U.S. patent application number 12/761096 was filed with the patent office on 2011-08-18 for repair of nucleic acids for improved amplification.
This patent application is currently assigned to NEW ENGLAND BIOLABS, INC.. Invention is credited to LIXIN CHEN, THOMAS C. EVANS, CHUDI GUAN, BARTON SLATKO, ROMALDAS VAISVILA.
Application Number | 20110201056 12/761096 |
Document ID | / |
Family ID | 35809781 |
Filed Date | 2011-08-18 |
United States Patent
Application |
20110201056 |
Kind Code |
A1 |
EVANS; THOMAS C. ; et
al. |
August 18, 2011 |
Repair of Nucleic Acids for Improved Amplification
Abstract
Methods and compositions are provided for repairing a
polynucleotide so that it can be synthesized efficiently with
improved fidelity and yield in, for example, an amplification
reaction. This involves the use of a reaction mixture that includes
a ligase and a cofactor selected from NAD+ or ATP and incubating
the polynucleotide with the reaction mixture in the absence of
Endonuclease VI. The reaction mixture may further contain an AP
endonuclease and a polymerase. These enzymes are optionally
selected according to their ability to withstand high temperatures
so they can be included in an amplification mixture. The reaction
mixture may be used prior to a polynucleotide synthesis reaction in
which case enzymes that are not thermophilic may be used. The
repair reaction is not time sensitive with respect to seconds,
minutes or hours of incubation in the enzyme mixture.
Inventors: |
EVANS; THOMAS C.;
(TOPSFIELD, MA) ; SLATKO; BARTON; (IPSWICH,
MA) ; CHEN; LIXIN; (BEVERLY, MA) ; VAISVILA;
ROMALDAS; (IPSWICH, MA) ; GUAN; CHUDI;
(WENHAM, MA) |
Assignee: |
NEW ENGLAND BIOLABS, INC.
IPSWICH
MA
|
Family ID: |
35809781 |
Appl. No.: |
12/761096 |
Filed: |
April 15, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11255290 |
Oct 20, 2005 |
7700283 |
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12761096 |
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60620896 |
Oct 21, 2004 |
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60646728 |
Jan 24, 2005 |
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60673925 |
Apr 21, 2005 |
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Current U.S.
Class: |
435/91.2 ;
435/91.5 |
Current CPC
Class: |
C12Q 1/6844 20130101;
C12Q 1/6844 20130101; C12Q 1/686 20130101; C12Q 1/6848 20130101;
C12Q 1/686 20130101; C12Q 2521/331 20130101; C12Q 2521/301
20130101; C12Q 2521/101 20130101; C12Q 1/6848 20130101; C12Q
2521/501 20130101; C12Q 2521/101 20130101; C12Q 2521/514 20130101;
C12Q 2521/331 20130101; C12Q 2521/501 20130101; C12Q 2521/501
20130101 |
Class at
Publication: |
435/91.2 ;
435/91.5 |
International
Class: |
C12P 19/34 20060101
C12P019/34 |
Claims
1. A method for enhancing at least one of fidelity and yield of an
amplification product of a damaged polynucleotide, comprising the
steps of: (a) incubating the polynucleotide containing one or more
damaged sites with a reaction mixture comprising an effective
amount of a ligase in the absence of ATP, and in the absence of
Endonuclease (Endo) VI, for repairing the damaged sites in the
polynucleotide; and (b) amplifying the polynucleotide in the
reaction mixture.
2. A method according to claim 1, wherein the reaction mixture in
step (a) further comprises a polymerase and an AP endonuclease.
3. A method according to claim 2, wherein the AP endonuclease is a
class II AP endonuclease.
4. A method according to claim 3, wherein the AP endonuclease is
selected from the group consisting of a T4 endonuclease, an E. coli
endonuclease, Tth Endo IV, and human AP endonuclease.
5. A method according to claim 3, wherein the AP endonuclease is E.
coli Endo IV.
6. A method according to claim 2, wherein the polymerase is
selected from the group consisting of Taq DNA polymerase, Bst DNA
polymerase, T4 DNA polymerase, T7 DNA polymerase, E. coli DNA
polymerase I and an archaeal DNA polymerase or modifications
thereof.
7. A method according to claim 2, wherein the polymerase is an
archaeal DNA polymerase and the archaeal polymerase is selected
from Pfu, Vent.RTM., Deep Vent.RTM., 9.degree. North, and GBD DNA
polymerase.
8. A method according to claim 2, wherein the polynucleotide is DNA
and the reaction mixture in step (a) comprises 1-100 units of the
AP endonuclease, 0.05-0.25 units of the polymerase and 5-500 units
of the ligase.
9. A method according to claim 1, wherein the reaction mixture in
(a) further comprises T4 pyrimidine dimer glycosylase (pdg).
10. A method according to claim 1, wherein the reaction mixture in
step (a) further comprises [fapy]-DNA glycosylase (Fpg).
11. A method according to claim 1, wherein the reaction mixture in
(a) further comprises at least one of UvrA, UvrB, UvrC, UvrD and
Cho.
12. A method according to claim 1, wherein the reaction mixture in
(a) further comprises at least one glycosylase/lyase
13. A method according to claim 12, wherein the at least one
glycosylase/lyase is selected from the group consisting of Endo
III, Endo VIII, Fpg, OGGI, and T4 pdg.
14. A method according to claim 1, wherein the reaction mixture in
(a) further comprises at least one glycosylase.
15. A method according to claim 1, wherein the at least one
glycosylase is selected from the group consisting of UDG, AlkA and
Aag.
14. A method according to claim 2, wherein the polymerase is a Bst
DNA polymerase, the AP endonuclease is Endo IV, the ligase is Taq
ligase, the reaction mixture in (a) further comprising one or more
glycosylases and one or more glycosylase/lyases.
15. A method according to claim 1, wherein the amplification is PCR
amplification, helicase-dependent amplification,
transcription-mediated amplification, strand-displacement
amplification, rolling circle amplification or whole genome
amplification.
16. A method according to claim 14, wherein the at least one
glycosylase is UDG.
17. A method according to claim 2, wherein the polymerase is an E.
coli Y family DNA polymerase.
Description
CROSS REFERENCE
[0001] This application is a divisional application of U.S.
application Ser. No. 11/255,290 filed Oct. 20, 2005, which claims
priority from U.S. Provisional Application Ser. No. 60/620,896
filed Oct. 21, 2004, U.S. Provisional Application Ser. No.
60/646,728 filed Jan. 24, 2005 and U.S. Provisional Application
Ser. No. 60/673,925 filed Apr. 22, 2005.
BACKGROUND
[0002] Various approaches have been reported to repair DNA using
base excision enzymes. Unfortunately, these approaches in different
ways cause further damage to the DNA. Conventional PCR techniques
have been modified to improve amplification in some aspects. U.S.
Pat. No. 5,035,996 describes a process for controlling
contamination of nucleic acid amplification reactions that uses the
modified nucleotide, dUTP, in the amplification reaction. This
process uses uracil glycosylase to eliminate those PCR products
containing uracil to prevent contaminating subsequent PCR
reactions. U.S. patent publication no. 2004-0067559 A1 also relies
on modified bases in primer DNA prior to amplification and uses,
for example, dUTP for incorporation into the amplicon. The amplicon
can then be fragmented by adding, for example, Uracil-DNA
Glycosylase (UDG) and Endonucleaese (Endo) IV.
[0003] Hot start nucleic acid amplification has been used to lower
mis-priming during PCR. One type of hot start amplification relies
on the presence of a PCR primer with a blocked 3' terminus to
prevent extension by the polymerase present in the PCR reaction
(see for example US 2003-0119150). The primer is unblocked by a
thermostable 3'-5' exonuclease that is active at >37.degree. C.
Therefore, the polymerase will only extend the PCR primers once the
exonuclease unblocks the 3' end at >37.degree. C. Alternatively
the Taq polymerase is blocked and then activated at amplification
temperatures.
[0004] Barnes, W. M. Proc. Natl. Acad. Sci. USA 91:2216-2220 (1994)
describes the use of vent polymerase and Taq polymerase as an
improvement over the use of Taq polymerase only in amplification.
Ghadessy et al. reported a mutant Taq polymerase that is not halted
by damaged or abasic sites (Ghadessy et al. Nature Biotechnol.
22(6) :755-9 (2004)).
[0005] It has been reported that conventional amplification
techniques are compromised if the DNA is substantially damaged
(DiBernardo et al. Nucl. Acids Res. 30:e16 (2002)). Degradation
and/or fragmentation of DNA resulting from exposure to the
environment and microorganisms which contain DNA endonucleases is a
frequent problem in forensics, diagnostic tests and routine
amplification and affects fidelity and yield of the amplification
product. In addition, the problem of degraded DNA is also faced by
researchers who are analyzing the DNA obtained from frozen, extinct
or extremely rare organisms.
[0006] Fromenty, B., et al. Nucl. Acids Res. 28(11):e50 (2000) and
International Publication No. WO/0151656 reported that Exonuclease
(Exo) III improved yields of long PCR. Fromenty also reported
decreased yields of amplicon for DNA<500 bp. One of the problems
associated with the use of Exo III is that it degrades template and
primers.
[0007] Di Benardo et al. Nucl. Acids Res. 30(4):e16 (2002)
described the use of T4 DNA ligase (T4 ligase) and an E. coli
polymerase to amplify short regions of single-stranded DNA between
cross-linked regions of double-stranded DNA.
[0008] Another approach to amplification of damaged DNA has been
described in U.S. Publication No. 2003-0077581. Degraded nucleic
acid was hybridized to undegraded nucleic acid having a sequence
homologous to the degraded nucleic acid. Regions of the degraded
nucleic acid were then filled in with nucleotide precursors. The
fragmented strands were then covalently linked using a polymerizing
and/or ligating enzyme.
[0009] Preparations for improving amplification of damaged DNA can
be obtained commercially from Sigma, St. Louis, Mo. and Qbiogene,
now MP Biomedicals, Irvine, Calif. Although the compositions of
these preparations are not provided, it is assumed that Exo III is
contained in the preparation. The preparations are not recommended
for DNA templates less than 500 base pairs in length.
[0010] Others report the use of a combination of E. coli DNA PolI
and T4 ligase for pre-amplification repair (Pusch, et al., Nucl.
Acids Res.
[0011] 26:857 (1998)). However, according to Pusch et al. the
preamplification product is purified before initiation of
amplification.
SUMMARY
[0012] In an embodiment of the invention, a method is provided for
enhancing at least one of fidelity and yield of an amplification
product of a damaged polynucleotide, that includes the steps of:
(a) incubating the polynucleotide in a reaction mixture comprising
a ligase and a cofactor selected from NAD+ or ATP and excluding
Endo VI; (b) permitting amplification of the polynucleotide to
occur in the reaction mixture by the addition of amplification
reagents to the reaction mixture during or after step (a); and (c)
enhancing at least one of fidelity or yield of the amplification
product in the presence of step (a) compared to in the absence of
step (a).
