U.S. patent application number 14/171369 was filed with the patent office on 2014-08-07 for methods for single-molecule analysis.
This patent application is currently assigned to Bionano Genomics, Inc.. The applicant listed for this patent is Bionano Genomics, Inc.. Invention is credited to Han Cao, Alex Hastie, Henry B. Sadowski, Michael G. Saghbini, Ming Xiao.
Application Number | 20140221218 14/171369 |
Document ID | / |
Family ID | 51259712 |
Filed Date | 2014-08-07 |
United States Patent
Application |
20140221218 |
Kind Code |
A1 |
Cao; Han ; et al. |
August 7, 2014 |
METHODS FOR SINGLE-MOLECULE ANALYSIS
Abstract
Methods for single-molecule preparation and analysis are
disclosed herein. The methods can, for example, be used for
isolating and analyzing DNA from various biological samples.
Inventors: |
Cao; Han; (San Diego,
CA) ; Xiao; Ming; (Huntingdon Valley, PA) ;
Hastie; Alex; (San Diego, CA) ; Saghbini; Michael
G.; (Poway, CA) ; Sadowski; Henry B.; (San
Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Bionano Genomics, Inc. |
Philadelphia |
PA |
US |
|
|
Assignee: |
Bionano Genomics, Inc.
Philadelphia
PA
|
Family ID: |
51259712 |
Appl. No.: |
14/171369 |
Filed: |
February 3, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61761189 |
Feb 5, 2013 |
|
|
|
Current U.S.
Class: |
506/2 ; 435/6.1;
435/6.11 |
Current CPC
Class: |
C12Q 1/6806 20130101;
C12Q 1/6869 20130101; C12Q 1/6809 20130101; C12Q 1/6809 20130101;
C12Q 2521/301 20130101; C12Q 2565/1025 20130101 |
Class at
Publication: |
506/2 ; 435/6.1;
435/6.11 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1-36. (canceled)
37. A method of characterizing a DNA, the method comprising:
nicking a first DNA at a first sequence motif, wherein the first
DNA is double stranded, and wherein the first DNA remains
double-stranded adjacent to the nicks; labeling the nicks on the
first DNA with a first label; marking the labeled first DNA with a
third label, wherein the third label is non-sequence-specific, and
wherein the third label is different from the first label;
linearizing the first DNA following labeling with the first and
third labels; and detecting the pattern of the first label on the
linearized first DNA.
38. The method of claim 37, further comprising repairing the nicks
on the first DNA.
39. The method of claim 37, further comprising: nicking a second
DNA at the first sequence motif; labeling the nicks on the second
DNA with the first label; repairing the nicks on the second DNA;
marking the repaired second DNA with the third label; linearizing
the second DNA following labeling with the first and third labels;
and detecting the pattern of the first label on the linearized
second DNA.
40. The method of claim 37, further comprising: nicking the
repaired first DNA at a second sequence motif, wherein the repaired
first DNA remains double-stranded adjacent to the nicks; labeling
the nicks at the second sequence motif on the first DNA with a
second label, wherein the third label is different from the second
label; and repairing the nicks on the first DNA following labeling
with the second label.
41. The method of claim 40, wherein the first label and second
label comprise the same label.
42. The method of claim 37, wherein the first DNA and the second
DNA are each from the same source.
43. The method of claim 37, wherein the linearizing comprises
transporting the DNA into a nanochannel.
44. The method of claim 37, further comprising comparing the
pattern of the first labels to a pattern of labels on a reference
DNA.
45. The method of claim 37, wherein nicking the first DNA comprises
nicking with Nt.BpsQI.
46. The method of claim 37, wherein the first label is selected
from the group consisting of a fluorophore, a quantum dot, a
dendrimer, a nanowire, a bead, a hapten, a streptavidin, an avidin,
a neutravidin, a biotin, and a reactive group.
47. The method of claim 37, wherein the first label does not
comprise a fluorophore, and wherein the first label does not
comprise a quantum dot.
48. A method of characterizing DNA, the method comprising: nicking
one strand of a first DNA at a recognition sequence with a first
nicking endonuclease, wherein the first DNA is double stranded, and
wherein the first DNA remains double-stranded adjacent to the
nicks; labeling the first DNA at the nicking sites with a first
label; repairing the nicks on the first DNA; nicking a
complementary strand of a second DNA at the recognition sequence
with a second nicking endonuclease, wherein the complementary
strand of the second DNA is complementary to the one strand of the
first DNA, wherein the second DNA is double stranded, and wherein
the second DNA remains double-stranded adjacent to the nicks;
labeling the second DNA at the nicking sites with a second label;
repairing the nicks on the second DNA; marking the repaired first
and second DNA with a third label, wherein the third label is
non-sequence specific; linearizing the marked first DNA and marked
second DNA; and detecting a pattern of the first and second label
on the linearized first DNA and linearized second DNA.
