U.S. patent application number 12/169399 was filed with the patent office on 2010-01-14 for method and kits for repairing nucleic acid sequences.
This patent application is currently assigned to GENERAL ELECTRIC COMPANY. Invention is credited to Wei Gao, John Richard Nelson.
Application Number | 20100009411 12/169399 |
Document ID | / |
Family ID | 41505489 |
Filed Date | 2010-01-14 |
United States Patent
Application |
20100009411 |
Kind Code |
A1 |
Nelson; John Richard ; et
al. |
January 14, 2010 |
Method and Kits for Repairing Nucleic Acid Sequences
Abstract
Methods and kits for DNA repair are provided. The methods and
kits described herein repair multiple types of DNA damage. The kit
may include a plurality of enzymes to repair a greater variety of
lesions than any single enzyme is capable of repairing. Repair of
damaged DNA may include releasing damaged bases from the DNA
strand, nicking the DNA at the damaged sites, translating the nicks
via 5'-3' exonuclease activity, and sealing the nicks. The enzymes
employed in the repair process may then be heat-inactivated,
thereby obviating a purification process. The repaired DNA may then
be analyzed using a variety of DNA analysis methods.
Inventors: |
Nelson; John Richard;
(Clifton Park, NY) ; Gao; Wei; (Clifton Park,
NY) |
Correspondence
Address: |
GENERAL ELECTRIC COMPANY (PCPI);C/O FLETCHER YODER
P. O. BOX 692289
HOUSTON
TX
77269-2289
US
|
Assignee: |
GENERAL ELECTRIC COMPANY
Niskayuna
NY
|
Family ID: |
41505489 |
Appl. No.: |
12/169399 |
Filed: |
July 8, 2008 |
Current U.S.
Class: |
435/89 ;
435/200 |
Current CPC
Class: |
C12N 9/1252 20130101;
C12N 9/93 20130101 |
Class at
Publication: |
435/89 ;
435/200 |
International
Class: |
C12P 19/30 20060101
C12P019/30; C12N 9/24 20060101 C12N009/24 |
Claims
1. A DNA repair kit, comprising: a thermally labile DNA polymerase
having 5'-3' exonuclease activity; and a thermally labile DNA
ligase.
2. The method of claim 1, wherein the thermally labile DNA
polymerase and the thermally labile DNA ligase are substantially
inactivated by incubation at a temperature between about 42.degree.
C. and about 75.degree. C.
3. The DNA repair kit of claim 1, wherein the DNA polymerase is
Escherichia coli DNA polymerase I.
4. The DNA repair kit of claim 1, further comprising at least one
additional DNA repair enzyme.
5. The DNA repair kit of claim 1, further comprising at least one
of 8-oxoguanine DNA glycosylase, an endonuclease IV, an
endonuclease VIII, or a T4 endonuclease V.
6. The DNA repair kit of claim 1, wherein the DNA polymerase and
the DNA ligase are a premixed single solution.
7. The DNA repair kit of claim 1, further comprising a solution
containing a glycosylase enzyme and/or an endonuclease enzyme.
8. A DNA repair kit, comprising a combination of enzymes, wherein
the combination exhibits N-glycosylase, AP-lyase, 5'-3' DNA
polymerase, and 5'-3' exonuclease nick translation activities, and
wherein the combination of enzymes is heat-inactivatable.
9. A DNA repair kit, comprising: a DNA glycosylase; a DNA
endonuclease; a DNA polymerase I; and a T4 DNA ligase.
10. The DNA repair kit of claim 9, wherein the DNA glycosylase
comprises 8-oxoguanine DNA glycosylase.
11. The DNA repair kit of claim 9, wherein the endonuclease
comprises edonuclease IV, endonuclease VIII, T4 endonuclease V, or
a combination thereof.
12. The DNA repair kit of claim 9, further comprising a buffer
solution having a pH of about 7.9.
13. The DNA repair kit of claim 9, wherein the DNA polymerase I
comprises both 5'-3' DNA polymerase activity and 5'-3' exonuclease
activity.
14. A method for repairing damaged DNA, comprising: incubating
damaged DNA with a DNA repair enzyme blend at a first temperature
to generate repaired DNA, wherein the DNA repair enzyme blend
comprises a thermally labile DNA polymerase having 5'-3'
exonuclease activity and a thermally labile DNA ligase.
