# Patent

## Title
Methods for repairing degraded DNA

## Abstract

The present invention provides methods and kits for repair of degraded DNA which may then be used as a template for efficient amplification by a number of different amplification reactions. The method relies upon a series of enzymatic activities provided by DNA repair enzymes.

## Background
FIELD OF THE INVENTION
This invention relates generally to the field of amplification of DNA and provides methods and kits for repair of degraded DNA in order to improve DNA amplification processes.
BACKGROUND OF THE INVENTION
Successful DNA amplification relies on the integrity of the DNA template but aged samples or samples stored under sub-optimal conditions often results in failed amplification reactions.
DNA both in the living cells and isolated in vitro is subject to many chemical alterations (a fact often forgotten in the excitement of being able to do DNA sequencing on dried and/or frozen specimens). If the genetic information encoded in the DNA is to remain uncorrupted, any chemical changes must be corrected. The recent publication of the human genome has already revealed 130 genes whose products participate in DNA repair.
Agents that damage DNA include: ionizing radiation such as gamma rays and x-rays ultraviolet rays, especially the UV-C rays ( ̃260 nm) that are absorbed strongly by DNA but also the longer-wavelength UV-B, highly-reactive oxygen radicals produced during normal cellular respiration as well as by other biochemical pathways, many hydrocarbons, including some found in cigarette smoke, some plant and microbial products and chemicals used in chemotherapy, especially chemotherapy of cancers
All four of the bases in DNA (A, T, C, G) can be covalently modified at various positions. One of the most frequent is deamination, resulting for example, in a C being converted to a U. Mismatches of the normal bases because of a failure of proofreading during DNA replication. A common example is incorporation of the pyrimidine U (normally found only in RNA) instead of T.
Nicks in the DNA backbone can be limited to one of the two strands (a single-stranded break, SSB) or on both strands (a double-stranded break (DSB). Ionizing radiation is a frequent cause, but some chemicals produce breaks as well.
Covalent cross-linkages can be formed between bases on the same DNA strand (“intra-strand”) or on the opposite strand (“inter-strand”).
Various types of DNA damage can occur during improper storage, aging, freeze-thawing, or exposure to acid, heat or light. This will adversely affect the performance of the DNA as template in PCR because the damage will block the progression of the DNA polymerase.
Enzyme reagents for use in the modification and repair of degraded DNA are available commercially, for example from New England BioLabs (neb.com/nebecomm/products/category12.asp#16). However, only recently has attention been focused on combining such enzyme reagents for the purpose of repairing DNA and improving the DNA template integrity for subsequent amplification reactions. For example, the RestoraseTM (Sigma Aldrich—sigmaaldrich.com/Area_of Interest/Life_Science/Molecular_Biology/PCR/Specialty_Enzymes.html) has been demonstrated to repair AP sites, but not other types of DNA damage such as backbone nicks.
There remains a need for combinations of enzymes to effect repair of the many different classes of DNA damage for subsequent amplification reactions, particularly since alternative amplification methods to PCR are gaining prominence. One particularly useful method is whole genome amplification by multiple displacement amplification (MDA). MDA has been developed for the whole genome amplification of human DNA samples. MDA relies on priming the genomic DNA with exonuclease resistant random primers and the use of a strand displacing DNA polymerase such as Φ29 DNA polymerase. Φ29 DNA polymerase is highly processive, strand displacing and has a very low error rate of 1 in 106-107 nucleotides compared to an error rate of 3 in 104 for native Taq polymerase and 1.6 in 106 for Pfu polymerase. Due to the high processivity and strand displacing properties of Φ29 DNA polymerase the MDA reaction is performed under isothermal conditions at 30° C. The combination of a highly processive enzyme and random priming makes MDA amplification ideal approach for the amplification of DNA where there is currently insufficient material for use standard DNA typing technologies.