49. The method of claim 48, further comprising: nicking one strand
of a third DNA at a recognition sequence with the first nicking
endonuclease, wherein the third DNA is double stranded, and wherein
the third DNA remains double-stranded adjacent to the nicks;
labeling the third DNA at the nicking sites; repairing the nicks on
the third DNA; nicking a complementary strand of a fourth DNA at
the recognition sequence with the second nicking endonuclease,
wherein the complementary strand of the fourth DNA is complementary
to the one strand of the third DNA; labeling the fourth DNA at the
nicking sites; repairing the nicks on the fourth DNA; and marking
the repaired third and fourth DNAs with a third label, wherein the
third label comprises a non-sequence-specific label.
50. The method of claim 48, wherein the first DNA and second DNA
are each from the same source.
51. The method of claim 48, wherein the first DNA and second DNA
are each from a first source, and wherein the second and third DNA
are each from a second source.
52. The method of claim 48, wherein the first label and second
label comprise the same label.
53. The method of claim 48, wherein the first label is selected
from the group consisting of a fluorophore, a quantum dot, a
dendrimer, a nanowire, a bead, a hapten, a streptavidin, an avidin,
a neutravidin, a biotin, and a reactive group.
54. The method of claim 48, wherein the first label does not
comprise a fluorophore, and wherein the first label does not
comprise a quantum dot.
55. The method of claim 48, wherein the linearizing includes
transporting the DNA into a nanochannel.
56. A method of characterizing a DNA comprising a double-stranded
DNA comprising at least one base flap on either strand of the DNA,
the method comprising: treating the double-stranded DNA with a 5'
to 3' exonuclease activity of a polymerase under conditions wherein
at least one species of dNTP is present in limited concentration or
omitted compared to other dNTPs that are present; ligating the
nicks to restore strand integrity at flap regions; and
characterizing the DNA.
Description
BACKGROUND
[0001] 1. Technical Field
[0002] The present invention relates to the field of nanotechnology
and to the field of single molecule genomic analysis.
[0003] 2. Description of the Related Art
[0004] Next-generation sequencing (NGS) technologies have enabled
high-throughput and low-cost generation of sequence data. However,
de novo genome assembly remains a great challenge, particularly for
large genomes. NGS short reads are often insufficient to create
large contigs that span repeat sequences and facilitate unambiguous
assembly. Plant genomes are notorious for containing high
quantities of repetitive elements, which combined with huge genome
sizes, makes accurate assembly of these large and complex genomes
intractable.
[0005] Accurate de novo assembly of sequence reads represents the
weak link in genome projects despite advances in high-throughput
sequencing [1,2]. There are two general steps in genome sequence
assembly, generation of sequence contigs and scaffolds, and their
anchoring on genome-wide, lower resolution maps. NGS platforms
generate sequence reads ranging from 25 to more than 500 bases [3],
while reads of up to 1000 bases can be obtained by Sanger
sequencing with high accuracy. NGS reads are often too short for
unambiguous assembly. Paired-end reads can bridge contigs into
scaffolds, but there are often gaps within the scaffolds. To order
contigs and scaffolds, high-resolution genomic maps from an
independent technology platform are needed. They may be of
chromosomal scale, i.e., genetic maps, or regional scale, i.e.,
contigs of bacterial artificial chromosomes (BACs) or fosmids [4].
Contigs and scaffolds may be difficult to map if they are too short
compared to the map resolution. For example, maps may have a
resolution of 50-150 kb, while many contigs and scaffolds may only
span a few kilobases. Additionally, there are errors in the contigs
and scaffolds themselves, often due to misassembly of repeat
sequences. Typical medium to large genomes contain 40-85%
repetitive sequences [5-8], dramatically hindering effective de
novo sequence assembly.
[0006] Genome finishing has relied on guidance of a physical map
for large and complex genomes, including human, arabidopsis [9],
rice [10] and maize [11,12]. BAC-based restriction fragment
physical mapping of complex genomes is fairly robust because even
in the presence of interspersed repeat sequences along the BAC
inserts (typically 100-220 kb long), a unique pattern of
restriction fragments is generated. State of the art technologies
for physical map construction include SNaPshot [13,14],
whole-genome profiling [15,16], optical mapping [17,18], and genome
mapping [19]. SNaPshot is a restriction fingerprinting method which
uses one or more restriction enzymes and fluorescent labels
followed by separation of fragments by capillary electrophoresis.
SNaPshot has been used for physical mapping of wheat and other
genomes [14,20]. Optical mapping provides an additional layer of
information by retaining the physical order of restriction sites
along DNA molecules immobilized on a surface [18]. It has been
applied to the maize and the rice genome [11,21]. One can validate
a sequence assembly by comparing in silico sequence motif maps to
consensus optical maps [22-25]. However, information density for
optical maps is only about one site per 20 kb, and the technology
is limited in utility by high error-rates, non-uniform DNA
linearization, and low throughput. Therefore, a high-resolution
(e.g., <5 kb) DNA sequencing-independent mapping method that can
overcome these constraints of optical mapping is much needed.
SUMMARY OF THE INVENTION
[0007] Methods for preparing samples and performing single molecule
analysis, including methods of mitigating the effects of fragile
sites and improving information density for genome mapping, are
provided herein.
