 0 3.7 7.2 16.1 2 0 0 0 0 4.4 9.5 18.0 CTA5
- 0.01 nM 1 0 5.7 8.6 19.0 45.4 106.5 187.6 2 0 5.4 11.0 18.0 40.5
83.9 166.5 CTA5 - 0.1 nM 1 17.7 34.0 65.9 129.3 252.1 408.3 530.5 2
15.9 31.3 70.1 125.4 243.9 419.4 502.6 LB - 0.001 nM 1 0 0 0 0 0 0
0 2 0 0 0 0 0 0 0 LB - 0.01 nM 1 0 0 0 3.2 6.4 15.5 28.2 2 0 0 0
4.0 6.0 12.0 24.5 LB - 0.1 nM 1 3.5 6.4 14.1 32.9 62.8 119.4 194.1
2 3.2 6.1 15.7 32.2 67.1 113.3 173.8
TABLE-US-00004 TABLE 4 C.sub.ME values for FIG. 5c RCA RCA RCA RCA
RCA RCA RCA Name Rpt 5M 10M 20M 40M 80M 160M 320M CTA5 1 0 11.1
20.1 35.9 68.9 127.2 278.5 2 0 9.6 17.9 28.8 57.7 104.3 225.1 CDT1
1 0 0 0 0 0 13.8 10.1 2 0 0 0 0 0 24.2 19.8
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Sequence CWU 1
1
40135DNAArtificial SequenceSynthetic 1aacaataccc atcaagcacc
aaccacattt cacac 35235DNAArtificial SequenceSynthetic 2cacacatact
acaactaccc aacacaatat cccac 35335DNAArtificial SequenceSynthetic
3acaaacctca cacacataca aacccattat ctcca 35435DNAArtificial
SequenceSynthetic 4caagaacaac acacacccat aacaattcct ctaac
35535DNAArtificial SequenceSynthetic 5cacacatcaa agcccatact
acaacaacta caaca 35635DNAArtificial SequenceSynthetic 6ctcacataaa
acaaacacca cttaaaacac acacc 35735DNAArtificial SequenceSynthetic
7aaaagaacaa caagcacaca cataccccca aatac 35835DNAArtificial
SequenceSynthetic 8acacccaccg caataataaa aaccacaact tacca
35935DNAArtificial SequenceSynthetic 9ccaaaatagc acaaacacac
acacatacct taaac 351035DNAArtificial SequenceSynthetic 10aagaacaaac
acttccccat accacacaac atcaa 351160DNAArtificial SequenceSynthetic
11atctcgacta gtcagnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn tgtctcggat
601220DNAArtificial SequenceSynthetic 12tagtcgagat atccgagaca
201320DNAArtificial SequenceSynthetic 13tgtctcggat atctcgacta
201451DNAArtificial SequenceSynthetic 14ttcggaagag atggcgacgc
cgaactatct ctcgagctga tcctgatgga a 511560DNAArtificial
SequenceSynthetic 15atctcgacta gtcagaacaa tacccatcaa gcaccaacca
catttcacac tgtctcggat 601660DNAArtificial SequenceSynthetic
16atctcgacta gtcagcacac atcaaagccc atactacaac aactacaaca tgtctcggat
601760DNAArtificial SequenceSynthetic 17atctcgacta gtcagaagaa
caaacacttc cccataccac acaacatcaa tgtctcggat 601860DNAArtificial
SequenceSynthetic 18atctcgacta gtcagcacat acaacacaca aatcaccaac
aacacaacat tgtctcggat 601960DNAArtificial SequenceSynthetic
19atctcgacta gtcagaataa tcaaacaaca ccacaactat caaaatacca tgtctcggat
602060DNAArtificial SequenceSynthetic 20atccgagaca gtgtgaaatg
tggttggtgc ttgatgggta ttgttctgac tagtcgagat 602160DNAArtificial
SequenceSynthetic 21atccgagaca tgttgtagtt gttgtagtat gggctttgat
gtgtgctgac tagtcgagat 602260DNAArtificial SequenceSynthetic
22atccgagaca ttgatgttgt gtggtatggg gaagtgtttg ttcttctgac tagtcgagat
602360DNAArtificial SequenceSynthetic 23atccgagaca atgttgtgtt
gttggtgatt tgtgtgttgt atgtgctgac tagtcgagat 602460DNAArtificial
SequenceSynthetic 24atccgagaca tggtattttg atagttgtgg tgttgtttga
ttattctgac tagtcgagat 602560DNAArtificial SequenceSynthetic
25atctcgacta gtcagacaac atcaaagccc atactcacac aactacaaca tgtctcggat
602660DNAArtificial SequenceSynthetic 26atctcgacta gtcagcacac
aactaagccc atactacaac atcaacaaca tgtctcggat 602760DNAArtificial
SequenceSynthetic 27atctcgacta gtcagcacac atcaacaaca atactacaac
aactaagccc tgtctcggat 602860DNAArtificial SequenceSynthetic
28atctcgacta gtcagatact atcaaagccc cacacacaac aactacaaca tgtctcggat
602960DNAArtificial SequenceSynthetic 29atctcgacta gtcagcaaaa
acatgtcaac ccaacaaacc atccactcaa tgtctcggat 603060DNAArtificial
SequenceSynthetic 30atctcgacta gtcaggtttc ctttccttga aacttcttcc
tttcctttac tgtctcggat 603137DNAArtificial SequenceSynthetic
31ggttggtgtg gttggaatag tcgagatatc cgagaca 373220DNAArtificial
SequenceSynthetic 32gcctcaactt atccgagaca 203320DNAArtificial
SequenceSynthetic 33atctcgacta gtcaggcact 203478DNAArtificial
SequenceSynthetic 34aatgatacgg cgaccaccga gatctacact ctttccctac
acgacgctct tccgatctgc 60ctcaacttat ccgagaca 783584DNAArtificial
SequenceSynethic 35caagcagaag acggcatacg agatttcttg gtgactggag
ttcagacgtg tgctcttccg 60atctatctcg actagtcagg cact
843635DNAArtificial SequenceSynthetic 36acaacatcaa agcccatact
cacacaacta caaca 353735DNAArtificial SequenceSynthetic 37cacacaacta
agcccatact acaacatcaa caaca 353835DNAArtificial SequenceSynthetic
38cacacatcaa caacaatact acaacaacta agccc 353935DNAArtificial
SequenceSynthetic 39atactatcaa agccccacac acaacaacta caaca
354035DNAArtificial SequenceSynthetic 40caaaaacatg tcaacccaac
aaaccatcca ctcaa 35
* * * * *