15. The method of claim 14, further comprising inactivating the
enzyme blend by incubating the repaired DNA and the DNA repair
enzyme blend at a second temperature, wherein the second
temperature is higher than the first temperature.
16. The method of claim 14, further comprising amplifying the
repaired DNA.
17. The method of claim 14, further comprising performing a
polymerase chain reaction on the repaired DNA.
18. The method of claim 14, further comprising analyzing the
repaired DNA for comparison to a database of known DNA samples.
19. The method of claim 14, wherein the second temperature is in
the range of about 42.degree. C. to about 75.degree. C.
20. A method for DNA repair, comprising: removing a base from a
damaged site on a DNA strand via a glycosylase; nicking the DNA
strand at the damaged site via an endonuclease; translating the
nick down the DNA strand via a DNA polymerase having an associated
5'-3' exonuclease activity; and sealing the nick with a ligase.
21. The method of claim 20, wherein the DNA polymerase and the
ligase are thermally labile.
22. The method of claim 20, wherein the DNA polymerase comprises
both a 5'-3' DNA polymerase activity and a 5'-3' DNA exonuclease
activity.
Description
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
[0001] The present application contains a Sequence Listing of SEQ
ID NOS 1-28 in file "197952-1_sequence_listing.sub.--07JUL2008.txt"
(5.1 kilobytes), created on Jun. 13, 2008, concurrently submitted
with the specification by electronic filing, which is herein
incorporated by reference in its entirety.
BACKGROUND
[0002] The invention relates generally to forensic analysis, and,
more specifically, to repair of damaged nucleic acid sequences.
[0003] In criminal investigations, investigators often look for
physical evidence left behind at the scene of a crime to link a
suspect to the crime. For example, physical evidence has
traditionally included fingerprints, hairs, fibers, and so forth. A
modern forensic analysis of physical evidence may include analysis
of evidence, such as blood or other bodily fluids, which contains
deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) specimens.
Forensic laboratories analyze DNA and/or RNA samples in an attempt
to determine the identity of the person to whom the sample belongs.
For example, in criminal forensics, a subject may leave blood or
other evidence containing DNA at the scene of the crime. By
determining from whom the DNA evidence came, the government can use
such evidence in court to prosecute a suspect.
[0004] DNA analysis performed at forensic laboratories may be
hampered by DNA samples that have been exposed to uncontrolled
environmental conditions. Environmentally damaged samples may
contain shortened DNA fragments (i.e., double-stranded breaks in
the DNA), which are refractive to standard analysis methods, or may
contain somewhat intact DNA with single-stranded nicks and lesions.
Some examples of damaging exposure include acid and heat exposure,
which leads to missing bases and/or apurinic and apyrimidinic sites
(AP sites); ionizing radiation and electrophilic agents, including
alkylating agents, which modify bases or the sugar backbone;
oxidative damage, which leads to altered bases; and UV irradiation,
which produces cyclobutane dimers. In the event of such damage, the
DNA sample often cannot be analyzed effectively and thus cannot be
used as evidence in criminal prosecutions.
BRIEF DESCRIPTION
[0005] In certain embodiments, there is provided a DNA repair kit
including a thermally labile DNA polymerase having 5'-3'
exonuclease activity and a thermally labile DNA ligase.
[0006] In other embodiments, there is provided a DNA repair kit
having a combination of enzymes, where the combination exhibits
N-glycosylase, AP-lyase, 5'-3' DNA polymerase, and 5'-3'
exonuclease nick translation activities. The combination of DNA
enzymes may be heat inactivable.
[0007] There is also provided a DNA repair kit including a DNA
glycosylase, a DNA endonuclease, a DNA polymerase I, and a T4 DNA
ligase.
[0008] Further embodiments provide a method, including incubating
damaged DNA with a DNA repair enzyme blend at a first temperature
to generate repaired DNA. The DNA repair enzyme blend may include a
thermally labile DNA polymerase having 5'-3' exonuclease activity
and a thermally labile DNA ligase.
[0009] Still further embodiments provide a method, including
removing a base from a damaged site on a DNA strand via a
glycosylase, nicking the DNA strand at the damaged site via an
endonuclease, translating the nick down the DNA strand via a DNA
polymerase having an associated 5'-3' exonuclease activity, and
sealing the nick with a ligase.