[0008] In an embodiment, a method of characterizing a DNA is
provided, comprising: nicking a first DNA at a first sequence
motif, wherein the first DNA is double stranded, and wherein the
first DNA remains double-stranded adjacent to the nicks; labeling
the nicks on the first DNA with a first label; repairing the nicks
on the first DNA; marking the repaired first DNA with a second
label, wherein the second label is non-sequence-specific, and
wherein the second label is different from the first label;
linearizing the first DNA following labeling with the first and
second labels; and detecting the pattern of the first label on the
linearized first DNA.
[0009] In an embodiment, a method of characterizing DNA is
provided, comprising: nicking a first DNA at a first sequence
motif, wherein the first DNA is double stranded, and wherein the
first DNA remains double-stranded adjacent to the nicks; labeling
the nicks on the first DNA with a first label; repairing the nicks
on the first DNA following labeling with the first label; nicking
the repaired first DNA at a second sequence motif, wherein the
repaired first DNA remains double-stranded adjacent to the nicks;
labeling the nicks at the second sequence motif on the first DNA
with a second label; repairing the nicks on the first DNA following
labeling with the second label; marking the first DNA with a third
label, wherein the third label is non-sequence-specific, and
wherein the third label is different from the first and second
labels; linearizing the first DNA following labeling with the third
label; detecting the pattern of at least one of the first and
second labels on the first linearized DNA.
[0010] In an embodiment, a method of characterizing DNA is
provided, comprising: nicking one strand of a first DNA at a
recognition sequence with a first nicking endonuclease, wherein the
first DNA is double stranded, and wherein the first DNA remains
double-stranded adjacent to the nicks; labeling the first DNA at
the nicking sites with a first label; repairing the nicks on the
first DNA; nicking the complementary strand of a second DNA at the
recognition sequence with a second nicking endonuclease, wherein
the second DNA is double stranded, and wherein the second DNA
remains double-stranded adjacent to the nicks; labeling the second
DNA at the nicking sites with a second label; and repairing the
nicks on the second DNA.
[0011] In some embodiments, the methods described herein further
comprise: nicking one strand of a second DNA at a recognition
sequence with the first nicking endonuclease, wherein the second
DNA is double stranded, and wherein the second DNA remains
double-stranded adjacent to the nicks; labeling the second DNA at
the nicking sites repairing the nicks on the second DNA; nicking
the complementary strand of the second DNA at the recognition
sequence with the second nicking endonuclease; labeling the second
DNA at the nicking sites; repairing the nicks on the second DNA;
and marking the repaired first and second DNAs with a third label,
wherein the third label is a non-sequence-specific label.
[0012] In an embodiment, a method of characterizing DNA is
provided, comprising: nicking a first DNA at a first sequence
motif, wherein the first DNA is double stranded, and wherein the
first DNA remains double-stranded adjacent to the nicks; labeling
the nicks on the first DNA with a first label; repairing the nicks
on the first DNA; tagging the first DNA at a second sequence motif
with a second label, wherein the second label does not cut DNA;
marking the first DNA with a third label, wherein the third label
is a non-sequence-specific label, and wherein the third label is
different from the first and second labels; linearizing the first
DNA following labeling with the first, second, and third labels;
and detecting the first and second labels on the linearized first
DNA.
[0013] In an embodiment, a method of characterizing DNA is
provided, comprising: treating a double-stranded DNA comprising at
least one flap on either strand of the DNA with a 5' to 3'
exonuclease activity of a polymerase under conditions wherein at
least one species of dNTP is in present in limited concentration
compared to other dNTPs that are present; ligating the nicks to
restore strand integrity at flap regions; and characterizing the
DNA.
[0014] In an embodiment, a method of characterizing DNA is
provided, comprising: treating a double-stranded DNA comprising at
least one flap on either stand of the DNA with a 5' to 3'
exonuclease activity of a polymerase under conditions wherein at
least one species of dNTP is omitted; ligating the nicks to restore
strand integrity at the flap regions; and characterizing the
DNA.
[0015] In some embodiments, the methods described herein further
comprise: nicking a second DNA at the first sequence motif;
labeling the nicks on the second DNA with the first label;
repairing the nicks on the second DNA; marking the repaired second
DNA with the second label; linearizing the second DNA following
labeling with the first and second labels; and detecting the
pattern of the first or second label on the linearized second
DNA.
[0016] In some embodiments, the methods described herein further
comprise: nicking a second DNA at the first sequence motif, wherein
the second DNA is double stranded, and wherein the second DNA
remains double-stranded adjacent to the nicks; labeling the nicks
on the second DNA with the first label; repairing the nicks on the
second DNA following labeling with the first label; nicking the
repaired second DNA at the second sequence motif, wherein the
repaired second DNA remains double-stranded adjacent to the nicks;
labeling the nicks at the second sequence motif on the second DNA
with the second label; repairing the nicks on the second DNA
following labeling with the second label; marking the second DNA
with the third label; linearizing the second DNA following labeling
with the third label; and detecting the pattern of at least one of
the first and second labels on the second linearized DNA.