[0013] The above method is not particularly time sensitive in
respect to whether the incubation occurs in seconds, minutes or
hours. The ligase used in embodiments of the method may be
mesophilic or thermophilic and does not exclude cryophilic ligases,
which might be useful under particular circumstances. The choice of
ligase with respect to temperature sensitivity depends on what is
best suited for a particular set of reaction conditions. For
example, if the amplification reagents are added during the
incubation step (a), then it may be desirable to employ a
thermophilic ligase to withstand temperatures utilized during
amplification. Examples of thermophilic ligases are Taq DNA ligase
(Taq ligase) and 9.degree. N ligase. Taq ligase is more effective
with a NAD.sup.+ cofactor while 9.degree. N DNA ligase (9.degree. N
ligase) is more effective with an ATP cofactor. Examples of a
mesophilic ligase are T4 ligase (using an ATP cofactor) and E. coli
DNA ligase (E. coli ligase) (using an NAD.sup.+ cofactor).
[0014] The reaction mixture may further include an AP endonuclease
such as Type II endonuclease, T7 Endonuclease (Endo) I or mutant
thereof or Endo IV. The reaction mixture may alternatively or also
include a polymerase for example Taq polymerase, an E. coli
polymerase, a Thermomicrobium sp. polymerase or an archaeal
polymerase or mutant thereof such as Pfu, Vent.RTM., Deep
Vent.RTM., 9.degree. N or GBD polymerase.
[0015] In embodiments of the invention, enzymes that may be
additionally added to the reagent mixture include T4 pyrimidine
dimer glycosylase, [fapy]-DNA glycosylase (Fpg), at least one of
UvrA, UvrB, UvrC, UvrD, Cho, UDG, Aag, Endo III and Endo V in
various combinations depending on the type of damage sustained by
the polynucleotide.
[0016] In an embodiment of the invention, a reaction mixture is
used containing about 1-100 units of endonuclease, about 0.05-0.25
units of polymerase and about 5-500 units of ligase optionally
added to 1-1000 ng DNA.
[0017] Types of damage that may affect a polynucleotide include
apurinic/apyrimidinic (AP) sites, mutagenized nucleotides, modified
nucleotides, nicks, gaps and DNA-DNA or DNA-protein
cross-links.
[0018] The damaged polynucleotide may be obtained from natural
sources, preserved biological material, forensic evidence, ancient
polynucleotides, a tissue biopsy or routine biological
manipulation.
[0019] According to embodiments of the method, amplification of DNA
is achieved by any of PCR amplification, helicase-dependent
amplification, transcription-mediated amplification,
strand-displacement amplification, rolling circle amplification and
whole genome amplification.
[0020] Where the polynucleotide is a single-stranded RNA, the
amplification may be a reverse transcriptase dependent
amplification.
[0021] In an embodiment of the method, the polynucleotide is
capable of producing an amplicon in a size range of 50 nucleotides
to 100,000 nucleotides for PCR amplification.
[0022] In an embodiment of the invention, an amplification kit is
provided that includes instructions for use and one or more enzymes
wherein at least one of the enzymes is a ligase, the one or more
enzyme being formulated for addition to an amplification mixture to
enhance amplification or for use prior to addition of the
amplification mixture to enhance amplification.
[0023] In another embodiment of the invention, a composition is
provided that contains an effective amount of a ligase, a
polymerase, and an AP endonuclease not including Endo VI, the
mixture being capable of enhancing at least one of yield and
fidelity of amplification of a polynucleotide compared with
amplification of the polynucleotide in the absence of the
composition. For example, concentrations of reagents in the
composition include: an AP endonuclease at a concentration of 1-100
units of endonuclease, a polymerase at 0.05-0.25 units, and 5-500
units of ligase contained for example in a reaction volume of
10-100 .mu.l. This formulation may be applied to 1-1000 ng DNA for
repairing the DNA. For larger concentrations of DNA, the amounts of
enzymes should be increased proportionally. In embodiments of the
invention, additional enzymes may be included in the composition
including one or more of T4 pyrimidine dimer glycosylase,
[fapy]-DNA glycosylase (Fpg), UvrA, UvrB, UvrC, UvrD, Cho, UDG,
Aag, Endo III and Endo V in various combinations depending on the
type of damage sustained by the polynucleotide.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIGS. 1A-1D show enhanced amplicon yield from heat-damaged
lambda DNA after preincubation with specified enzymes.
[0025] FIG. 1A shows DNA template damaged by heat to differing
extents and the effect of this damage on amplification of a 5 kb
segment of lambda DNA where 5 ng, 2 ng and 1 ng of heat-treated
lambda DNA was amplified after prior damage by 99.degree. C. heat
treatment for 0 sec, 30 sec, 60 sec, 90 sec, 120 sec or 180 sec.
The damaged DNA was not subjected to enzyme treatment prior to
amplification. The amount of amplification was determined after
electrophoresis and was found to be substantially reduced by 120
sec heat treatment. The first and last lanes on the gel contain 1
pg of a 2-log ladder size standard (NEB#N3200, New England Biolabs,
Inc., Ipswich, Mass.).
[0026] FIG. 1B shows increased amplicon yields from heat-damaged
lambda DNA using Taq ligase, E. coli Endo IV and E. coli PolI on
amplification of a 5 kb segment of lambda DNA. DNA was heat damaged
as described in FIG. 1A but the damaged DNA was subjected to enzyme
treatment prior to amplification. The results of amplification are
shown after a 10-minute pretreatment reaction with Taq ligase, E.
coli Endo IV and E. coli PolI. The amplicon yield was increased
throughout but was especially noticeable with 120 sec and 180 sec
heat damaged DNA.
[0027] FIG. 1C shows increased amplicon yields from heat-damaged
lambda DNA using Taq ligase, Tth Endo IV and E. coli PolI. The
amplification was performed according to FIG. 1B but the enzyme
treatment prior to amplification contained Thermus thermophilus
(Tth) Endo IV in place of E. coli Endo IV. The results of
amplification are shown after a 10-minute pretreatment reaction
with Thermus aquaticus (Taq) ligase, Tth Endo IV and E. coli PolI.
The amplicon yield was increased throughout but was especially
noticeable with 120 sec and 180 sec heat-damaged DNA. Only the
first lane contains the size ladder.
[0028] FIG. 1D shows increased amplicon yields from heat-damaged
lambda DNA using E. coli ligase, E. coli Endo IV and E. coli DNA
poll. The amplification was performed according to FIG. 1B but the
enzyme treatment prior to amplification contained E. coli ligase in
place of Taq ligase. The lambda DNA subjected to 99.degree. C. for
180 sec was used as a template. The amount of template DNA used is
indicated above each lane. The yield of amplicon is increased for
each of the template amounts by enzyme pretreatment.
[0029] FIGS. 2A-2B show the effect of citrate treatment of template
DNA on amplicon yield.
[0030] FIG. 2A shows the results of amplification of a 5 kb segment
of lambda DNA where lambda DNA was heated to 70.degree. C. in
citrate buffer for 0, 20, 40, 80, 120, and 160 minutes. 50 ng, 10
ng and 5 ng of each heat-treated sample were amplified and the
resulting products were visualized on a gel to determine the extent
of amplification. The DNA was not treated with selected enzymes
prior to amplification. The last lane on the right contains 1 .mu.g
of 2-log ladder.
[0031] FIG. 2B shows the increase in yield of a 5 kb amplicon of
lambda DNA regardless of which polymerase was used in the enzyme
mixture. 120-minute heat/citrate-damaged lambda DNA was treated
with various enzymes prior to amplification.
[0032] Lane 1: 1 .mu.g 2-log ladder (NEB# N3200, New England
Biolabs, Inc., Ipswich, Mass.).
[0033] Lane 2: no pretreatment.
[0034] Lane 3: Pretreatment with Taq ligase, Taq DNA polymerase and
E. coli Endo IV.
[0035] Lane 4: Pretreatment with Taq ligase, E. coli PolI, and E.
coli Endo IV.
[0036] Lane 5: Pretreatment with Taq ligase, Taq:Vent DNA
polymerase mix, and E. coli Endo IV.
[0037] FIG. 3 shows the results of amplification of a 200 by
segment of krill genome that has been extracted from an ethanol
stored sample of krill and pretreated with an enzyme mixture
containing one of various polymerases, a ligase and an AP
endonuclease that enhances amplification yields.
[0038] Lane 1: No pretreatment of krill DNA with enzymes.
[0039] Lane 2: Pretreatment of krill DNA with Taq ligase, E. coli
Endo IV, and Taq polymerase.
[0040] Lane 3: Pretreatment of krill DNA with Taq ligase, E. coli
Endo IV, and Vent.RTM. polymerase.
[0041] Lane 4: Pretreatment of krill DNA with Taq ligase, E. coli
Endo IV, and 50:1 Taq:Vent.RTM. polymerase.
[0042] FIG. 4 shows an increase in yield of a 10 kb amplicon from
heat-damaged DNA. 180 sec heat-damaged DNA was pretreated with an
enzyme mixture and then amplified.
[0043] Lane 1: 1 .mu.g of a 2-log ladder size standard (NEB#N3200,
New England Biolabs, Inc., Ipswich, Mass.).
[0044] Lane 2: Pre-treatment with Taq ligase, E. coli Endo IV, and
E. coil PolI.
[0045] Lane 3: Pre-treatment with Taq ligase and E. coli Endo
IV.
[0046] Lane 4: Pretreatment with Taq ligase.
[0047] Lane 5: Control--untreated DNA.
[0048] FIG. 5 shows that ligase pretreatment increases amplicon
yield from environmental DNA (soil sample extract).
[0049] Lane 1: A 2-log ladder size standard (NEB# N3200, New
England Biolabs, Inc., Ipswich, Mass.).
[0050] Lane 1: No enzyme pretreatment.
[0051] Lane 2: Pre-treatment with T4 ligase.
[0052] Lane 3: No enzyme pre-treatment.
[0053] Lane 4: Pretreatment with Taq ligase.
[0054] FIG. 6: Genbank search revealing proteins with sequence
homology with T4 ligase.
[0055] FIG. 7: DNA sequence of Tth Endo IV (SEQ ID NO:11).
[0056] FIG. 8 shows the effect of UV light on amplicon yield using
lambda DNA as a template by gel electrophoresis.
[0057] A: Lambda DNA is subjected to UV irradiation for up to 50
sec and a slight reduction in yield of a 2 Kb amplicon produced is
shown.
[0058] B: Lambda DNA is subjected to UV irradiation for up to 50
seconds and the reduction in yield of a 5 kb amplicon is shown.
[0059] C: The effect of various reaction mixtures added to lambda
DNA on yield of a 5 kb amplicon after UV irradiation is shown.
[0060] Lanes 2-7 are controls in the absence of a reaction
mixture.
[0061] Lanes 8-13 show the increased beneficial effect of adding
ligase, polymerase and AP endonuclease plus 10 Units of T4 PDG.
[0062] Lanes 14-19 show the increased beneficial effect of adding
ligase, polymerase and AP endonuclease plus 80 units of T4 PDG.
Lanes 1 and 20: A 2-log ladder size standard (NEB#N3200, New
England Biolabs, Inc., Ipswich, Mass.).
[0063] FIGS. 9A-9B show that adding ligase to T7 Endo I expands the
useful range of the enzyme:DNA ratio to facilitate the removal of
heteroduplexes from the amplification mixture so as to increase the
ratio of correct sequences. Taq ligase and T7 Endo I were added to
supercoiled DNA in varying amounts as indicated for each lane.
