Method and Kits for Repairing Nucleic Acid Sequences

Info

Publication number: 20100009411
Type: Application
Filed: Jul 8, 2008
Publication Date: Jan 14, 2010
Applicant: GENERAL ELECTRIC COMPANY (Niskayuna, NY)
Inventors: John Richard Nelson (Clifton Park, NY), Wei Gao (Clifton Park, NY)
Application Number: 12/169,399

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.

Description

Description

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The present application contains a Sequence Listing of SEQ ID NOS 1-28 in file “197952-1_sequence_listing_—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

The invention relates generally to forensic analysis, and, more specifically, to repair of damaged nucleic acid sequences.

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.

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

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.

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.

There is also provided a DNA repair kit including a DNA glycosylase, a DNA endonuclease, a DNA polymerase I, and a T4 DNA ligase.

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.

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

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:

FIG. 1 is a flow chart of a method for analyzing DNA in accordance with aspects of the present technique;

FIG. 2 is a diagrammatical illustration of a method for repairing damaged DNA in accordance with aspects of the present technique;

FIG. 3 is an image of a gel run using undamaged DNA;

FIG. 4 is an image of a gel run using oxidative damaged DNA;

FIG. 5 is an image of a gel run using repaired DNA;

FIG. 6 is an image of a gel run using UV damaged DNA; and

FIG. 7 is an image of a gel run using repaired DNA.

DETAILED DESCRIPTION

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.

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).

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™ 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™ 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).

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.

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.

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.

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.

In some embodiments, the repair enzymes are thermally labile and may be permanently inactivated by heating the sample to between 40-80° 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.

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 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.

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.

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 2 Final Catalog Amout Conc. Conc. Component Vendor No. (μl) (units/μl) (units/μl) 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

A 10× preparation of the RepairBuffer solution may include, for example, 100 mM Tris-HCl, 100 mM MgCl₂, 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 3 Catalog Amout Component Vendor No. (μl) 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)

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 μl of damaged DNA may be combined with the repair solution described in Table 3 and incubated at approximately 20-42° C. for 25-60 minutes, then at 42-75° 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° 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.

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 β-nicotinamide adenine dinucleotide (NAD) to function, and therefore its presence is not necessary.

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

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/μl. FIG. 3 is a slide of the undamaged DNA.

In order to damage the DNA, approximately 100 μl of an Fe-EDTA solution that is 9 mM in iron chloride (FeCl₃) and 18 mM in EDTA was prepared using 0.37 M FeCl₃and 0.5 EDTA diluted in water. In addition, approximately 1 ml of 30 mM hydrogen peroxide (H₂O₂) solution was prepared by adding about 3.4 μl of stock H₂O₂(i.e., 30 percent H₂O₂at approximately 8.8 M) to 1 ml of water. The H₂O₂solution was put on ice.

A reaction mix described in Table 4 was prepared in a tube. The total reaction volume was approximately 30 μl. The H₂O₂solution was added to start the damage reaction. The mix was incubated at 37° 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 4 Components Volume gDNA (130 ng/ul) 1 μl Fe-EDTA (9 mM-18 mM) 5 μl dd H2O 19 μl 30 mM H2O2 4 μl

Oxidative damage DNA samples were prepared using the Fenton reaction. Approximately 2 μl of the genomic DNA extracted from the HT-29 cell line with a concentration of about 130 ng/μl was mixed with 5 μl of the Fe-EDTA solution and 23 μl of double distilled water. About 4 μl of the 30 mM H₂O₂solution was added to start the reaction. Eight tubes of the reaction mixture were prepared and incubated at 37° 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/μl. FIG. 4 is a slide of the oxidative damaged DNA.

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° C. for approximately 30 minutes, and then at 65° C. for approximately 20 minutes.

TABLE 5 Amount Component Conc. (μl) Final Conc. Repair Buffer 10 X 3 1 X ATP 10 mM 3 1 mM dNTP 1 mM 3 100 μM BSA 1 mg/ml 3 0.1 mg/ml E. Coli Pol I 10 U/μl 1 10 U T4 DNA Ligase 400 U/μl 1 400 U Oxidative 2 ng/μl 15 30 ng (approx. 1 ng/μl) Damaged DNA Endo IV 0.1 U/μl 1 0.1 U Endo VIII 0.1 U/μl 1 0.1 U T4-PDG 0.1 U/μl 1 0.1 U

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° C. for 5 minutes; thirty-eight cycles of 95° C. for 30 seconds, 58.5° C. for 30 seconds, and 72° C. for 90 seconds; and 72° 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 6 Components Conc. Volume Repaired DNA (1 ng/ul) 1 ng/μl 2 μl P-Set-14 (2.5 uM) 2.5 μM 1.5 μl ddH2O 21.5 μl ReadyToGo-PCR beads 1

In addition to the oxidative damaged samples, UV damaged samples were prepared. Approximately 100 μl of the genomic DNA extracted from the HT-29 cell line with a concentration of about 130 ng/μl was placed in a quartz cuvette and exposed to UVC energy at approximately 245 nm and 4 mW/cm²for 5 minutes. The UV damaged DNA was then diluted to approximately 2 ng/μl. FIG. 6 is a slide of the UV damaged DNA.

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° C. for approximately 30 minutes, and then at 65° C. for approximately 20 minutes.

TABLE 7 Components Conc. Volume (ul) Final Conc. RepairBuffer 10X 3 1X ATP 10 mM 3 1 mM dNTP 1 mM 3 100 μM BSA 1 mg/ml 3 0.1 mg/ml E. Coli Pol I 10 U/μl 1 10 U T4 DNA Ligase 400 U/μl 1 400 U UV Damaged DNA 2 ng/μl 15 30 ng or ~1 ng/μl EndoIV 0.1 U/μl 1 0.1 U EndoVIII 0.1 U/μl 1 0.1 U T4-PDG 0.1 U/μl 1 0.1 U

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).

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 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

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.

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° C. and about 75° 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° C. to about 75° 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.