[0017] In some embodiments, the methods described herein further
comprise comparing the pattern of the first label on the first DNA
to the pattern of the first label on the second DNA. In some
embodiments, the methods described herein further comprise:
assembling a plurality of first DNAs using overlap of the labeled
sequence motifs to construct a first DNA map; assembling a
plurality of second DNAs using overlap of the labeled sequence
motifs to construct a second DNA map; and comparing the first DNA
map to the second DNA map.
[0018] In some embodiments, the methods described herein further
comprise: marking the repaired first and second DNAs with a third
label, wherein the third label is a non-sequence-specific label. In
some embodiments, the methods described herein further comprise:
linearizing the first and second DNAs; detecting the first and
second labels on the linearized DNA; and assembling the labeled DNA
molecules using overlap of the labeled sequence motifs to construct
a DNA map. In some embodiments, the first and second labels are the
same label.
[0019] In some embodiments, the methods described herein further
comprise: nicking a second DNA at the first sequence motif, wherein
the second DNA is double stranded, and wherein the second DNA
remains double-stranded adjacent to the nicks; labeling the nicks
on the second DNA with the first label; repairing the nicks on the
second DNA; tagging the second DNA at the second motif with the
second label; marking the second DNA with the third label;
linearizing the second DNA following labeling with the first and
second labels; and detecting the first and second labels on the
linearized second DNA.
[0020] In some embodiments, the linearizing includes transporting
the DNA into a nanochannel. In some embodiments, the methods
described herein further comprise comparing the pattern of at least
one of the first or second labels on the first DNA to a pattern of
labels on a reference DNA. In some embodiments, the methods
described herein further comprise comparing the pattern of the
first label on the first DNA to a pattern of labels on a reference
DNA. In some embodiments, the methods described herein further
comprise comparing the pattern of the second label on the first DNA
to a pattern of labels on a reference DNA, wherein the second label
is a sequence specific label. In some embodiments, the methods
described herein further comprise assembling the labeled first DNA
using the pattern of labeled motifs to construct a first DNA map.
In some embodiments, the methods described herein further comprise
assembling the labeled second DNA using the pattern of labeled
motifs to construct a first DNA map. In some embodiments, the
second label is a non-sequence-specific label. In some embodiments,
the second sequence motif includes at least one binding site for a
DNA binding entity selected form the group consisting of a
non-cutting restriction enzyme, a zinc finger protein, an antibody,
a transcription factor, a transcription activator like domain, a
DNA binding protein, a polyamide, a triple helix forming
oligonucleotide, and a peptide nucleic acid, wherein the tagging is
effected with the binding entity comprising the second label, and
wherein the second label is selected form the group consisting of a
fluorophore, a quantum dot, a dendrimer, a nanowire, a bead, a
hapten, streptavidin, avidin, neutravidin, biotin, and a stabilized
reactive group. In some embodiments, the second sequence motif
includes at least one binding site for a peptide nucleic acid,
wherein the tagging is performed with the peptide nucleic acid
comprising the second label, and wherein the second label is a
fluorophore or a quantum dot. In some embodiments, the second
sequence motif includes at least one binding site for a
methyltransferase, and wherein tagging is performed with the
methyltransferase comprising a modified cofactor which includes the
second label. In some embodiments, the first and second labels are
independently selected from the group consisting of a fluorophore,
a quantum dot, a dendrimer, a nanowire, a bead, a hapten, a
streptavidin, an avidin, a neutravidin, a biotin, and a reactive
group. In some embodiments, the first and second labels are
independently selected from the group consisting of a fluorophore
or a quantum dot. In some embodiments, the labeling is carried out
with a polymerase. In some embodiments, the labeling is carried out
with a polymerase in the presence of dNTPs comprising the label. In
some embodiments, the polymerase has a 5' to 3' exonuclease
activity. In some embodiments, the polymerase leaves a flap region,
and wherein the flap region is removed to restore a ligatable nick
prior to the repairing with a ligase. In some embodiments, the flap
region is removed using the 5' to 3' exonuclease activity of a
polymerase under conditions wherein at least one nucleotide is
present in limited concentration. In some embodiments, the flap
region is removed using the 5' to 3' exonuclease activity of a
polymerase under conditions wherein at least one nucleotide is
omitted from the reaction. In some embodiments, the flap region is
removed with a flap endonuclease. In some embodiments, the labeling
is carried out with a polymerase in the presence of at least one
species of dNTP. In some embodiments, the at least one species of
dNTP is a single species of dNTP. In some embodiments, activity of
the polymerase is modulated by adjusting the temperature, dNTP
concentration, cofactor concentration, buffer concentration, or any
combination thereof, during labeling.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 shows fragmentations that can occur at fragile sites
as a result of nicking, where nicks are closer to one another (FIG.
1A) or farther apart (FIG. 1B).
[0022] FIG. 2 shows DNA length corresponding to the midpoint in a
size histogram showing molecules arranged from smallest to largest
in length (or mass).(shown as "center of mass") the percent of DNA
molecules that are mapped against a reference genome (shown as
"mapping to reference genome"), and the false positive and false
negative rates for mapping to a sequenced reference genome compared
to a simulation for the same (shown as "false positive" and "false
negative") rates in E. coli subjected to the following treatments:
1.) no repair, 2.) repair with PreCR as recommended by manufacture
(New England BioLabs), 3.) repair with PreCR under conditions of
omitting dGTP, 4.) repair with PreCR under conditions of omitting
dATP and dGTP, and 5.) repair with Taq polymerase under conditions
of omitting dGTP.