[0064] FIG. 9A is the control in which no Taq ligase has been added
but increasing amounts of T7 Endo I are used. The supercoiled DNA
is predominantly cleaved into fragments of various sizes with
12.5-25 units of T7 Endo I.
[0065] FIG. 9B shows how the addition of 100 units of Taq ligase
protects DNA from non-specific cleavage in the presence of T7 Endo
I such that even at 200 units of T7 Endo I, there is a clear band
corresponding to linear DNA not present in the absence of
ligase.
[0066] FIGS. 10A-10B show the effect of repair enzyme treatment on
amplicon yield from oxidatively damaged DNA or undamaged
template.
[0067] FIG. 10A shows that the addition of repair enzymes to an
undamaged template, pWB407 has no effect on amplicon yield.
[0068] FIG. 10B shows that the addition of Fpg to a damaged
template, plasmid pWB407, incubated in the presence of methylene
blue, gives inconsistent effects on yield. The addition of Taq
ligase, E. coli DNA polymerase, and E. coli Endo IV in the presence
or absence of Fpg increases amplicon yield consistently.
[0069] FIG. 11 shows increased PCR reaction accuracy from damaged
DNA after treatment with repair enzymes. Repair enzyme treatment of
undamaged template, plasmid pWB407, prior to PCR has no significant
effect on reaction accuracy. Treatment of a damaged template,
plasmid pWB407 incubated with methylene blue, with Fpg alone or
also with Taq ligase, E. coli DNA polymerase I, and E. coli
endonuclease increases the accuracy of PCR. The measure of accuracy
is the number of white colonies verses the number of blue colonies
after cloning a lacZ-containing amplicon as discussed below. The
higher the percentage of white colonies the greater the error
rate.
[0070] FIG. 12 shows a flow diagram for treating DNA with unknown
damage to increase at least of one of fidelity and yield.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0071] Embodiments of the invention describe methods for improving
at least one of yield or fidelity for synthesis of damaged
polynucleotides. Where polynucleotide synthesis leads to
polymerase-dependent amplification, short amplicons that are less
than about 500 bases in length (as short as 100 nt) or long
amplicons that are greater than 500 bases or as much as about 100
kb may be amplified (for PCR amplification). Other types of
polynucleotide synthesis include primer extension reactions such as
amplification (for example PCR, RT-PCR, and QPCR), genome
amplification, rolling circle amplification (RCA) and
helicase-dependent amplification (HDA); and DNA sequencing
reactions. Embodiments of the methods have wide utility in
molecular biology research and in solving problems in applied
biology, for example, in forensics, in biological archeology in
which it is desirable to analyze DNA from ancient sources, for
taxonomy where it is desirable to analyze DNA from environmental
samples such as required for the Barcode of Life Project, for
diagnostic assays including tissue biopsies to determine a disease
susceptibility or status and for molecular biology research.
Source and Extent of Damage
[0072] Damage sustained by polynucleotide molecules is common even
in "normal" polynucleotides although damage is more severe in
preserved tissues, dried specimens or polynucleotides that are
exposed to the environment. Damage can occur as a result of the age
of the sample or its length, its source or its preparation. In
addition, damage can occur during the application of a methodology
for polynucleotide synthesis such as occurs during PCR
amplification, which involves a high temperature step.
[0073] Polynucleotides can sustain damage in a variety of ways.
Various types of damage include: (a) apurinic or apyrimidinic
damage caused for example by heat, storage of polynucleotides in
ethanol, and exposure to factors in the environment such as
H.sub.2O, pH etc; (b) modification of individual nucleotides,
caused for example by deamination, alkylation, oxidation and
dimerization; (c) nicks and gaps caused for example by heat,
storage of polynucleotide in ethanol, and exposure to factors in
the environment such as H.sub.2O, pH etc; (d) cross-linking caused
for example, by formaldehyde, environmental factors, and ethanol
storage; and (e) mismatched DNA caused by for example
misincorporation of a nucleotide by a polymerase.
[0074] Different polynucleotide preparations will experience
different types of damage resulting from, for example, storage or
handling of the polynucleotide preparation in vitro, and may depend
on how prokaryotic cells, archaea or eukaryotic cells containing
the polynucleotides are stored and the characteristics of the cells
from which the polynucleotide is extracted.
Definitions
[0075] The term "polynucleotide" refers to double-stranded DNA,
double-stranded RNA, hybrid DNA/RNA duplex, single-stranded DNA and
single-stranded RNA.
[0076] A "repair enzyme" refers to a cryophilic, mesophilic or
thermophilic enzyme that participates in the process of repair of a
polynucleotide. For example, a repair enzyme may induce breakage of
the polynucleotide at a bond, thereby facilitating removal of
damaged regions of the polynucleotide. Enzymes with a synthetic
role such as ligases and polymerases are also repair enzymes.
[0077] DNA repair enzymes are described in the scientific
literature, for example, see Wood, R. D., et al. Mutat. Res.
577(1-2):275-83 (2005) and Eisen, J. A. and Hanawalt, P. C. Mutat.
Res. 435(3):171-213 (1999). A list of human repair enzymes is
provided in Table 1. Although not described in Table 1, the
homologs of the listed enzymes and other functionally related
enzymes are included in the definition of repair enzymes. Any of
the above enzymes may be naturally occurring, recombinant or
synthetic. Any of the enzymes may be a native or in vitro-created
chimera with several activities. In addition to the enzymes
described above, it is known to a person of ordinary skill in the
art how to search the databases to identify other related enzymes
that share conserved sequence motifs and have similar enzyme
activity. For example, the NCBI web site (www.ncbi.com) provides a
conserved domain database. If, for example, the database is
searched for homologs of Endo IV, 74 sequence matches are
recovered. (Also see FIG. 6 for ligases).
[0078] A "polynucleotide cleavage enzyme" used in enzyme mixtures
for repairing damaged DNA is a class of repair enzymes and includes
AP endonucleases, glycosylases and lyases responsible for base
excision repair.
[0079] A damaged base can be removed by a DNA glycosylase enzyme
which hydrolyses an N-glycosylic bond between the deoxyribose sugar
moiety and the base. The product of this reaction is an apurinic or
apyrimidinic site (AP site) that must be correctly filled. This can
be achieved by an endonuclease, which nicks the sugar phosphate
backbone adjacent to the AP site. The abasic sugar is removed and a
new nucleotide is inserted by polymerase/ligase activity. Some
enzymes having applicability herein have glycosylase and AP
endonuclease activity in one molecule. These repair enzymes are
found in prokaryotic and eucaryotic cells. Abasic sites can be
recognized and cleaved by AP endonucleases and/or AP lyases. Class
II AP endonucleases cleave at AP sites to leave a 3' OH that can be
used in polynucleotide polymerization. Furthermore, AP
endonucleases can remove moieties attached to the 3' OH that
inhibit polynucleotide polymerization. For example a 3' phosphate
can be converted to a 3' OH by E. coli Endo IV. AP endonucleases
can work in conjunction with glycosylases.
[0080] Examples of glycosylase specificities include Uracil,
Hypoxanthine, 3-methyladenine (3-mAde), Formamidopyrimidine and
Hydroxymethyluracil. The presence of Uracil in DNA occurs due to
mis-incorporation or deamination of cytosine by bisulfate, nitrous
acids, or spontaneous deamination. Hypoxanthine occurs due to
deamination of adenine by nitrous acids or spontaneous deamination.
3-mAde is a product of alkylating agents. E. coli has two 3-mAde
glycosylase called TagI and TagII. Formamidopyrimidine (FAPY)
(7-mGua) is the most common product of methylating agents of DNA.
Gamma radiation produces 4.6-diamino-5-FAPY. An E. coli glycosylase
that repairs this lesion is Fpg endonuclease. Hydroxymethyuricil is
created by ionizing radiation or oxidative damage to thymidine.
[0081] Lyases break the phosphodiester bond in a
polynucleotide.
[0082] Examples of AP endonucleases belong to 4 classes.
[0083] (I) cleaves 3'.fwdarw.3'-OH+5'-P--and has associated
glycosylase activity.
[0084] (II) cleaves 5.fwdarw.3'-OH+5'-P
[0085] (III) cleaves 3.fwdarw.3'-P+5'-OH
[0086] (IV) cleaves 5.fwdarw.3'-P+5'-OH
[0087] Several enzymes have been isolated that appear to have AP
endonuclease or lyase and glycosylase activities that are
coordinated in a concerted manner (i.e., without causing AP site
formation) or sequentially.
[0088] Examples of polynucleotide cleavage enzymes for use in
enhancing at least one of yield and fidelity in an amplification
reaction include: 1) AP endonucleases, such as E. coli Endo IV, Tth
Endo IV, and human AP endonuclease; 2) glycosylases, such as UDG,
E. coli AIkA and human Aag; and 3) glycosylase/lyases, such as E.
coli Endo III, E. coli Endo V, E. coli Endo VIII, E. coli Fpg,
human OGG1, T4 pyrimidine dimer glycosylase (T4 pdg) and human AP
endonuclease.
[0089] Endo VI (also termed Exo III) is capable of degrading a
substantial portion of a polynucleotide outside the damaged regions
in a polynucleotide under normal reaction conditions in a few hours
and is not included in enzyme mixtures for treating damaged
polynucleotides.
[0090] A "polymerase" as used in enzyme mixtures herein refers to
an enzyme that contains polymerase activity. The repair and
amplifying polymerases can be the same or different.
[0091] Examples of polymerases include thermostable bacterial
polymerases such as Taq polymerase and archeal polymerases such as
Vent.RTM., deep Vent.RTM. and Pfu; and thermolabile enzymes such as
Bst polymerase, E. coli PolI, thermomicrobium roseum polymerase and
thermomicrobium thermophilus, phage polymerases such as phi29
polymerase, T7 polymerase and T4 polymerase etc., or mutants,
derivatives or modifications therefrom. Examples of derivatives
include Pfusion.TM. enzyme (Finnzymes, Espoo, Finland) and other
polymerases that combine a double strand binding protein with
polymerase sequences from one or several sources.
[0092] A "ligase" as used in the enzyme mixtures described here
refers to an enzyme that joins a 5' end of a single strand of a
polynucleotide to a 3' end of another single strand of a
polynucleotide. Such ligases are found in substantially all
eukaryotic cells as well as prokaryotic cells, viruses and archaea.
Any of these ligases can be used in repair as described herein.
Examples of ligases include 9.degree. N ligase, E. coli ligase, T4
ligase and Taq ligase. Other ligases include LIGA (NP-416906.1),
TthDNALGS (AAA27486.1), LIG3 (NM-013975) and LIG4 (NM-002312).
[0093] Other ligases or ligase-like proteins that may have utility
herein are revealed by a Genbank search using T4 ligase or E. coli
ligase to search the database (see FIG. 6) in which any enzyme
sharing at least 6 contiguous amino acids with these known ligases
may be included in a repair mixture according to embodiments of the
invention.
[0094] Contrary to a published use of ligase in combination with
Exo III in the absence of any cofactors (U.S. Publication No.
2005-0026147), it has been found here that NAD+ or ATP is required
in enzyme mixtures that include ligase. More specifically, Taq
ligase and E. coli ligase require NAD+ while T4 ligase and
9.degree. N ligase require ATP.