DRAWINGS
[0010] These and other features, aspects, and advantages of the
present invention will become better understood when the following
detailed description is read with reference to the accompanying
drawings in which like characters represent like parts throughout
the drawings, wherein:
[0011] FIG. 1 is a flow chart of a method for analyzing DNA in
accordance with aspects of the present technique;
[0012] FIG. 2 is a diagrammatical illustration of a method for
repairing damaged DNA in accordance with aspects of the present
technique;
[0013] FIG. 3 is an image of a gel run using undamaged DNA;
[0014] FIG. 4 is an image of a gel run using oxidative damaged
DNA;
[0015] FIG. 5 is an image of a gel run using repaired DNA;
[0016] FIG. 6 is an image of a gel run using UV damaged DNA;
and
[0017] FIG. 7 is an image of a gel run using repaired DNA.
DETAILED DESCRIPTION
[0018] Damaged DNA may contain nucleotide modifications and/or
breaks (single- or double-stranded) in the DNA that are a block to
the standard DNA replication machinery of the cell. The pathways to
the repair of damaged nucleic acids may be broken down into four
categories: ligation to seal nicks; direct reversal of a nucleotide
modification to normal state; recombinational repair using a second
DNA copy as template; and excision repair. The methods described
herein may employ enzymes to perform one or more of these repair
techniques to repair damaged DNA. Methods for DNA repair described
herein may be used, for example, for detection of pathogenic
organisms, forensic purposes, medical diagnostic purposes, or
clinical purposes. Embodiments are also provided that relate
generally to methods applicable in analytical, diagnostic, or
prognostic applications.
[0019] FIG. 1 illustrates an exemplary process 10 by which the
methods described herein for DNA repair may be effectively used in
analyzing the damaged DNA. Cells containing DNA may be acquired
from various sources (block 12). Samples suspected or known to
contain DNA may be obtained from a variety of sources, such as, for
example, blood or semen samples from a crime scene. The sample may
be, but is not limited to, a biological sample, a food or
agricultural sample, or an environmental sample. Such samples may
be derived from biological tissue, body fluid, or exudate (e.g.,
blood, plasma, serum or urine, milk, cerebrospinal fluid, pleural
fluid, lymph, tears, sputum, saliva, stool, lung aspirates, throat
or genital swabs, and the like); whole cells; cell fractions; or
cultures. In certain circumstances, it may be desirable to treat
the sample to release and/or extract the DNA. A purified DNA sample
may be acquired by gently extracting DNA from the cells without
denaturing the DNA (e.g., not boiling or using potassium hydroxide)
(block 14). Any method for effective extraction and purification of
DNA from the source cells may be employed. An exemplary method for
DNA purification may be utilization of the illustra tissue and
cells genomicPrep Midi Flow Kit, available from GE Healthcare
(Catalog No. 28-9042-73).
[0020] The extracted DNA may then be repaired by the methods
described herein (block 16). The methods for DNA repair are
described in greater detail below. As will be demonstrated, the
repair method may utilize certain enzymes that can be easily
inactivated without denaturing the DNA (block 18). Accordingly, a
second purification step may not be required in certain
embodiments. The repaired DNA sample may then be amplified (block
20), such as by using isothermal amplification techniques, to
increase the amount of sample and facilitate analysis. For example,
the repaired DNA may be amplified using a whole genome
amplification kit, such as the illustra GenomiPhi DNA Amplification
Kit (Catalog No. 25-6600), or a circular amplification kit, such as
the illustra TempliPhi.TM. Amplification Kit (Catalog No. 25-6400),
both available from GE Healthcare. Various analysis methods may
then be employed to match the DNA sample to an individual (block
22). For example, polymerase chain reaction (PCR) amplification and
analysis may be utilized to determine the number of repeating
minisatellites at each of thirteen core loci where base pairs are
known to repeat uniquely. Other analysis techniques may be
employed, such as, for example, amplified fragment length
polymorphism (AmpFLP) analysis, short tandem repeats (STR)
analysis, or Y-chromosome (Y-STR) analysis. An exemplary
quantification analysis technique may employ the Quantifiler.TM.
Human DNA Quantification Kit (Part No. 4343895) available from
Applied Biosystems. Analysis produces a unique code, which may be
compared to a database of known DNA samples (e.g., the Combined DNA
Index System (CODIS) maintained by the Federal Bureau of
Investigation (FBI)) (block 24).