[0023] FIG. 3 shows center of mass, percent mapping to a reference
genome, and false positive and false negative rates in E. coli
subjected to the following treatments: 1.) no repair, or 2)
treatment with FEN I to remove flaps followed by a ligase to repair
the translated nicks.
[0024] FIG. 4 shows center of mass, percent mapping to a reference
genome, and false positive and false negative rates in Drosophila
subjected to the following treatments: 1.) nicking with Nt.BspQI
and PreCR repair, and 2.) nicking with Nb.BbVCI and PreCR
repair.
[0025] FIG. 5 shows two-color genome mapping with two enzymes,
including the layout of an IrysChip (5A), linearization in
nanochannels (5B), distribution of labels at sequence-specific
locations (5C), and the alignment of consensus maps (5D) as
described in Example 4.
DETAILED DESCRIPTION
[0026] Maintaining and restoring the integrity of DNA strands is
essential for obtaining long labeled molecules that are useful for
complex genome mapping and information density. The methods
described herein provide approaches to minimize the formation of
fragile DNA sites and fragmentation of DNA, restore the structural
integrity of DNA following the use of nicking approaches, and
maximize the information content of DNA in order to generate
high-resolution maps.
[0027] Described herein are approaches that can be used in
conjunction with a nanochannel array to reproducibly and uniformly
linearize DNA. In addition to improved noise characteristics (e.g.,
by virtue of keeping DNA in solution rather than affixed), these
approaches can entail cycles of channel-loading and imaging to
generate high-throughput DNA reads. Genome mapping on nanochannel
arrays at the single-molecule level overcomes many of the
limitations of preexisting technologies and is described in depth
in Lam ET et al. (Genome mapping on nanochannel arrays for
structural variation analysis and sequence assembly, Nat Biotechnol
30: 771-776, 2012), which is hereby incorporated by reference in
its entirety. In some embodiments described herein, a genome
mapping approach allows multiple motifs to be labeled with
different colors is employed, significantly increasing information
density.
[0028] In some embodiments, a high-resolution physical map is
constructed. The physical map can be used to validate or correct a
physical map generated using another method, such as SNaPshot
fingerprinting technology. In some embodiments, the physical map is
used to validate assembled regions and correct inaccuracies in
sequence scaffolds. The physical map can also be used to facilitate
de novo sequence assembly of a region by anchoring sequence
scaffolds. In some embodiments, the physical map is used to produce
a highly accurate and complete sequence assembly.
[0029] In some embodiments provided herein, nick labeling is used
to prepare DNA for analysis. As part of the nick labeling process,
nicks can move closer to one another (as shown in FIG. 1A) or
farther apart (as shown in FIG. 1B). It has been discovered that
fragile sites occur when two nicks are <1 Kb apart on opposite
DNA strands. Fragmentation can occur at fragile sites due, for
example, to: 1) mechanical manipulation, 2) heat required for
labeling, 3) strand extension associated with labeling and certain
kinds of repair (e.g., using the exonuclease activity of
polymerases), or 4) shear forces associated with linearizing DNA
molecules. In general, the shorter the distance between nicks, the
more frequent the fragmentation, particularly if labeling decreases
the original distance (FIG. 1A). As described herein, it has been
found that repairing nicks can ameliorate the breakage of DNA.
[0030] In some embodiments, the methods described herein utilize
nicking enzymes to create sequence-specific nicks that are
subsequently labeled, for example by a fluorescent nucleotide
analog. In some embodiments, the nick-labeled DNA is stained with
the intercalating dye, loaded onto a nanofluidic chip by an
electric field, and imaged. In some embodiments, the DNA is
linearized by confinement in a nanochannel array, resulting in
uniform linearization and allowing precise and accurate measurement
of the distance between nick-labels on DNA molecules comprising a
signature pattern. In some embodiments, DNA loading and imaging can
be repeated in an automated fashion. In some embodiments, a second
nicking enzyme is used. In some embodiments, this second nicking
enzyme is used with a second label color.
[0031] In some embodiments, methods are provided to mitigate
fragile site-based fragmentation. In some embodiments, reduced
driving conditions are used to limit the rate of incorporation of a
label, and therefore minimize fragmentation at the fragile sites.
In some embodiments, reduced driving conditions are used to
minimize shearing stress forces associated with DNA elongation. In
some embodiments, drive is reduced by lowering the concentration of
dNTPs, lowering reaction temperature, lowering cofactor
concentration, adjusting buffer and salt concentration, or a
combination thereof. Drive can be also be reduced at the level of
repair by stimulating the exonuclease activity of a polymerase with
a high concentration of dNTPs, then limiting extension by
restricting or omitting at least one nucleotide (which can be
referred to as "choked repair"). In a preferred embodiment, a
single species of dNTP (e.g., dATP) is incorporated at the nick
site, the flap is removed with a flap nuclease without extension,
and ligation is performed.