[0095] Exemplified ligases, polymerases and endonucleases are
available from New England Biolabs Inc. where pages 107-117 of the
2005-2006 catalog are incorporated by reference (pp. 102-108 for
ligases), U.S. Provisional Application No. 60/717,296 and
International Publication No. WO 2005/052124. In addition,
thermostable repair enzymes can be used interchangeably with
thermolabile repair enzymes in a preamplification mixture.
Thermostable enzymes are active at above 40.degree. C. or more
particularly 65.degree. C. or above.
[0096] Embodiments of present methods improve the yield or fidelity
of products resulting from polynucleotide amplification or other
synthesis reaction. This can be achieved, for example, when a
damaged polynucleotide is treated with a preparation of enzyme(s)
in a pre-incubation mixture and/or during amplification.
[0097] Amplification protocols that may benefit from the above
described pre-incubation include polymerase chain reaction (PCR),
Strand-Displacement Amplifcation (SDA) (U.S. Pat. Nos. 5,455,166
and 5,470,723); HDA (U.S. Publication No. 2004-0058378-A1);
Transcription-Mediated Amplification (TMA) (Guatelli et al., Proc.
Natl. Acad. Sci. USA 87:1874-1878 (1990)); Rolling Circle
Amplification (RCA) which generates multiple copies of a sequence
for the use in in vitro DNA amplification adapted from in vivo
rolling circle DNA replication (see, for example, Fire and Xu,
Proc. Natl. Acad Sci. USA 92:4641-4645 (1995); Lui, et al., J. Am.
Chem. Soc. 118:1587-1594 (1996); Lizardi, et al., Nature Genetics
19:225-232 (1998)) and whole genome amplification methods.
[0098] A universal enzyme mixture has been found to be useful in a
reaction mixture for repairing damaged polynucleotides prior to or
during amplification regardless of the type of damage to the
polynucleotide. The mixture repairs damaged DNA without causing
further damage.
[0099] The universal enzyme mixture contains a ligase and a
cofactor such as NAD+ or ATP. The mixture preferably additionally
includes a polymerase and an AP endonuclease as defined above
within a suitable buffer such as Thermopol (New England Biolabs,
Inc., Ipswich, Mass.), AccuTaq LA DNA polymerase buffer (Takara Bio
Inc., Shiga, Japan) or any other standard Taq buffer. In various
embodiments, the universal enzyme mixture contains E. coli PolI or
Taq polymerase and an AP endonuclease such as a mesophilic Endo IV,
e.g., E. coli Endo IV or a thermophilic Endo IV, e.g., Tth Endo IV
and a ligase selected from E. coli ligase, Taq ligase or an
archaeal ligase such as 9.degree. N ligase. In a particular
embodiment, the enzyme mixture contains 1-100 units Endo IV,
0.05-0.25 units E. coli PolI, and 5-500 units of a ligase suitable
for repairing 1-1000 ng DNA prior to or during amplification. It
will be understood that the concentration range for endonucleases
and polymerases other than those specified in the universal mixture
above may vary with the enzyme used and the temperature of the
reaction. However, the concentration range can be readily
ascertained using the assays described in the Examples. For
example, a standard preparation of lambda DNA can be heat treated
according to Example 1. The DNA can then be subjected to a series
of enzyme mixtures containing ligase and cofactors. An additional
enzyme is titrated to determine a preferred concentration for that
enzyme in the mixture. In this way, DNA repair can be optimized.
After amplification of each sample, the amount of the amplified DNA
can be determined by gel electrophoresis revealing the preferred
concentration range for the test enzyme.
[0100] The universal enzyme mixture can be used prior to or during
polynucleotide amplification or other synthesis.
[0101] As demonstrated in the Examples, depending on the type of
damage, it may be desirable to supplement the universal enzyme
mixture with additional repair enzymes depending on the nature of
the DNA damage. The utility of individual repair enzymes or
mixtures of repair enzymes can be determined using the assays
described in the Examples and in the Figures to determine their
suitability for repairing a particular polynucleotide.
Repair of General or Specific Damage to Polynucleotides
(a) General Damage
[0102] Determining the nature of damage in a polynucleotide is
time-consuming. If some form of damage to a polynucleotide is
suspected, for example, the polynucleotide is poorly amplified, it
is preferable not to have to identify the lesion or lesions. In
these circumstances, a universal mix of enzymes such as described
above may be utilized to determine whether improved amplification
is obtained. If the improvement is sufficient using the universal
mixture then no further action is required. If the improvement is
not sufficient, additional enzymes can be added to the mixture as
described herein until the preferred result is obtained. The entire
assay may be achieved in a single reaction vessel such as a 96 well
dish. Each micro-well in the dish is available for a different
enzyme mixture including the universal mixture plus enzymes
selected to address each class of damage outlined below.
[0103] The protocol for selecting enzymes for repair of general
damage or unknown damage of DNA is provided in FIG. 12 (flow chart)
and in the assays described in the Examples.
(b) Specific Damage
[0104] In some circumstances, the nature of the damage to a
polynucleotide might be known. In these circumstances, a mixture of
enzymes can be selected without undertaking the analysis of FIG.
12.
[0105] (i) AP Sites
[0106] The loss of a base is the most common spontaneous form of
DNA damage. Polymerases and polymerase-based techniques are
adversely affected by the presence of these abasic sites. The
effectiveness of primer extension reactions is enhanced by
repairing any abasic sites found in a polynucleotide. This is
achieved in one embodiment by Endo IV activity that cleaves the
phosphate backbone at the abasic site. This leaves an extendable 3'
OH on the DNA fragment 5' to the cleaved abasic site. It also
leaves a deoxyribose-5'-phosphate (dR5P) on the DNA fragment 3' to
the cleaved abasic site. A polymerase can extend from the free 3'
OH replacing the cleaved abasic site with a correct nucleotide. The
dR5P may be removed by an enzyme that specifically targets dR5Ps by
a flap endonuclease activity present in certain polymerases such as
E. coli DNA polymerase I or a separate flap endonuclease such as
FENI. The removal of dR5P can also occur by cleavage downstream of
this group by the flap endonuclease activity. After removal of the
dR5P and the generation of a 5' phosphate adjacent to the 3' OH, a
ligase can seal this nick finishing the repair (see Examples 1-3
and 7).
[0107] (ii) Modified Nucleotides
[0108] (a) Thymidine Dimers
[0109] Light can damage DNA by inducing the formation of pyrimidine
dimers. Pyrimidine dimers block the DNA extension reaction
catalyzed by DNA polymerases such as Taq DNA polymerase and hence
inhibit DNA amplification (Wellinger, et al. Nucleic Acids Res.
24(8):1578-79 (1996)). Consequently it is desirable to repair
pyrimidine primers prior to or during amplification. This can be
achieved by adding a pyrimidine dimer glycosylase/lyase (Vande
Berg, et al. J. Biol. Chem. 273(32):20276-20284 (1998)) to the
universal enzyme mixture. The DNA backbone is cleaved 5' to the
pyrimidine dimer and leaves a 3' hydroxyl moiety that is extendable
by a DNA polymerase. In certain embodiments, extension at the 3'
hydroxyl and subsequent formation and then cleavage of the
lesion-containing flap generated during DNA extension results in a
nick that is sealed by an enzyme capable of sealing the nick.
Cleavage of the flap can be achieved by the extending polymerase
for example, E. coli polymerase I or by the action of a flap
endonuclease ((Xu, Y., et al. J. Biol. Chem. 275(27):20949-20955
(2000), Liu, Y., et al., Annu. Rev. Biochem. 73:589-615
(2004)).
[0110] (b) Oxidative Damage
[0111] Inaccuracies can be introduced into the products of DNA
amplification reactions because of undesired nucleotide
incorporation opposite a damaged base (Gilbert, et al. Am. J. Hum.
Gen. 72:48-61 (2003); Hofreiter et al. Nucl. Acids Res. 29:4793-9
(2001)). These inaccuracies can be discovered by amplifying,
cloning and sequencing the same sample many times. Inaccuracies due
to base damage can also be identified by comparing sequence data
before and after sample treatment with an enzyme such as UDG, which
removes one of the common types of mutagenic DNA lesions
(Hofreiter, et al. Nucl. Acids Res 29:4793-9 (2001)). However,
treatment with UDG creates an abasic site within the DNA that
inhibits DNA amplification by primer extension. This creates
problems for rare DNA samples that may be made refractory to
amplification by UDG treatment.
[0112] Modified nucleotides that are the product of oxidative
damage can be removed from the polynucleotide by Fpg or hOGG to
leave a blocked polynucleotide where the blocked polynucleotide is
repairable by an AP endonuclease such as Endo IV.
[0113] The effectiveness of enzyme pretreatment to repair oxidative
damage to a polynucleotide prior to amplification is illustrated in
Example 9 where the universal enzyme mixture is supplemented with
Fpg in the pre-incubation mixture.
[0114] Other modified nucleotides such as alkylated bases or
deaminated bases where cytosine is converted to uracil, guanine to
xanthine or adenine to hypoxanthine give rise to miscoding. Removal
of these modified nucleotides is desirable. These modified bases
can be removed by any of AlkA, UDG or Aag as described in Example
10, leaving an AP site. This AP site can then be repaired by a
reaction mixture containing a ligase and preferably also an AP
endonuclease and a polymerase. Removal of a uracil enables a
polymerase in an amplification reaction that would normally be
stopped at this site to continue amplifying the DNA. For example,
Vent.RTM. polymerase activity is inhibited by an incorrect uracil
inserted into the DNA. The ability to remove the uracil permits the
polymerase to have enhanced effectiveness.
[0115] (iii) Nicks and Gaps
[0116] Nicks and gaps in the DNA backbone can lead to truncated
primer extension products and inhibit amplification reactions. The
concerted action of a ligase and a polymerase in the universal
enzyme mixture repairs nicks and gaps in the DNA thus enhancing DNA
amplification reactions.
[0117] (iv) Cross-Links
[0118] Additional nucleotide excision repair (NER) proteins (Minko
et al. Biochemistry 44:3000-3009 (2005); Costa et al. Biochimie
85(11):1083-1099 (2003); Sancar Ann. Rev. Biochem 65:43-81 (1996))
can be added to the Universal enzyme mixture to repair damage
resulting from exposure of polynucleotides to formaldehyde and
bulky adducts as well as damage that results in chemically-modified
bases that form DNA-protein cross-links. At least one of E. coli
UvrA, UvrB, mutant UvrB, UvrC, UvrD or Cho (Moolenar et al. Proc.
Natl Acad. Sci USA 99:1467-72 (2002)) can be used to make incisions
at the 5' end and optionally the 3' end around a damaged site.
Details about the properties and purification protocols of these
enzymes can be obtained from (Zou, Y., et al. Biochemistry
43:4196-4205 (2004)). The repair process can be completed by means
of a DNA polymerase, a DNA ligase and optionally a flap
endonuclease.
[0119] The generation of a 3' hydroxyl at a 5' incision site can be
useful if the NER enzyme(s) cleave the DNA but leave a blocked 3'
end on the DNA that inhibits primer extension. An example would be
if the NER enzyme(s) cleaved the DNA and left a 3' phosphate. This
would not be extendable by known DNA polymerases unless the 3'
phosphate was removed by for example, E. coli Endo IV.