[0021] For DNA repair (block 16), there are separate classes of
protein enzymes that may be used. The first class of enzymes
directly repairs damage. This class includes ligase, which seals
single-stranded nicks in the double-stranded template, and
photolyase, which catalyzes the reversal of certain light-induced
damage. A second class of enzymes that may be used for DNA repair
operates by identifying base-damaged nucleotides and removing the
base section of the nucleotide (e.g., glycosylase activity),
leaving an intact sugar backbone. This area of sugar-only backbone
is referred to as an apurinic or apyrimidinic (AP) site and is
itself a block to replication or amplification. The next class of
enzymes includes endonucleases that identify damaged bases and nick
the DNA backbone next to the site of damage. In vivo, AP sites may
be removed by AP endonucleases. In some cases, the glycosylase
activity is associated with an AP endonuclease, and both steps are
accomplished by one enzyme. The product of this removal may be
either nicked DNA, with the AP site still associated with the
strand, or a single base gap with the AP site eliminated. Either
product is a block to replication or amplification.
[0022] Finally, a nick translation enzyme may also be utilized for
DNA repair. In the nick translation process, a DNA polymerase
having the ability to both replicate DNA and simultaneously remove
blocking single-stranded DNA (e.g., 5'-3' DNA polymerase and 5'-3'
exonuclease activities) may be utilized to move down a strand of
DNA, essentially eliminating damaged bases in front of it while
synthesizing an undamaged version. For the process of nick
translation, any DNA polymerase having intrinsic 5'-3' polymerase
activity which is functionally coordinated with a 5'-3' exonuclease
may be utilized. Classically, this coordination is available using
E. coli DNA polymerase I; however, any DNA ligase that catalyzes
the formation of a phosphodiester bond between juxtaposed 5'
phosphate and 3' hydroxyl termini in duplex DNA may be used,
including T4 DNA ligase, T7 DNA ligase, and bacterial DNA
ligase.
[0023] Nick translation by E. coli DNA polymerase I is accomplished
by simultaneously removing DNA ahead of the enzyme while adding DNA
by the polymerase activity. During this process, after a short
segment of DNA is replaced by nick translation, the nick
translation enzyme dissociates, leaving a nick that may be sealed
with ligase. This process is referred to as nick translation
because the DNA polymerase is translating a nick down the DNA
strand. The process of nick translation may be used to eliminate
damaged DNA located to the 5' side of the nick, while
simultaneously replacing the damaged nucleotides with an undamaged
version. Because the 5'-3' exonuclease activity may be desirable in
this repair process, the Klenow fragment (i.e., the large fragment
of DNA polymerase I) may not be utilized as the sole polymerase in
such embodiments.
[0024] The simultaneous use of both the nick translation and ligase
enzymes enables completion of short patch repair. Nick translation
alone without ligase may result in double-stranded breaks. DNA
containing damage on both strands may contain two opposing sites of
nick translation. If this occurs, the product of the reaction may
be two shorter DNA strands containing a double-stranded DNA break,
which is generally undesirable. The inclusion of ligase in the nick
translation reaction may prevent double-stranded breakage.
[0025] In some embodiments, the repair enzymes are thermally labile
and may be permanently inactivated by heating the sample to between
40-80.degree. C. so that the enzymes do not continue functioning
during subsequent handling steps where their activity may be
inappropriate. When proteins are exposed to increasing temperature,
loss of solubility or enzymatic activity may occur over a fairly
narrow range. Depending upon the protein studied and the severity
of the heating, these changes may not be reversible. As the protein
enzyme's tertiary structure is broken, hydrophobic groups are
exposed to the solvent in which the enzymes are disposed. The
protein may then attempt to minimize its free energy by burying as
many hydrophobic groups as possible while exposing as many polar
groups as possible to the solvent. While this is analogous to what
generally occurs when proteins fold, the much higher temperature
may greatly weaken the short-range interaction that initially
directs protein folding, and the resulting structures may be vastly
different from the native protein. Thus, exposure of many proteins
to high temperatures may result in irreversible denaturation.
[0026] A combination of enzymes may be employed to simultaneously
repair different types of DNA damage. For example, one enzyme may
be effective at repairing a particular type of lesion, while
another enzyme is preferable for repairing a different lesion. By
combining several enzymes into one DNA repair kit, a more robust
repair process may be implemented. Exemplary enzymes which may be
employed to perform the described functions are listed in Table 1,
along with the lesions repaired by each enzyme and the activity
performed by each enzyme.