[0032] In some embodiments, a suboptimal temperature for a
thermophilic polymerase is used to reduce driving conditions. In
some embodiments, the reaction temperature is about 35.degree. C.
to about 75.degree. C., such as 35 .degree. C., 36.degree. C.,
37.degree. C., 38.degree. C., 39.degree. C., 40.degree. C.,
41.degree. C., 42.degree. C., 43.degree. C., 44.degree. C.,
45.degree. C., 46.degree. C., 47.degree. C., 48.degree. C.,
49.degree. C., 50.degree. C., 51.degree. C., 52.degree. C.,
53.degree. C., 54.degree. C., 55.degree. C., 56.degree. C.,
57.degree. C., 58.degree. C., 59.degree. C., 60.degree. C.,
61.degree. C., 62.degree. C., 63.degree. C., 64.degree. C.,
65.degree. C., 66.degree. C., 67.degree. C., 68.degree. C.,
69.degree. C., 70.degree. C., 71.degree. C., 72.degree. C.,
73.degree. C., 74.degree. C., or 75.degree. C. In preferred
embodiments, the temperature is between about 50.degree. C. and
about 55.degree. C., between about 55.degree. C. and about
60.degree. C., between about 60.degree. C. and about 65.degree. C.,
or between about 50.degree. C. and about 65.degree. C.
[0033] In some embodiments, the polymerase used herein is
thermostable. In some embodiments, the polymerase is mesophilic. In
some preferred embodiments, the polymerase does not have a
proofreading capability. In some preferred embodiments, the
polymerase has a strand displacement capability. In some preferred
embodiments, the polymerase has a 5' to 3' exonuclease activity. In
some preferred embodiments, the polymerase does not have
proofreading ability, but does have a strand-displacement
capability and a 5' to 3' exonuclease activity.
[0034] In some embodiments, nickases that target the same sequence
motif but nick at opposite strands are used to target specific DNA
strands to minimize the formation of fragile sites. In some
embodiments, nickases have been modified to only bind to one strand
of a double-stranded DNA. In some embodiments, nickases are used to
target a single strand from a first DNA molecule, and a single
strand from a second DNA molecule. In some of these embodiments, a
single strand from the first DNA is targeted by a first nickase,
and the complementary strand from the second DNA molecule is
targeted with a second nickase that recognizes the same sequence
motif as the first nickase. In some embodiments, the orientation of
extension is reversed for one of the strands. For example, in some
embodiments, extension from the site of nicking occurs in one
direction for a first DNA molecule, and in the opposite direction
for a second DNA molecule. In some embodiments, extension from the
site of nicking occurs in one direction for a top strand of a DNA
molecule, and in the opposite direction for the bottom strand for
the same DNA molecule.
[0035] In some embodiments, a reference map is used for assembly as
described herein.
[0036] In some embodiments, a plurality of nickases are used to
maximize information density. In some embodiments, molecules nicked
by the plurality of nickases are assembled using a reference
map.
[0037] In some embodiments, more than one nicking step is used to
maximize information density. In some embodiments, the molecule or
molecules subjected to more than one nicking step are assembled
using a reference map.
[0038] In some embodiments, DNA is linearized. Means of linearizing
DNA can include the use of shear force of liquid flow, capillary
flow, convective flow, an electrical field, a dielectrical field, a
thermal gradient, a magnetic field, combinations thereof (e.g., the
use of physical confinement and an electrical field), or any other
method known to one of skill in the art. In some embodiments, the
channel(s) described herein have a cross sectional dimension in the
micrometer range. In some preferred embodiments, channels have a
cross sectional dimension in the nanometer range. Examples of
nanochannels and methods incorporating the use of nanochannels are
provided in U.S. Publication Nos. 2011/0171634 and 2012/0237936,
which are hereby incorporated by reference in their entireties.
[0039] In some embodiments, a second motif is investigated in a
molecule of interest. In some embodiments, the second motif
includes at least one binding site for a binding entity selected
from a non-cutting restriction enzyme, a zinc finger protein, an
antibody, a transcription factor, a transcription activator like
domain, a DNA binding protein, a polyamide, a triple helix forming
oligonucleotide, and a peptide nucleic acid. In some embodiments,
marking or tagging of the second motif is effected with a binding
entity comprising a second label. In some embodiments, marking is
performed with a label that does not cut or nick the DNA. In some
embodiments, tagging is performed with a label that does not cut or
nick the DNA.
[0040] In some preferred embodiments, the second motif includes at
least one binding site for a peptide nucleic acid. In some
embodiments, tagging is effected with a peptide nucleic acid
comprising a second label. In other embodiments, the second motif
includes at least one recognition sequence for a methyltransferase.
In some embodiments, tagging is performed with a methyltransferase.
In some embodiments, tagging is performed with a methyltransferase
comprising a modified cofactor which includes a second label.
[0041] In some embodiments, a modified cofactor is used. In some
embodiments, the modified cofactor contains a second label that
functions as a transferable tag which becomes covalently coupled to
a methyltransferase recognition sequence. In other embodiments, the
modified cofactor contains a second label that is directly coupled
to a methyltransferase recognition sequence.