[0120] If the NER enzyme or enzymes cleaves 5' and 3' to the DNA
lesion then the damage is removed when the newly released
oligonucleotide dissociates from the DNA. A polymerase can simply
fill in the excised region of DNA leaving a nick which ligase then
seals to complete the repair. In certain cases the polymerase may
fill in the DNA and then proceeds to displace the remaining DNA
strand. In these circumstances, an enzyme with flapase activity
permits a nick to be formed that a ligase can seal. In cases in
which the NER enzyme or enzymes only cleaves 5' to the damage, the
polymerase preferably displaces the original DNA strand until it is
past the damage at which point a flapase cleaves the DNA flap to
create a ligatable nick. The flapase may be active before and after
the DNA lesion is reached. Preferably, the polymerase and flapase
activities work to eventually displace and remove the DNA lesion
leaving a ligatable nick, thus repairing the DNA template. An
example of the effectiveness of the above approach is provided in
Example 7.
[0121] (v) Mismatched Polynucleotides
[0122] Heteroduplex DNA can be a problem in multi-template PCR and
in homogeneous template PCR (Lowell, J. L. & Klein, D. A.
Biotechniques 28:676-681 (2000); Thompson, J. R., et al. Nucl.
Acids Res. 30(9):2083-2088 (2002); Smith, J. & Modrich, P.
Proc. Natl. Acad. Sci. USA 94:6847-6850 (1997)). T7 Endo I or
mutant thereof can be used together with a ligase to remove
mismatch regions. This approach does not require quantitation of
DNA and avoids the extra steps after the PCR reaction required by
Lowell, et al. Biotechniques 28:676-681 (2000); and Smith, et al.
Proc. Natl. Acad. Sci. USA 94:6847-6850 (1997). An example of the
use of these enzymes is provided in Example 8. The useful range of
the T7 endonuclease or mutant:DNA ratio can be expanded by
including a DNA ligase activity to minimize non-specific cleavage
in the heteroduplex cleavage reaction.
Discussion of the Examples and Figures
[0123] Example 1 and FIGS. 1A-1D show that amplicon yields obtained
from PCR amplification are substantially negatively affected when
the template DNA is damaged beyond a certain threshold of damage
(e.g., about 90 seconds heat treatment) (see FIG. 1A). The effect
of this damage on amplification can be reversed and amplicon yields
enhanced by incubating the DNA with a mixture of enzymes before
amplification (see FIGS. 1B, 1C and 1D). In addition, amplicon
yields of "undamaged" DNA can be enhanced by adding the enzyme
mixture described.
[0124] Example 1 shows that the effect of the enzyme mixture on
amplification of DNA is not dependent on a single type of AP
endonuclease or ligase, but instead endonucleases or ligases from
multiple alternative sources can be used. For example, thermostable
Tth Endo IV was found to be as effective as E. coli Endo IV and E.
coli ligase was as effective as the thermostable Taq ligase.
[0125] Example 2 and FIGS. 2A-2B show the negative effect on
amplification yields of another type of DNA damage--depurination,
which is induced in the presence of heat and citrate. Moreover, the
example shows that the effect of a mixture of enzymes on
amplification of DNA is not dependent on a single type of
polymerase but rather polymerases from multiple alternative sources
can be used. For example, E. coli PolI can be substituted by Taq
DNA polymerase or a mixture of Taq and Vent.RTM. DNA polymerases to
produce enhanced yields.
[0126] Example 3 and FIG. 3 show that the enhancement of
amplification yields can be observed with short (200 bp) fragments.
In fact, enhancement of amplification yields are observed for a
wide range of sizes of DNA templates from as short as 100 bases to
as long as 100 kb and it is believed that amplification yields for
DNA even larger than 100 kb can be achieved. The upper limit of
size is limited only by the polymerase in the amplification
mixture.
[0127] FIG. 3 also shows that even when the DNA has been damaged
through storage in a crude form (for example, within the cells of
an organism that has itself been stored), amplification yields are
significantly enhanced by the addition of a mixture of enzymes
prior to amplification. Although the mixture of enzymes was added
to template DNA prior to amplification, a similar yield effect can
be seen when the template DNA is incubated with the mixture of
enzymes that are thermostable equivalents during amplification or
during a pre-amplification step.
[0128] Example 4 and FIG. 4 show that ligase alone can enhance
amplicon yield, but adding an AP endonuclease helps more. The best
result was observed in this example when a ligase, an AP
endonuclease, and a DNA polymerase were used prior to
amplification. Furthermore, this example demonstrates that repair
is not DNA size dependent. For example, similar results were
obtained with 5 kb and 10 kb amplicons.
[0129] Example 5 and FIG. 5 show that an enhanced yield from
amplification can be achieved using a ligase and that this effect
can be achieved without limitation to a single source of ligase.
FIG. 5 shows that Taq ligase and T4 ligase are both effective in
enhancing amplification yield even when used without additional
enzymes in a pre-incubation mix. This effect is also believed to
occur if the ligase is added to the amplification mix (if
thermostable). FIG. 5 also shows the benefit of this approach to
amplifying environmental DNA obtained directly from soil samples
that has been exposed in nature to a variety of damaging
agents.
[0130] All references cited herein are incorporated by
reference.
EXAMPLES
Example 1
Enhancing Amplification Yields for DNA with Various Extents of
Damage
[0131] An assay was developed for optimizing the use of selected
reagents to repair DNA prior to amplification.
[0132] Generation of Various Extents of Heat Damage
[0133] Various amounts of DNA damage were induced by heat
treatment. This was achieved as follows: 100 .mu.L lambda DNA
(NEB#N3011, New England Biolabs, Inc., Ipswich, Mass.) at 0.5 mg/ml
was aliquoted into separate tubes for heat treatment at 99.degree.
C. for 30 sec, 60 sec, 90 sec, 120 sec, and 180 sec, respectively
in a PE2700 thermal cycler. A sample was used as a template for
amplification without pretreatment.
[0134] The remaining damaged DNA was pretreated by the mixture of
enzymes as follows: The damaged DNA templates were incubated at
room temperature in the following mixture for 10 minutes:
[0135] DNA (5 ng, 2 ng and 1 ng);
[0136] 100 .mu.M dNTPs (NEB#M0447, New England Biolabs, Ipswich,
Mass.);
[0137] 1 mM NAD.sup.+ (Sigma#N-7004, Sigma, St. Louis, Mo.);
[0138] 80 units Taq ligase (NEB#M0208, New England Biolabs,
Ipswich, Mass.) or 40-100 units of E. coli ligase;
[0139] 0.1 units E. coli DNA polymerase I (E. coli pPolI)
NEB#M0209, New England Biolabs, Inc., Ipswich, Mass.);
[0140] 10 units E. coli Endo IV (NEB#M0304, New England Biolabs,
Inc., Ipswich, Mass.) or 10 units of Tth Endo IV;
[0141] 1.times. thermopol buffer (NEB#B9004, New England Biolabs,
Inc., Ipswich, Mass.) to a final volume of 96 .mu.L.
[0142] At the end of the reaction, the samples were transferred to
ice and then amplified.
DNA Amplification Reaction
[0143] DNA amplification of lambda was performed using the
following primers: CGAACGTCGCGCAGAGAAACAGG (L72-5R) (SEQ ID NO:1)
and CCTGCTCTGCCGCTTCACGC (L30350F) (SEQ ID NO:2) according to the
method of Wang et al. Nucl. Acids Res. 32: 1197-1207(2004).
[0144] 4 .mu.l of amplification mixture was added to 96 .mu.l of
the above repair mixture. The amplification mixture contained 100
.mu.M dNTPs, 5 units Taq DNA polymerase, 0.1 units Vent.RTM. (exo+)
DNA polymerase, 5.times.10-7 M primer L72-5R and 5.times.10-7 M
primer L30350F in 1.times. thermopol buffer.
[0145] To correct for any enzyme storage buffer effects, when a
repair enzyme was omitted from a reaction, the appropriate volume
of its storage buffer was added to the reaction. In all cases, the
amplification reactions were placed into a thermal cycler using the
following parameters: 20 sec at 95.degree. C. for 1 cycle followed
by 5 sec at 94.degree. C., then 5 min at 72.degree. C. for 25
cycles. The size of the amplicon being amplified was 5 kb.
[0146] The results of amplification of DNA (5 kb) were determined
by 1% agarose gel elecrophoresis. 6.times. loading dye (Molecular
Cloning:A Laboratory Manual, 3.sup.rd ed., eds. Sambrook and
Russell, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 2001,
pp. 5.4-5.17) was added to the 100 .mu.l amplification reactions.
20 .mu.l of this solution was then loaded onto the agarose gel
along with 1 .mu.g of 2-log ladder (NEB#N3200, New England Biolabs,
Inc., Ipswich, Mass.) as a size standard.
[0147] The amount of amplified DNA for each sample was compared by
gel electrophoresis and the results are shown in FIG. 1A.
[0148] When the samples were treated with a mixture of enzymes
after heat treatment but prior to amplification, significant
enhancement of amplification yields were achieved (FIGS. 1B, 1C and
1D).
Example 2
Increased Amplicon Yields from DNA with Induced Abasic Sites (After
Citrate Treatment) Following Pretreatment with an Enzyme
Mixture
[0149] Generation of Various Extents of Damage Resulting from
Abasic Site
[0150] To assay the extent of repair of damaged DNA, various
amounts of DNA damage was first induced by citrate treatment. This
was achieved as follows:
[0151] DNA was depurinated as described by Ide, H., et al.
Biochemistry 32(32):8276-83 (1993). Lambda DNA (NEB#N3011, New
England Biolabs, Inc., Ipswich, Mass.) was ethanol precipitated.
The DNA was resuspended in depurination buffer (100 mM NaCl, 10 mM
citrate, pH 5.0) at a concentration of 0.5 mg/ml and incubated at
70.degree. C. for 0, 20, 40, 80, 120, and 160 minutes. The sample
was then ethanol precipitated and resuspended in EB buffer (Qiagen,
Inc., Valencia, Calif.). The DNA concentration was determined by
measuring the A260 of the DNA containing solutions.
Pretreatment of DNA with a Mixture of Enzymes
[0152] The damaged DNA was incubated at room temperature for 10
minutes in the following mixture:
[0153] DNA (2.5 ng/120 minute damage);
[0154] 100 .mu.M dNTPs;
[0155] 1 mM NAD+;
[0156] 80 units Taq ligase;
[0157] 0.1 units Taq DNA polymerase or 0.1 units E. coli PolI
(NEB#M0209, New England Biolabs, Inc., Ipswich, Mass.)) or 0.1
units Taq:0.002 units of Vent Pol, (NEB#M0254, New England Biolabs,
Inc., Ipswich, Mass.));
[0158] 10 units E. coli Endo IV;
[0159] 1.times. thermopol buffer to a final volume of 96 .mu.l.
[0160] The above mixture was incubated at room temperature for 10
minutes and then transferred to ice prior to amplification.
DNA Amplification Reaction
[0161] Amplification was performed as described in Example 1 to
generate a 5 kb amplicon. Amplicon yields were increased as
compared with negative controls (FIG. 2A) by treating lambda DNA
containing abasic sites with the mixture of enzymes. The results
are shown in FIG. 2B for a series of pretreatments using different
enzyme mixtures. The enzyme mixtures were varied with respect to
the polymerase (E. coli PolI or Taq:Vent.RTM.).