TABLE-US-00001 TABLE 1 Enzyme Lesion Activity FPG (E. coli
8-oxoguanine 7,8-dihydro-8-oxoguanine (8- The N-glycosylase
activity releases DNA glycosylase) oxoguanine) damaged purines from
double- 8-oxoadenine stranded DNA, generating an AP Fpy-guanine
site. The AP-lyase activity cleaves Methy-fapy-guanine both 3' and
5' to the AP site, thereby Fapy-adenine removing the AP site and
leaving a Aflatoxin B1-fapy-guanine 1-base gap. 5-hydroxy-cytosine
5-hydroxy-uracil Endonuclease IV Apurinic/apyrimidinic site AP
sites are cleaved at the first phosphodiester bond that is 5' to
the lesion, leaving a hydroxyl group at the 3' terminus and a
deoxyribose 5'-phosphate at the 5' terminus E. coli Endonuclease
VIII Urea The N-glycosylase activity releases 5,6-dihydroxythymine
damaged pyrimidines from double- Thymine glycol stranded DNA,
generating an AP 5-hydroxy-5-methylhydanton site. The AP-lyase
activity cleaves Uracil glycol 3' and 5' to the AP site, leaving a
6-hydroxy-5,6-dihydrothymine 5' phosphate and a 3' phosphate.
Methyltartronylurea T4 Endonuclease V Cis-syn isomer of cyclobutane
The N-glycosylase activity releases pyrimidine dimer damaged bases
from double-stranded DNA, generating an AP site. The associated AP
lyase activity nicks the duplex DNA to produce single- strand gaps.
E. coli DNA polymerase I N/A 5''-3'' DNA polymerase, 5''-3''
exonuclease (nick translation) activity, and 3''-5'' exonuclease
proofreading activity. T4 DNA ligase N/A Joins blunt end and
cohesive end termini as well as repairing single- stranded nicks in
duplex DNA.
[0027] Turning to FIG. 2, an illustration of the repair of damaged
DNA in accordance with the present technique is illustrated. A DNA
strand 50 may have one or more damaged (e.g.,
amplification-blocking) sites 52 and 54. A combination of enzymes
may be utilized to perform several repair functions on the DNA
strand 50. This combination may include, for example, a glycosylase
(e.g., FPG) and/or an endonuclease (e.g., endonuclease IV,
endonuclease VII), which removes the damaged base section 52 and
leaves an AP site 56. The combination may further include an
endonuclease (e.g., T4 endonuclease V) which nicks the DNA at the
damaged sites 52 and 54 and/or at the AP site 56, leaving nicks 58
and 60. A DNA polymerase (e.g., E. coli DNA polymerase I) included
in the combination may then translate the nicks 58 and 60 down the
DNA strand 50, repairing the damaged DNA as the nicks 58 and 60 are
translated. The nick translation may be accomplished via 5'-3'
exonuclease activity. Finally, the nicks 58 and 60 may be sealed
with a ligase (e.g., T4 DNA ligase) present in the combination.
[0028] It should be understood that the exemplary technique
described in reference to FIG. 2 may be carried out via combination
of multiple enzymes in a single or multiple mixtures. An exemplary
DNA repair kit may include the enzyme mixture described in Table
2.
TABLE-US-00002 TABLE 2 Final Catalog Amout Conc. Conc. Component
Vendor No. (.mu.l) (units/.mu.l) (units/.mu.l) FPG NEB M0240 1 0.08
0.008 Endonuclease IV NEB M0304 1 1 0.1 E. coli NEB M0299 1 1 0.1
Endonuclease VIII T4 Endonuclease V NEB M0308 1 1 0.1 RepairBuffer
6 1x
[0029] A 10.times. preparation of the RepairBuffer solution may
include, for example, 100 mM Tris-HCl, 100 mM MgCl.sub.2, 500 mM
NaCl, and 10 mM dithiothreitol, resulting in a pH of 7.9. The
enzymes in the mixture described in Table 2, as well as other
enzymes, may be concurrently applied to damaged DNA in a single
repair solution. For example, repair of the damaged DNA may be
carried out via incubation in a repair solution composed of the
mixture described in Table 3.
TABLE-US-00003 TABLE 3 Catalog Amout Component Vendor No. (.mu.l)
Conc. Final Conc. RepairBuffer 3 10x 1x rATP 3 10 mM 1 mM BSA 3 1
mg/ml 0.1 mg/ml dNTP 3 2 mM 0.2 mM E. coli NEB M0209 1 Polymerase I
T4 DNA NEB M0202 1 Ligase Enzyme Mix 1 (Table 2)
[0030] Accordingly, an exemplary DNA repair kit may contain some or
all of the components described in Table 3 in either a pre-mixed
solution or as separate components available for combination.