[0042] In some embodiments, the labels described herein are
selected from a fluorophore, a quantum dot, a dendrimer, a
nanowire, a bead, a hapten, a streptavidin, an avidin, a
neutravidin, a biotin, or a reactive group. In some preferred
embodiments, the first and second labels described herein are
selected from a fluorophore or a quantum dot.
[0043] In some embodiments, labeling is carried out with a
polymerase in the presence of at least one labeled dNTP using the
process of nick translation. The labeled dNTP preferably contains a
fluorophore or a quantum dot. In some embodiments, labeling is
carried out as described in U.S. Provisional Application No.
61/713,862, which is hereby incorporated by reference in its
entirety.
[0044] In some embodiments, the polymerase used herein leaves a
flap region that is removed to generate a ligatable nick prior to
repair. In some preferred embodiments, repair is carried out with a
DNA ligase. Examples of DNA ligases include Taq DNA ligase, E. coli
DNA ligase, T7 DNA ligase, T4 DNA ligase, and 9.degree. N DNA
ligase (New England Biolabs). In some embodiments, the flap region
is removed with an endonuclease. For example, in some preferred
embodiments, the flap region is removed with a flap endonuclease
(e.g., FEN I). In some embodiments, the flap region is removed with
an exonuclease. In some preferred embodiments, the flap region is
removed using the 5' to 3' exonuclease activity of a polymerase. In
some preferred embodiments, the flap region is removed using the 5'
to 3' exonuclease activity of a polymerase under conditions where
at least one of four nucleotides (e.g., dATP, dGTP, dCTP,
dTTP/dUTP) is provided in limited concentration. In some preferred
embodiments, the flap region is removed using the 5' to 3'
exonuclease activity of a polymerase under conditions where at
least one of the four nucleotides is omitted. In some preferred
embodiments, the flap region is removed using the 5' to 3'
exonuclease activity of a Taq polymerase. In some embodiments, the
flap is removed to restore ligatability of the translated nick. In
some embodiments, the flap region is removed and the nick is
repaired using a mixture of enzymes that perform these functions,
such as PreCR enzyme mix (New England BioLabs). In some
embodiments, the PreCR enzyme mix is used under conditions where at
least one of the four nucleotides is provided in limited
concentration or omitted.
[0045] Nucleotides that are not omitted during the flap removal
process can be present at a concentration of about 25 nM to about
50 nM each, about 50 nM to about 100 nM, about 100 nM to about 200
nM, about 200 nM to about 400 nM, about 400 nM to about 800 nM,
about 800 nM to about 1.6 uM, about 1.6 uM to about 3.2 uM, about
3.2 uM to about 6.4uM, about 6.4 uM to about 12.8 uM, about 12.8 uM
to about 25.6 uM, about 25.6 uM to about 51.2 uM, about 51.2 uM to
about 102.4 uM, about 102.4uM to about 204.8 uM, about 204.8 uM to
about 409.6 uM, and about 409.6 uM to about 819.2 uM, about 819.2
uM to about 1638.4 uM, or about 1638.4 uM to about 3276.8 uM. In
some preferred embodiments, the concentration of nucleotides that
are not omitted is about 50 uM to about 500 uM each. In some
preferred embodiments, the nucleotides that are present are present
in equimolar amounts.
[0046] In some embodiments, the at least one nucleotide that is
limited in concentration is at a concentration at least 2.times.
less, at least 5.times. less, at least 10.times. less, at least
20.times., at least 30.times. less, at least 60.times. less, at
least 100.times., at least 500.times. less, at least 1000.times.
less, or at least 3000.times. less than at least one of the other
nucleotides that is present. In some embodiments, the at least one
nucleotide that is limited in concentration is at a concentration
that is negligible compared to the nucleotides that are present. In
some preferred embodiments, the at least one nucleotides that is
limited in concentration is at a concentration at least 100.times.
less that the nucleotides that are present.
[0047] In some embodiments, a method for repairing flap-containing
DNA is provided. In some embodiments, at least one nucleotide is
omitted prior to DNA characterization. For example, in some
embodiments, the method entails treating a double stranded DNA
containing at least one flap on either stand of the DNA with a 5'
to 3' exonuclease activity of a polymerase under conditions wherein
at least one nucleotide is omitted, ligating the nicks to restore
strand integrity at the flap regions, and characterizing the DNA.
In some embodiments, at least one nucleotide is limited in
concentration prior to DNA characterization. For example, in some
embodiments, the method entails treating a double stranded DNA
comprising at least one flap on either stand of the DNA with a 5'
to 3' exonuclease activity of a polymerase under conditions wherein
at least one nucleotide is limited in concentration, ligating the
nicks to restore strand integrity at the flap regions, and
characterizing the DNA.
[0048] Methods for characterizing the molecules described herein
include any method for determining the information content of the
DNA, such as sequencing, mapping, single nucleotide polymorphism
(SNP) analysis, copy number variant (CNV) analysis, haplotyping, or
epigenetic analysis.