Example 3
Improved Amplification Yield of DNA Extracted from an Intact
Organism After Storage in a Preservative
[0162] Genomic DNA was isolated from Meganyctiphanes norvegica
(Krill) as described in Bucklin, A. & Allen, L. D. Mol.
Phylogenet. Evoi. 30(3):879-882 (2004). The Krill had been stored
in ethanol since 1999.
[0163] Pretreatment of the Krill DNA by a mixture of enzymes was
carried out as follows:
[0164] 50 ng of M. norvegica genomic DNA;
[0165] 100 .mu.M dNTPs;
[0166] 1 mM NAD+;
[0167] 40 units of Taq ligase;
[0168] 0.5 units Taq DNA polymerase, 0.2 units Vent (exo+) DNA
polymerase, or a Taq:Vent.RTM. (exo+) mix containing 0.05 units of
Taq DNA polymerase and 0.001 units of Vent (exo+);
[0169] 10 units E. coli Endo IV;
[0170] 1.times. Thermopol buffer to a final volume of 96 .mu.l.
This reaction was incubated 15 minutes at room temperature before
proceeding to the amplification step.
DNA Amplification Reaction
[0171] The amplification primers corresponded to 52F and 233R as
described in Bucklin, A. & Allen, L. D. Mol. Phylogenet. Evol.
30(3):879-82 (2004) generating a 200 by amplicon. [0172] 52F:
TTTTTAGCAATACACTACACAGCAA (SEQ ID NO:3) [0173] 233R:
ATTACGCCAATCGATCACG (SEQ ID NO:4)
[0174] Primers were added to a final concentration of 0.5 .mu.M,
and each dNTP to a final concentration of 200 .mu.M. 1 .mu.l of the
50:1 Taq:Vent.RTM. mix (5 units Taq DNA polymerase and 0.1 units
Vent.RTM. (exo+) DNA polymerase added to the reaction) was then
added to each reaction to a final volume of 100 .mu.L.
[0175] For the control reaction (lane 1), no Endo IV, Taq ligase or
pretreatment polymerase was added. Volumes were adjusted
accordingly. In reactions in which repair enzymes were omitted, the
appropriate volume of enzyme storage buffer was added to control
for buffer effects.
[0176] Cycling conditions were as follows: 30 sec at 94.degree. C.,
30 sec at 52.degree. C. and 1 min 40 sec at 72.degree. C. for 40
cycles. 25 .mu.L (one quarter of the reaction) was loaded on a 1%
agarose gel, prepared, electrophoresed and visualized as described
above.
[0177] Increased amplicon yield from krill genomic DNA was observed
after preincubation of the samples using the enzyme mixtures
described above (FIG. 3).
Example 4
Increased Yields of 10 kb Amplicon Using Heat-Damaged DNA
[0178] Heat-damaged DNA was prepared as described in Example 1.
Lambda DNA was heated to 99.degree. C. for 180 sec.
[0179] Pretreatment of damaged DNA by a mixture of enzymes was
carried out as follows:
[0180] Lambda DNA (1 .mu.g of 180 sec heat-treated DNA);
[0181] 100 .mu.M dNTPs;
[0182] 1 mM NAD+;
[0183] 80 units of Taq ligase;
[0184] 0.1 unit of E. coli PolI;
[0185] 100 units of E. coli Endo IV;
[0186] 1.times. thermopol buffer to a volume of 96 .mu.L.
[0187] The mixture was incubated for 10 minutes prior to
amplification.
[0188] DNA amplification was performed as described in Example 1
except where specified below. Primers were added to the above 96
.mu.l of preincubation mixture. Primer L71-10R (sequence
GCACAGAAGCTATTATGCGTCCCCAGG) (SEQ ID NO:5) replaced L72-5R in
Example 1. The icycler thermal cycler program was: 20 sec at
95.degree. C. for 1 cycle, 5 sec at 95.degree. C., 10 min at
72.degree. C. for 25 cycles and then 10 min at 72.degree. C. for 1
cycle. Amplicon size was 10 kb.
[0189] The DNA was visualized as described in Example 1 with the
following exceptions. 20 .mu.l of 6.times. loading buffer was added
to the 100 .mu.l amplification reaction. 10 .mu.l of this solution
was diluted to 100 .mu.l with H.sub.2O and 1.times. loading buffer.
20 .mu.l of this was loaded into each lane. The gel was a 0.8%
agarose gel. The results are shown in FIG. 4.
Example 5
Improved Amplification Yield of DNA Extracted from Soil Samples
[0190] Environmental DNA was isolated from the soil using an
UltraClean Soil DNA Kit from MoBio Laboratories, Inc., Carlsbad,
Calif. (catalog #12800-50).
[0191] Pretreatment of DNA with a Ligase
[0192] A final volume of 100 .mu.l containing 0.6 .mu.g of
environmental DNA isolated from soil and one of the two ligases
described below in (a) and (b) formed the reaction mixture. This
reaction mixture was then incubated at room temperature for 15
min.
[0193] (a) 1.times. Taq ligase buffer (New England Biolabs, Inc.,
Ipswich, Mass.) and 80 units of Taq ligase.
[0194] (b) 1.times. T4 ligase buffer (New England Biolabs, Inc.,
Ipswich, Mass.) and 800 units of T4 ligase (NEB#M0202, New England
Biolabs, Inc., Ipswich, Mass.).
[0195] 1 .mu.l of reaction mixture was used in the amplification
reaction described below.
[0196] DNA Amplification Reaction
[0197] DNA amplification was performed using primers:
TABLE-US-00001 GGGGGXAGAGTTTGATCMTGGCTCA (SEQ ID NO: 6) and
GGGGGXTACGGYTACCTTGTTACGACTT (SEQ ID NO: 7)
(M=C or A, Y=C or T, X=8-oxo-Guanine). These primers target 16S
rDNA having an amplicon size of 1.6 Kb.
[0198] The 50 .mu.l reaction contained 10 pmol of each of the
primers, 1 .mu.l of the repaired environmental DNA, 200 .mu.M
dNTPs, 1.times. thermopol buffer, and 1.25 units Taq DNA
polymerase. The amplification was performed using the following
cycling parameters: 5 min at 94.degree. C. for 1 cycle, 30 sec at
94.degree. C., 1 min at 55.degree. C., 1 min 40 sec at 72.degree.
C. for 32 cycles, then 5 min at 72.degree. C. for 1 cycle.
[0199] Gel electrophoresis was performed as described in Example 1.
The results are shown in FIG. 5.
Example 6
Improved Amplification Yield of Ultraviolet Light-Damaged DNA
[0200] To determine conditions for assaying the effectiveness of
DNA repair, 50 .mu.g lambda DNA (NEB#N3011, New England Biolabs,
Inc., Ipswich, Mass.) was diluted in TE buffer (10 mM Tris-HCl, 1
mM EDTA, pH 7.5) to a concentration of 50 .mu.g/ml and irradiated
with 36 J/m.sup.2 UV light for 0, 10, 20, 30, 40 and 50 sec.
[0201] Pretreatment of damaged DNA by a mixture of enzymes was
carried out as follows:
[0202] The damaged DNA was incubated at room temperature for 15
minutes in the following mixture:
[0203] DNA (50 ng of lambda DNA-damaged for 0, 10, 20, 30, 40, or
50 seconds);
[0204] 200 .mu.M dNTPs;
[0205] 1 mM NAD.sup.+;
[0206] 400 units Taq ligase;
[0207] 0.1 units E. coli DNA polymerase I;
[0208] 10 units E. coli Endo IV;
[0209] 80 units or 10 units T4 pdg (also referred to as T4 Endo V).
(Trevigen, Gaithersburg, Md.);
[0210] Thermopol buffer to a volume of 50 .mu.l.
[0211] After the 15 minutes incubation, the 50 .mu.l reaction
mixture was added to 50 .mu.l of an amplification solution. The
amplification solution consisted of 40 pmol of each primer (L72-5R
and L30350F as described in Example 1 or L72-2R (the DNA sequence
was CCATGATTCAGTGTGCCCGTCTGG) (SEQ ID NO:8), 1.times. Thermopol
buffer, 1 mM NAD.sup.+, 200 .mu.M dNTPs, 2.5 units Taq DNA
polymerase (NEB#M0267, New England Biolabs, Inc., Ipswich, Mass.),
and H.sub.2O to a final volume of 50 .mu.L. Combining the 50 .mu.L
repair reaction with the 50 .mu.l amplification solution gave a
final volume of 100 .mu.l.
[0212] The 100 .mu.l solutions were placed into a thermal
cycler.
[0213] For the L72-5R and L30350F primer combination: [0214] 5 min
at 94.degree. C. for 1 cycle; 30 sec at 94.degree. C., 60 sec at
58.degree. C., and 4 min at 72.degree. C. for 30 cycles; 5 min at
72.degree. C. for 1 cycle.
[0215] For the L72-2R and L30350F primer combination: [0216] 5 min
at 94.degree. C. for 1 cycle; 30 sec at 94.degree. C., 60 sec at
58.degree. C., and 2 min at 72.degree. C. for 30 cycles; 5 min at
72.degree. C. for 1 cycle.
[0217] The presence of amplification product was visualized on a
1.8% agarose gel using ethidium bromide. The size of any band was
compared against a lane containing the 2-log ladder (NEB#N3200S,
New England Biolabs, Inc., Ipswich, Mass.) size standards. The
results are shown in FIG. 8.
Example 7
Improved Amplification Yield of DNA Using the Nucleotide Excision
Repair Proteins, UvrA, UvrB and UvrC
[0218] Increased amplicon yield from krill genomic DNA is
determined after preincubation of the samples using an enzyme
mixture containing proteins involved in nucleotide excision
repair.
[0219] Pretreatment of stored DNA by a mixture of enzymes is
carried out as follows:
[0220] Stored DNA is incubated for 1-180 minutes at 4-37.degree. C.
in the following mixture:
[0221] DNA: 50 ng of M. norvegica genomic DNA;
[0222] 100 .mu.M dNTPs;
[0223] 1 mM ATP;
[0224] 400 units of Taq ligase;
[0225] 0.1 units E. coli DNA polymerase I;
[0226] 10 nM E. coli UvrA, 250 nM E. coli UvrB (or mutant UvrB*),
plus or minus 50 nM E. coli UvrC
[0227] 1.times. Thermopol buffer to a final volume of 96 .mu.l.
* for mutant UvrB, see Zou, Y., et al. Biochemistry 43:4196-4205
(2004).
[0228] DNA amplification reactions are conducted as described in
Example 3.
Example 8
Increasing Sequence Accuracy of a DNA Amplification Reaction by
Enzyme Cleavage of Heteroduplexes
Experimental Conditions
[0229] A. Adding Taq ligase to T7 Endo I was demonstrated to
increase the T7 Endo I:DNA ration in a reaction mixture without
randomly degrading the DNA. This approach makes it possible to
reduce unwanted heteroduplexes resulting from mismatches in an
amplification reaction.
[0230] The assay relies on treating a supercoiled DNA containing a
cruciform structure with increasing amounts of T7 Endo I.