Approximately 15 .mu.l of damaged DNA may be combined with the
repair solution described in Table 3 and incubated at approximately
20-42.degree. C. for 25-60 minutes, then at 42-75.degree. C. for an
additional 15-25 minutes. The initial incubation period enables
repair of the damaged DNA, while the higher-temperature incubation
inactivates the enzymes. In an exemplary embodiment, inactivation
of the repair enzymes may be performed by incubating the repaired
DNA and enzyme mixture at approximately 65.degree. C. for about 15
minutes. This high-temperature incubation inactivates the thermally
labile DNA repair enzymes, including the ligase, without denaturing
the repaired DNA. Accordingly, a thermally stable DNA polymerase
(e.g., Bst DNA polymerase) and/or a thermally stable DNA ligase
(e.g., Taq DNA ligase) may not be desirable, and, in certain
embodiments, would not be used in the present process. Furthermore,
because certain or all of the enzymes may be heat-inactivatable,
purification of the repaired DNA may not be required before
amplification.
[0031] It should be noted that while the repair solution described
in Table 3 includes riboadenosine triphosphate (ribo-ATP), this
component may be omitted in practice. In addition, none of the
enzymes in the exemplary kit require .beta.-nicotinamide adenine
dinucleotide (NAD) to function, and therefore its presence is not
necessary.
[0032] Once the damaged DNA has been repaired in accordance with
presently-described techniques, the repaired DNA may be amplified
and analyzed, for example, to determine the identity of the subject
from whom the DNA came. As described above, a number of analysis
techniques may be employed, including PCR, AmpFLP, STR, Y-STR, and
so forth. The heat-inactivated enzymes do not affect the analysis
of the DNA and therefore need not be removed from the repaired DNA
before proceeding to amplification and analysis.
EXPERIMENTAL RESULTS
[0033] Genomic DNA was subject to oxidative and ultraviolet damage
then repaired in accordance with embodiments of the present
technique. The results are summarized below. The genomic DNA for
use in the experiments was extracted from the HT29 cell line with a
concentration of about 80-160 ng/.mu.l. FIG. 3 is a slide of the
undamaged DNA.
[0034] In order to damage the DNA, approximately 100 .mu.l of an
Fe-EDTA solution that is 9 mM in iron chloride (FeCl.sub.3) and 18
mM in EDTA was prepared using 0.37 M FeCl.sub.3 and 0.5 EDTA
diluted in water. In addition, approximately 1 ml of 30 mM hydrogen
peroxide (H.sub.2O.sub.2) solution was prepared by adding about 3.4
.mu.l of stock H.sub.2O.sub.2 (i.e., 30 percent H.sub.2O.sub.2 at
approximately 8.8 M) to 1 ml of water. The H.sub.2O.sub.2 solution
was put on ice.
[0035] A reaction mix described in Table 4 was prepared in a tube.
The total reaction volume was approximately 30 .mu.l. The
H.sub.2O.sub.2 solution was added to start the damage reaction. The
mix was incubated at 37.degree. C. for various times (e.g., 20-100
min) then desalted using a NAP-5 desalting column, available from
GE Healthcare (Catalog No. 17-0853-01), in equilibrium with TE. The
mixture was then eluted in 1 ml TE.
TABLE-US-00004 TABLE 4 Components Volume gDNA (130 ng/ul) 1 .mu.l
Fe-EDTA (9 mM-18 mM) 5 .mu.l dd H2O 19 .mu.l 30 mM H2O2 4 .mu.l
[0036] Oxidative damage DNA samples were prepared using the Fenton
reaction. Approximately 2 .mu.l of the genomic DNA extracted from
the HT-29 cell line with a concentration of about 130 ng/.mu.l was
mixed with 5 .mu.l of the Fe-EDTA solution and 23 .mu.l of double
distilled water. About 4 .mu.l of the 30 mM H.sub.2O.sub.2 solution
was added to start the reaction. Eight tubes of the reaction
mixture were prepared and incubated at 37.degree. C. for 80
minutes. The mixtures were then desalted using the NAP-5 desalting
column and eluted in 1 ml of TE so that the resulting damaged DNA
concentration was about 2 ng/.mu.l. FIG. 4 is a slide of the
oxidative damaged DNA.