[0049] Unless defined otherwise, technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art.
[0050] The DNA described herein can be of any length (e.g., 0.1 Kb
to a mega base). The DNA can be a highly pure preparation, crude,
or semi-crude material. The DNA can come from any biological source
or can be synthetic.
[0051] As used herein, the term "polymerase" refers to any enzyme,
naturally occurring or engineered, that is capable of incorporating
native and modified nucleotides in a template dependent manner
starting at a 3' hydroxyl end.
[0052] As used herein, the term "nicking endonuclease" refers to
any enzyme, naturally occurring or engineered, that is capable of
breaking a phosphodiester bond on a single DNA strand, leaving a
3'-hydroxyl at a defined sequence. Nicking endonucleases can be
engineered by modifying restriction enzymes to eliminate cutting
activity for one DNA strand, or produced by fusing a nicking
subunit to a DNA binding domain, for example, zinc fingers and DNA
recognition domains from transcription activator-like
effectors.
EXAMPLES
[0053] The following examples are intended to illustrate, but not
to limit, the invention in any manner, shape, or form, either
explicitly or implicitly. While they are typical of those that
might be used, other procedures, methodologies, or techniques known
to those skilled in the art may alternatively be used.
Example 1
[0054] E. coli genomic DNA was nicked with Nt.BspQI nicking
endonuclease. The nicked DNA was labeled with Taq polymerase by
nick translation using Atto dUTP or Alexa dUTP in the presence of
cold dATP, dGTP, and dCTP. The labeled nicks were: 1.) not
repaired, 2.) repaired with PreCR as recommended by manufacture
(New England BioLabs), 3.) repaired with PreCR under conditions of
omitting dGTP, 4.) repaired with PreCR under conditions of omitting
dATP and dGTP, or 5.) repaired with Taq polymerase under conditions
of omitting dGTP. Ligation was then performed with a ligase. The
resulting DNA was stained with YOYO-1 (Life Technologies) and
processed on the Irys system (BioNano Genomics). Briefly, DNA was
linearized in massively parallel nanochannels, excited with the
appropriate laser for backbone and label detection, and optically
imaged. Mapping to a reference genome, center of mass, and False
Positive (FP) and False Negative (FN) calculations were carried out
using nano Studio data analysis software (BioNano Genomics).
Results are shown in FIG. 2.
Example 2
[0055] E. coli genomic DNA was nicked with Nt.BspQI nicking
endonuclease. The nicked DNA was labeled with Taq polymerase by
nick translation using Atto dUTP. The labeled DNA was: 1.) left
unrepaired or 2.) treated with FEN Ito remove flaps followed by a
ligase to repair the translated nicks. The DNA was linearized in
massively parallel nanochannels, excited with the appropriate laser
for backbone and label detection, and optically imaged. Mapping to
a reference genome, center of mass, and False Positive (FP) and
False Negative (FN) calculations were carried out using nano Studio
data analysis software (BioNano Genomics). Results are shown in
FIG. 3.
Example 3
[0056] Drosophila genomic DNA was nicked with Nt.BspQI or Nb.BbVCI
nicking endonuclease. The nicked DNA was labeled with Taq
polymerase by nick translation using Atto dUTP. The labeled DNA was
treated with PReCR reagent (New England Biolabs) to repair the
nicks. The resulting DNA was stained with YOYO-1 (Life
Technologies) and processed on the Irys system (BioNano Genomics).
Mapping to a reference genome, center of mass, and False Positive
(FP) and False Negative (FN) calculations were carried out using
nano Studio data analysis software (BioNano Genomics). Results are
shown in FIG. 4.
Example 4
[0057] A genome map was constructed using two nicking enzymes,
Nt.BbvCI and Nt.BspQI, whose nick motifs were labeled with red and
green dyes, respectively, across 27 BACs making up an MTP of a
2.1-Mb region containing the prolamin multigene family in the Ae.
tauschii genome. FIG. 5A shows the layout of the IrysChip (BioNano
Genomics).
[0058] The YOYO-stained DNA was loaded into the port, unwound
within the pillar structures, and linearized inside 45 nm
nanochannels (FIG. 5B). After image processing, individual BAC
molecules with red and green labels distributed at
sequence-specific locations were compared and clustered into pools
with similar map patterns (FIG. 5C, top). Density plots for the BAC
clones were generated to determine the consensus peak locations
(FIG. 5C, bottom). The consensus maps of individual BAC clones were
aligned based on overlaps of consensus maps of adjacent BACs (FIG.
5D) to create a genome map of the entire region.
[0059] The two-color labeling strategy resulted in an average
information density of one label per 4.8 kb (437 labels in 2.1 Mb).
Since each motif was marked by its own color, peaks of different
motifs could be distinguished from each other even if their peaks
were almost overlapping (arrow in FIG. 5D). Peaks of the same motif
(i.e., the same color) could be resolved when they were at least
.about.1.5 kb apart. Taking advantage of the combination of long
molecule lengths (.about.140 kb average), high-resolution, accurate
length measurement, and multiple sequence motifs, a high-quality
genome map of the 2.1-Mb region for scaffold assembly was
generated.
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