[0231] 0, 1.6, 3.1, 6.2, 12.5, 25, 50, 100, 200, or 400 units of T7
Endo I (NEB#M0302, New England Biolabs, Inc., Ipswich, Mass.) was
added to 50 .mu.l reactions composed of 1 .mu.g of pUC(AT) (Guan,
C., et.al. Biochemistry 43:4313-4322 (2004)) and 1.times. NEBuffer
2 (NEB#B7002S, New England Biolabs, Inc., Ipswich, Mass.). Plasmid
pAT25tetA can be used in place of pUC(AT) (Parkinson, M. J. &
Lilley, D. M. J. Mol. Biol. 270:169-178 (1997)) and Bowater, R. P.,
et. al. Biochemistry 33:9266-9275 (1994)). Another set of reactions
were set up simultaneously and used the same components as
described above with the addition of 1 mM NAD.sup.+ (Sigma
catalog#N-7004, Sigma, St. Louis, Mo.) and 100 units of Taq ligase
(using a stock of NEB#M0208 at a concentration of 100 u/.mu.l). All
reactions were incubated at 37.degree. C. for 60 minutes.
[0232] The results were analyzed by running the reactions on a 0.9%
TBE agarose gel, stained with ethidium bromide, and visualized
using UV light (see FIG. 9). With no T7 Endo I present the pUC(AT)
plasmid produced 2 bands on the gel corresponding to the
supercoiled form (lower band) and the relaxed circular form (upper
band).
[0233] T7 Endo I resolved the supercoiled pUC(AT) into the relaxed
circular form and a linear form that ran intermediate to the
supercoiled and relaxed circular forms. At certain T7 Endo I:DNA
ratios, a smear was produced indicating that the T7 Endo I had
degraded the DNA by non-specific enzymatic activity. The presence
of Taq ligase significantly increased the usable T7 Endo I to DNA
ratio. This ratio is further improved by substituting T7 Endo I
with the mutant T7 Endo I described in International Publication
No. WO 2005/052124.
[0234] B. Experimental conditions for determining the effectiveness
of the T7 Endo I and ligase mix for removing heterduplexes from PCR
reactions.
[0235] Isolation of DNA from soil and amplification of the purified
DNA is performed as described in Example 5 with the optional
addition of 5 units T7 Endo I or mutant thereof. When T7 Endo 1 or
mutant thereof is added, an additional amplification cycle is added
(37.degree. C. for 15 minutes for 1 cycle). The last step is to
allow the AP endonuclease to cleave any heteroduplexes formed.
[0236] Gel electrophoresis is performed as described in Example 1.
Heteroduplex DNA is visualized on the gel as described in Lowell,
J. L. & Klein, D. A. Biotechniques 28:676-681 (2000)). Absence
of heteroduplex DNA in the presence of T7 Endo I or mutant thereof
shows the effectiveness of T7 Endo I or mutant thereof with
ligase.
[0237] Unit definitions are described with the product description
for each of the enzymes recited herein in the NEB catalog, New
England Biolabs, Inc., Ipswich, Mass. For example, unit definition
for T7 Endo I or mutant thereof is the amount of enzyme required to
convert greater than 90% of 1 .mu.g of supercoiled plasmid into
greater than 90% linear DNA in a reaction volume of 50 .mu.l in 1
hour at 37.degree. C.
[0238] The T7 Endo I:DNA ratio can be increased without increasing
non-specific cleavage of DNA in the presence of ligase.
Example 9
Increasing the Sequence Accuracy of a DNA Amplication Reaction
After Oxidative Damage
[0239] Generating DNA with Oxidative Damage
[0240] The DNA subject to oxidative damage was pWB407 (Kermekchiev,
M. B. et al. Nucl. Acids Res. 31:6139-47 (2003)). The damage was
incurred using a combination of methylene blue (MB) and visible
light as described previously (Sattler, et al. Arch. Biochem
Biophys. 376(1):26-3 (2000)). Plasmid DNA (200 .mu.g/ml in
distilled water) was spotted on parafilm stretches (50 .mu.l
drops). MB was added to the drops to a final concentration ranging
from 0 to 50 (0, 3, 6, 12.5, 25 and 50) .mu.g/ml (100 .mu.l final
volume). Plates with these parafilm stretches were placed on ice
and illuminated for 8 min. with a 1.times.100-W lamp. The
MB-light-treated DNA was precipitated, dried, and then resuspended
in 50 .mu.l of TE buffer (pH 8.0). Final DNA concentration was
determined by the absorbance of light at 260 nm.
DNA Amplification Conditions
[0241] A portion of pWB407 that contained the lacZ gene was
amplified using primers 316-138, TGTCGATCAGGATGATCTGGACGAAGAGC (SEQ
ID NO:9), and 316-137, CGAAAGCTTTCAAGGATCTTACCGCTGTTGAGA (SEQ ID
NO:10). Primers 316-138 and 316-137 were based on the
previously-described primers Kfd-29 and H3Bla34, respectively
(Kermekchiev, M. B. et al. Nucl. Acids Res. 31:6139-47 (2003)). The
100 .mu.L PCR reactions contained either 10 or 50 ng of template
DNA, indicated where appropriate, and 40 picomoles of each primer.
The cycling conditions utilized varied with the thermal stable
polymerase used for amplification.
[0242] Cycling conditions when using Taq DNA polymerase (NEB
cat#M0267S, New England Biolabs, Inc., Ipswich, Mass.) had an
initial denaturation step of 5 min at 94.degree. C. for 1 cycle,
then 30 sec at 94.degree. C., 60 sec at 58.degree. C., and 3 min 30
sec at 72.degree. C. for 30 cycles, and finally 5 min at 72.degree.
C.
[0243] Cycling conditions when using Phusion DNA polymerase (NEB
cat#F-5305, New England Biolabs, Inc., Ipswich, Mass.) had an
initial denaturation step of 30 sec at 98.degree. C. for 1 cycle,
then 10 sec at 98.degree. C., 30 sec at 62.degree. C., and 1 min 30
sec at 72.degree. C. for 30 cycles, and finally 5 min at 72.degree.
C.
[0244] The reaction outcomes were analyzed by loading 25 .mu.L of
the reaction on a 1.6% agarose gel, prepared, electrophoresed and
visualized as described above. The marker used was the 2-log DNA
ladder (NEB cat#N3200S, New England Biolabs, Inc., Ipswich,
Mass.).
Amplification Accuracy Determination
[0245] The accuracy of DNA amplification from the pWB407 template
was determined as described by Barnes, et al. Gene 112:29-35 (1992)
and Kermekchiev, et al. Nucl. Acids Res. 31:6139-47 (2003).
Amplicons containing the lacZ gene were generated from plasmids
pWB407 that had been subjected to differing amounts of oxidative
damage. The oxidative damage was performed using methylene blue as
described above. The PCR reactions were performed using 50 ng of
template as described above. After cycling, 10 units of the
restriction endonuclease DpnI was added to each 100 .mu.L PCR
reaction and incubated for 2 hours at 37.degree. C. This step
eliminated the original template plasmid. Next, the resulting
amplification products were extracted with phenol/chloroform
precipitation using isopropanol (Molecular Cloning: A Laboratory
Manual, 3.sup.rd ed., eds. Sambrook and Russell, Cold Spring Harbor
Press, Cold Spring Harbor, N.Y., 2001, pp. 6.25, A8.12-A8.24).
Precipitated products were resuspended in H.sub.2O and cut with the
restriction endonucleases StyI and HindIII using conditions
recommended by the manufacturer (New England Biolabs, Inc.,
Ipswich, Mass.). The DNA digestion reactions were stopped by
inactivating the HindIII and StyI enzymes by heating to 65.degree.
C. for 20 min. The restriction digestion products were purified
using a microcon YM-100 column (Millipore, Billerica, Mass.) to
eliminate short DNA fragments.
[0246] The repair reaction mixtures in a total of 50 .mu.l
contained 10 or 50 ng of pWB407 amplicons +/- methylene blue
incubation. The repair reactions contained 20 mM Tris-HCl (pH 8.8
at 25.degree. C.), 10 mM KCl, 10 mM (NH.sub.4).sub.2SO.sub.4, 2 mM
MgSO.sub.4, 0.1% Triton X-100, 1 mM NAD.sup.+, 200 .mu.M dNTPs
(dATP, dTTP, dCTP, and dGTP), and various repair enzyme
mixtures.
[0247] The repair enzyme mixtures used separately or in various
combinations in a total volume of 50 ul were:
[0248] 0.4 units Fpg, NEB cat#M0240S, New England Biolabs, Inc.,
Ipswich, Mass.);
[0249] 200 units Taq ligase;
[0250] 0.1 units E. coli DNA polymerase I;
[0251] 10 units E. coli Endo IV;
[0252] 1 mM NAD+;
[0253] 100 .mu.M dNTPs;
[0254] 1.times. Thermopol buffer.
[0255] The reactions were incubated at 25.degree. C. for 15
minutes. After the incubation, 50 pL of a PCR mix (20 mM Tris-HCl
(pH 8.8 at 25.degree. C.), 10 mM KCl, 10 mM
(NH.sub.4).sub.2SO.sub.4, 2 mM MgSO.sub.4, 0.1% Triton X-100, 1 mM
NAD.sup.+, 200 .mu.M dNTPs (dATP, dTTP, dCTP, and dGTP), 2.5 units
Taq DNA polymerase (NEB cat#M0267S, New England Biolabs, Inc.,
Ipswich, Mass.) was added to the 50 .mu.L repair reaction and this
new solution was subjected to thermal cycling conditions for PCR.
The amplicons from these reactions were purified and restriction
enzyme digested as described for other amplicons above.
[0256] The amplicons were cloned into the pWB407 plasmid. Plasmid
pWB407 was prepared by digestion with the restriction endonucleases
StyI and HindIII followed by a 30-minute incubation at 37.degree.
C. with 1 unit/.mu.g DNA of antarctic phosphatase (NEB cat#M0289S,
New England Biolabs, Inc., Ipswich, Mass.). The dephosphorylated
pWB407 vector backbone was purified by agarose gel electrophoresis.
Gel extraction was performed with a QlAquick Gel Extraction Kit
(Qiagen, Valencia, Calif.).
[0257] The digested amplicons were ligated into the prepared pWB407
plasmids in 30 .mu.L reactions using approximately 0.1 .mu.g vector
DNA and about 0.5 .mu.g amplicon. T4 ligase was used to perform the
ligation following the manufacturers recommended conditions (New
England Biolabs, Inc., Beverly, Mass.). Ligation products were
electroporated into E. coli strain WB441 (Barnes, W. Gene 112:29-35
(1992)). The selective indicator plates used were LB plates
containing 50 .mu.g/ml ampicilin and 80 ug/ml Xgal. Before plating,
the bacteria were incubated in rich broth for 1 hour at 37.degree.
C. to allow expression of the ampicilin resistance.
[0258] Control transformations lacking ligase treatment resulted in
zero colonies. Colonies were scored for blue color after one day at
37.degree. C., and one or two days at 25.degree. C. The results are
shown in FIGS. 10 and 11.
Example 10
Increasing the Sequence Accuracy of a DNA Amplification Reaction
After Deamination Damage
Generating Deaminated DNA
[0259] The DNA subject to deamination was pWB407 (Kermekchiev, et
al. Nucleic Acids Research, 2003, Vol. 31, 6139-6147). The damage
was incurred using random mutagenesis with nitrous acid as
described in Yan, W. et al. 3 Virol. 2003 February; 77(4):2640-50.