[0037] The oxidative damaged DNA was then combined with a repair
mixture containing multiple repair enzymes in a single tube in
accordance with embodiments of the present technique. The
components of the repair mixture are summarized in Table 5. The
repair mixture was incubated at 37.degree. C. for approximately 30
minutes, and then at 65.degree. C. for approximately 20
minutes.
TABLE-US-00005 TABLE 5 Amount Component Conc. (.mu.l) Final Conc.
Repair Buffer 10 X 3 1 X ATP 10 mM 3 1 mM dNTP 1 mM 3 100 .mu.M BSA
1 mg/ml 3 0.1 mg/ml E. Coli Pol I 10 U/.mu.l 1 10 U T4 DNA Ligase
400 U/.mu.l 1 400 U Oxidative 2 ng/.mu.l 15 30 ng (approx. 1
ng/.mu.l) Damaged DNA Endo IV 0.1 U/.mu.l 1 0.1 U Endo VIII 0.1
U/.mu.l 1 0.1 U T4-PDG 0.1 U/.mu.l 1 0.1 U
[0038] After incubation, PCR amplification was performed on the
mixture containing the repaired DNA. The PCR amplification was
carried out using the components listed in Table 6. The PCR beads
are available from GE Healthcare, Catalog No. 27-9558-01. The PCR
amplification was run at 95.degree. C. for 5 minutes; thirty-eight
cycles of 95.degree. C. for 30 seconds, 58.5.degree. C. for 30
seconds, and 72.degree. C. for 90 seconds; and 72.degree. C. for 10
minutes. FIG. 5 is a slide of the repaired DNA. Based on these
results, the DNA repaired using an embodiment of the
presently-disclosed technique (FIG. 5) appears to be closer to the
undamaged control DNA (FIG. 3) than is the oxidative damaged DNA
(FIG. 4).
TABLE-US-00006 TABLE 6 Components Conc. Volume Repaired DNA (1
ng/ul) 1 ng/.mu.l 2 .mu.l P-Set-14 (2.5 uM) 2.5 .mu.M 1.5 .mu.l
ddH2O 21.5 .mu.l ReadyToGo-PCR beads 1
[0039] In addition to the oxidative damaged samples, UV damaged
samples were prepared. Approximately 100 .mu.l of the genomic DNA
extracted from the HT-29 cell line with a concentration of about
130 ng/.mu.l was placed in a quartz cuvette and exposed to UVC
energy at approximately 245 nm and 4 mW/cm.sup.2 for 5 minutes. The
UV damaged DNA was then diluted to approximately 2 ng/.mu.l. FIG. 6
is a slide of the UV damaged DNA.
[0040] The UV damaged DNA was then combined with a repair mixture
containing multiple repair enzymes in a single tube in accordance
with embodiments of the present technique. The components of the
repair mixture are summarized in Table 7. The repair mixture was
incubated at 37.degree. C. for approximately 30 minutes, and then
at 65.degree. C. for approximately 20 minutes.
TABLE-US-00007 TABLE 7 Components Conc. Volume (ul) Final Conc.
RepairBuffer 10X 3 1X ATP 10 mM 3 1 mM dNTP 1 mM 3 100 .mu.M BSA 1
mg/ml 3 0.1 mg/ml E. Coli Pol I 10 U/.mu.l 1 10 U T4 DNA Ligase 400
U/.mu.l 1 400 U UV Damaged DNA 2 ng/.mu.l 15 30 ng or ~1 ng/.mu.l
EndoIV 0.1 U/.mu.l 1 0.1 U EndoVIII 0.1 U/.mu.l 1 0.1 U T4-PDG 0.1
U/.mu.l 1 0.1 U
[0041] After incubation, PCR amplification was performed on the
mixture containing the repaired DNA, as described above. FIG. 7 is
a slide of the repaired DNA. Based on these results, the DNA
repaired using an embodiment of the presently-disclosed technique
(FIG. 7) appears to be closer to the undamaged control DNA (FIG. 3)
than is the UV damaged DNA (FIG. 6).
[0042] As described above, 2 ng of DNA, damaged DNA, or repaired
DNA was amplified by PCR as described using primer sets as
indicated in Table 8.
TABLE-US-00008 TABLE 8 Lane on Gel 1 MW marker STR loci Primer ID
Primer name 2 CSF1PO Oligo Seq ID 1 CSF1PO-5'F-1 Oligo Seq ID 2
CSF1PO-3'R-1 3 D3S1358 Oligo Seq ID 3 D3S1358-5'F Oligo Seq ID 4
D3S1358-3'R 4 D5S818 Oligo Seq ID 5 D5S818-5'F Oligo Seq ID 6
D5S818-3'R 5 D7S820 Oligo Seq ID 7 D7S820-5'F Oligo Seq ID 8
D7S820-3'R 6 D8S1179 Oligo Seq ID 9 D8S1179-5'F Oligo Seq ID 10
D8S1179-3'R 7 D13S317 Oligo Seq ID 11 D13S317-5'F Oligo Seq ID 12
D13S317-3'R 8 D16S539 Oligo Seq ID 13 D16S539-5'F Oligo Seq ID 14
D16S539-3'R 9 D18S51 Oligo Seq ID 15 D18S51-5'F Oligo Seq ID 16
D18S51-3'R 10 D21S11 Oligo Seq ID 17 D21S11-5'F Oligo Seq ID 18
D21S11-3'R 11 FGA Oligo Seq ID 19 FGA-5'F Oligo Seq ID 20 FGA-3'R
12 TH01 Oligo Seq ID 21 TH01-5'F Oligo Seq ID 22 TH01-3'R 13 TPOX
Oligo Seq ID 23 TPOX-5'F-1 Oligo Seq ID 24 TPOX-3'R-1 14 vWR Oligo
Seq ID 25 vWR-5'F Oligo Seq ID 26 vWR-3'R 15 AMEL control Oligo Seq
ID 27 AMEL-5'F (undamaged DNA) Oligo Seq ID 28 AMEL-3'R
[0043] While only certain features of the invention have been
illustrated and described herein, many modifications and changes
will occur to those skilled in the art. It is, therefore, to be
understood that the appended claims are intended to cover all such
modifications and changes as fall within the true spirit of the
invention.
Sequence CWU 1
1
28124DNAArtificial SequencePRIMER 1ccggaggtaa aggtgtctta aagt
24222DNAArtificial SequencePRIMER 2atttcctgtg tcagaccctg tt
22319DNAArtificial SequencePRIMER 3actgcagtcc aatctgggt
19421DNAArtificial SequencePRIMER 4atgaaatcaa cagaggcttg c
21523DNAArtificial SequencePRIMER 5ggtgattttc ctctttggta tcc
23626DNAArtificial SequencePRIMER 6agccacagtt tacaacattt gtatct
26721DNAArtificial SequencePRIMER 7atgttggtca ggctgactat g
21824DNAArtificial SequencePRIMER 8gattccacat ttatcctcat tgac
24929DNAArtificial SequencePRIMER 9gcaacttata tgtatttttg tatttcatg
291028DNAArtificial SequencePRIMER 10accaaattgt gttcatgagt atagtttc
281123DNAArtificial SequencePRIMER 11acagaagtct gggatgtgga gga
231219DNAArtificial SequencePRIMER 12ggcagcccaa aaagacaga
191324DNAArtificial SequencePRIMER 13gggggtctaa gagcttgtaa aaag
241429DNAArtificial SequencePRIMER 14gtttgtgtgt gcatctgtaa
gcatgtatc 291520DNAArtificial SequencePRIMER 15ttcttgagcc
cagaaggtta 201624DNAArtificial SequencePRIMER 16ctaccagcaa
caacacaaat aaac 241722DNAArtificial SequencePRIMER 17atatgtgagt
caattcccca ag 221826DNAArtificial SequencePRIMER 18tgtattagtc
aatgttctcc agagac 261920DNAArtificial SequencePRIMER 19ggctgcaggg
cataacatta 202023DNAArtificial SequencePRIMER 20ttctatgact
ttgcgcttca gga 232121DNAArtificial SequencePRIMER 21gtgattccca
ttggcctgtt c 212220DNAArtificial SequencePRIMER 22tcctgtgggc
tgaaaagctc 202320DNAArtificial SequencePRIMER 23gcacagaaca
ggcacttagg 202418DNAArtificial SequencePRIMER 24cgctcaaacg tgaggttg
182533DNAArtificial SequencePRIMER 25gccctagtgg atgataagaa
taatcagtat gtg 332630DNAArtificial SequencePRIMER 26ggacagatga
taaatacata ggatggatgg 302719DNAArtificial SequencePRIMER
27ccctgggctc tgtaaagaa 192824DNAArtificial SequencePRIMER
28atcagagctt aaactgggaa gctg 24
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