Nitrous acid can deaminate guanine in DNA to xanthine, cytosine to
uracil, and adenine to hypoxanthine.
[0260] Plasmid DNA (2 mg) was treated with 0.7 M Nallo.sub.2 in 1M
acetate buffer, pH 4.6. The reaction was terminated at various time
points by addition of 4 volumes of ice-cold 1 M Tris-CI (pH 7.9.
The plasmid DNA was precipitated, dried and then resuspended in 100
ml of TE buffer.
Pretreatment Reaction to Repair Deaminated Bases
[0261] The repair enzyme mixtures used separately or in various
combinations in total volume of 50 l were:
[0262] (a)
[0263] 1 unit Human Aag, New England Biolabs, Inc., Ipswich,
Mass.;
[0264] 2 units Endo III (NEB cat #M0268S), New England Biolabs,
Inc., Ipswich, Mass.;
[0265] 2 units Endo V (NEB cat #M0305S), New England Biolabs, Inc.,
Ipswich, Mass.;
[0266] 2 units UDG (NEB cat #M0280S), New England Biolabs, Inc.,
Ipswich, Mass.;
[0267] 200 units E. coli Endo IV;
[0268] 0.1 units E. coli DNA polymerase I;
[0269] 10 units E. coli Endo IV;
[0270] 1mM NAD+;
[0271] 100 M dNTPs;
[0272] 1.times. Thermopol buffer.
[0273] (b)
[0274] 2 units Endo V (NEB cat #M0305S), New England Biolabs, Inc.,
Ipswich, Mass.;
[0275] 2 units UDG (NEB cat #M0280S), New England Biolabs, Inc.,
Ipswich, Mass.;
[0276] 200 units E. coli Endo IV;
[0277] 0.1 units E. coli DNA polymerase I;
[0278] 10 units E. coli Endo IV;
[0279] 1mM NAD+;
[0280] 100 M dNTPs;
[0281] 1.times. Thermopol buffer.
[0282] (c) 2 units Endo V (NEB cat #M0305S), New England Biolabs,
Inc., Ipswich, Mass.;
[0283] 200 units E. coli Endo IV;
[0284] 0.1 units E. coli DNA polymerase I;
[0285] 10 units E. coli Endo IV;
[0286] 1 mM NAD+;
[0287] 100 M dNTPs;
[0288] 1.times. Thermopol buffer.
[0289] (d)
[0290] 1 unit Human Aag, New England Biolabs, Inc., Ipswich,
Mass.;
[0291] 2 units Endo III (NEB cat #M0268S), New England Biolabs,
Inc., Ipswich, Mass.;
[0292] 200 units E. coli Endo IV;
[0293] 0.1 units E. coli DNA polymerase I;
[0294] 10 units E. coli Endo IV;
[0295] 1 mM NAD+;
[0296] 100 M dNTPs;
[0297] 1.times. Thermopol buffer.
[0298] (e)
[0299] 1 unit Human Aag, New England Biolabs, Inc., Ipswich,
Mass.;
[0300] 2 units UDG (NEB cat #M0280S), New England Biolabs, Inc.,
Ipswich, Mass.;
[0301] 200 units E. coli Endo IV;
[0302] 0.1 units E. coli DNA polymerase I;
[0303] 10 units E. coli Endo IV;
[0304] 1 mM NAD+;
[0305] 100 M dNTPs;
[0306] 1.times. Thermopol buffer.
[0307] (f)
[0308] 1 unit Human Aag, New England Biolabs, Inc., Ipswich,
Mass.;
[0309] 2 units Endo V (NEB cat #M0305S), New England Biolabs, Inc.,
Ipswich, Mass.;
[0310] 200 units E. coli Endo IV;
[0311] 0.1 unit E. coli DNA polymerase I;
[0312] 10 units E. coli Endo IV;
[0313] 1 mM NAD+;
[0314] 100 M dNTPs;
[0315] 1.times. Thermopol buffer.
[0316] The amplification reaction conditions and amplification
accuracy determination are performed as described in Example 9.
Example 11
Unit Definitions
Thermophilic Ligase Unit
[0317] One unit is defined as the amount of enzyme required to give
50% ligation of 1 .mu.g of BstE II-digested lambda DNA in a total
reaction volume of 50 .mu.l in 15 minutes at 45.degree. C.
Mesophilic Ligase Unit
[0318] One unit is defined as the amount of enzyme required to give
50% ligation of Hind III digested lambda DNA (5' DNA termini
concentration of 0.12 .mu.M, 300 .mu.g/ml) in a total reaction
volume of 20 .mu.l in 30 minutes at 16.degree. C.
AP Endonuclease Unit
[0319] One unit is defined as the amount of enzyme required to
cleave 1 pmol of a 34-mer oligonucleotide duplex containing a
single AP site in a total reaction volume of 10 .mu.l in 1 hour at
37.degree. C.
Mesophilic Polymerase Unit
[0320] One unit is defined as the amount of enzyme that will
incorporate 10 nmol of dNTP into acid-insoluble material in a total
reaction volume of 50 .mu.l in 30 minutes at 37.degree. C. with 33
.mu.M dNTPs including [3H]-dTTP and 70 .mu.g/ml denatured herring
sperm DNA.
Thermophilic PolymeraseuUnit
[0321] One unit is defined as the amount of enzyme that will
incorporate 10 nmol of dNTP into acid-insoluble material in a total
reaction volume of 50 pl in 30 minutes at 75.degree. C. with 200
.mu.M dNTPs including [3H]-dTTP and 200 .mu.g/ml activated Calf
Thymus DNA.
[0322] For unit definitions for UDG and Fpg, (see NEB catalog, New
England Biolabs, Inc., Ipswich, Mass.).
TABLE-US-00002 Gene Name Activity Accession Number UNG Uracil-DNA
glycosylase NM 080911 SMUG1 Uracil-DNA glycosylase NM 014311 MBD4
Removes U or T opposite G at NM 003925 CpG sequences TDG Removes U,
T or ethenoC NM 003211 opposite G OGG1 Removes 8-oxoG opposite C NM
016821 MUTYH (MYH) Removes A opposite 8-oxoG NM 012222 NTHL1 (NTH1)
Removes Ring-saturated or NM 002528 fragmented pyrimidines MPG
Removes 3-meA, ethenoA, NM 002434 hypoxanthine NEIL1 Removes
thymine glycol NM 024608 NEIL2 Removes oxidative products of NM
145043 pyrimidines XPC Binds damaged DNA as complex NM 004628
RAD23B XPC, RAD23B, CETN2 NM 002874 (HR23B) CETN2 NM 004344 RAD23A
Substitutes for HR23B NM 005053 (HR23A) XPA Binds damaged DNA in NM
000380 preincision complex RPA1 Binds DNA in preincision NM 002945
RPA2 complex NM 002946 RPA3 RPA1, RPA2, RPA3 NM 002947 ERCC5 (XPG)
3' incision NM 000123 ERCC1 5' incision subunit NM 001983 ERCC4
(XPF) 5' incision subunit NM 005236 LIG1 DNA joining NM 000234 CKN1
(CSA) Cockayne syndrome; Needed for NM 000082 ERCC6 (CSB)
transcription-coupled NER NM 000124 XAB2 (HCNP) CKN1, ERCC6, XAB2
NM 020196 DDB1 Complex defective in XP NM 001923 DDB2 group E NM
000107 DDB1, DDB2 MMS19L Transcription and NER NM 022362 (MMS19)
FEN1 (DNase IV) Flap endonuclease NM 004111 SPO11 endonuclease NM
012444 FLJ35220 incision 3' of hypoxanthine NM 173627 (ENDOV) and
uracil FANCA Involved in tolerance or NM 000135 FANCB repair of DNA
crosslinks NM 152633 FANCC FANCA, FANCB, FANCC, NM 000136 FANCD2
FANCD2, FANCE, NM 033084 FANCE FANCF, FANCG, FANCL NM 021922 FANCF
NM 022725 FANCG NM 004629 (XRCC9) FANCL NM 018062 DCLRE1A DNA
crosslink repair NM 014881 (SNM1) DCLRE1B Related to SNM1 NM 022836
(SNM1B) NEIL3 Resembles NEIL1 and NEIL2 NM 018248 ATRIP
ATR-interacting protein NM 130384 (TREX1) 5' alternative ORF of the
TREX1/ATRIP gene NTH Removes damaged pyrimidines NP 416150.1 NEI
Removes damaged pyrimidines NP 415242.1 NFI Deoxyinosine 3'
endonuclease NP 418426.1 MUTM Formamidopyrimidine DNA NP 418092.1
glycosylase UNG Uracil-DNA glycosylase NP 417075.1 UVRA DNA
excision repair enzyme NP 418482.1 UVRB complex NP 415300.1 UVRC
UVRA, UVRB, UVRC NP 416423.3 DENV Pyrimidine dimer glycosylase NP
049733.1
Sequence CWU 1
1
11123DNAartificialprimer 1cgaacgtcgc gcagagaaac agg
23220DNAartificialprimer 2cctgctctgc cgcttcacgc
20325DNAartificialprimer 3tttttagcaa tacactacac agcaa
25419DNAartificialprimer 4attacgccaa tcgatcacg
19527DNAartificialprimer 5gcacagaagc tattatgcgt ccccagg
27625DNAartificialprimer 6ggggggagag tttgatcmtg gctca
25728DNAartificialprimer 7ggggggtacg gytaccttgt tacgactt
28824DNAartificialprimer 8ccatgattca gtgtgcccgt ctgg
24929DNAartificialprimer 9tgtcgatcag gatgatctgg acgaagagc
291033DNAartificialprimer 10cgaaagcttt caaggatctt accgctgttg aga
3311813DNAunknownThermus thermophilus endonuclease IV 11atgccgcgct
acgggttcca cctttccatc gccgggaaaa agggcgtggc cggggcggtg 60gaggaggcca
ccgccctcgg cctcaccgct ttccagatct tcgccaaaag cccgcggagc
120tggcgcccaa gggccctctc cccggccgag gtggaggcct tccgcgcctt
aagggaggcc 180tccgggggcc tccccgccgt gatccacgcc tcctacctgg
tcaacctggg ggcggagggg 240gagctttggg agaagagcgt ggcgagcctg
gcggacgacc tggagaaggc cgccctcctc 300ggggtggagt acgtggtcgt
ccaccccggc tcgggccgcc ccgagcgggt caaggaaggg 360gccctcaagg
ccctgcgcct cgccggcgtc cgctcccgcc ccgtcctcct cgtggagaac
420accgccgggg gcggggagaa ggtgggggcg cggtttgagg agctcgcctg
gctcgtggcg 480gacacccccc tccaggtctg cctggacacc tgccacgcct
acgccgccgg gtacgacgtg 540gccgaggacc ccttgggggt cctggacgcc
ctggaccggg ccgtgggcct ggagcgggtg 600cccgtggtcc acctcaacga
ctccgtgggc ggcctcggaa gccgcgtgga ccaccacgcc 660cacctcctcc
agggaaagat cggggagggg ctcaagcgcg tcttcttgga cccgaggctc
720aaggaccggg tcttcatcct ggaaaccccc aggggaccgg aggaggacgc
ctggaacctc 780cgggtcctca gggcctggct cgaggaggcc taa 813
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