Other such alternatives to PCR amplification include isothermal helicase-dependent amplification, LATE-PCR assymmetric amplification, and single primer isothermal linear amplification.
Endonuclease VIII from E. coli acts as both an N-glycosylase and an AP-lyase. The N-glycosylase activity releases degraded pyrimidines from double-stranded DNA, generating an AP site. The AP-lyase activity cleaves 3′ to the AP site leaving a 5′ phosphate and a 3′ phosphate. Degraded bases recognized and removed by Endonuclease VIII include urea, 5, 6-dihydroxythymine, thymine glycol, 5-hydroxy-5-methylhydantoin, uracil glycol, 6-hydroxy-5,6-dihydrothymine and methyltartronylurea. While Endonuclease VIII is similar to Endonuclease III, Endonuclease VIII has β and δ lyase activity while Endonuclease III has β lyase activity (New England BioLabs—neb.com/nebecomm/products/productM0299.asp).
Fpg (formamidopyrimidine [fapy]-DNA glycosylase) (also known as 8-oxoguanine DNA glycosylase) acts both as an N-glycosylase and an AP-lyase. The N-glycosylase activity releases degraded purines from double stranded DNA, generating an apurinic (AP site). The AP-lyase activity cleaves both 3′ and 5′ to the AP site thereby removing the AP site and leaving a 1 base gap. Some of the degraded bases recognized and removed by Fpg include 7,8-dihydro-8-oxoguanine (8-oxoguanine), 8-oxoadenine, fapy-guanine, methyl-fapy-guanine, fapy-adenine, aflatoxin B1-fapy-guanine, 5-hydroxy-cytosine and 5-hydroxy-uracil (New England BioLabs—neb.com/nebecomm/products/productM0240.asp).
USER (Uracil-Specific Excision Reagent) enzyme generates a single nucleotide gap at the location of a uracil. USER Enzyme is a mixture of Uracil DNA glycosylase (UDG) and the DNA glycosylase-lyase Endonuclease VIII. UDG catalyses the excision of a uracil base, forming an abasic (apyrimidinic) site while leaving the phosphodiester backbone intact. The lyase activity of Endonuclease VIII breaks the phosphodiester backbone at the 3′ and 5′ sides of the abasic site so that base-free deoxyribose is released (New England BioLabs—neb.com/nebecomm/products/productM5505.asp).
Endonuclease IV can act on a variety of oxidative damage in DNA. The enzyme is apurinic/apyrimidinic (AP) endonuclease that will hydrolyze intact AP sites in DNA. 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. The enzyme also has a 3′-diesterase activity and can release phosphoglycoaldehyde, intact deoxyribose 5-phosphate and phosphate from the 3′ end of DNA (New England BioLabs—neb.com/nebecomm/products/productM0304.asp).
DNA Polymerase I, Large (Klenow) Fragment is a proteolytic product of E. coli DNA Polymerase I which retains polymerization and 3′→5′ exonuclease activity, but has lost 5′→3′ exonuclease activity. Klenow retains the polymerization fidelity of the holoenzyme without degrading 5′ termini. This enzyme is used in applications such as filling-in of 5′ overhangs to form blunt ends, removal of 3′ overhangs to form blunt ends, and second strand cDNA synthesis (New England BioLabs-neb.com/nebecomm/products/productM0210.asp).
T4 polynucleotide kinase catalyzes the transfer and exchange of a phosphate group from the γ position of ATP to the 5′-hydroxyl terminus of polynucleotides (double-and single-stranded DNA and RNA) and nucleoside 3′-monophosphates and also catalyzes the removal of 3′-phosphoryl groups from 3′-phosphoryl polynucleotides, deoxynucleoside 3′-monophosphates and deoxynucleoside 3′-diphosphates (New England BioLabs—neb.com/nebecomm/products/productM0201.asp).
T4 DNA ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5′ phosphate and 3′ hydroxyl termini in duplex DNA or RNA. This enzyme will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex DNA, RNA or DNA/RNA hybrids (New England BioLabs—neb.com/nebecomm/products/productM0202.asp).
The present invention satisfies the need for providing, inter alia, combinations of DNA repair enzymes that are useful for restoring the integrity of degraded DNA templates so that effective and efficient amplification can be obtained and subsequent analyses carried out. Examples of such subsequent analyses include, but are not limited to, methods for identification of bacterial and viral bioagents for biodefense, diagnostics and forensics investigations which are disclosed and claimed in U.S. patent application Ser. Nos. 09/798,007, 10/660,122, 10/728,486, 10/405,756, 10/660,998 and 10/807,019, each of which is commonly owned and incorporated herein by reference in its entirety.
SUMMARY OF THE INVENTION
The present invention provides methods for repairing degraded DNA in vitro comprising contacting the degraded DNA with an enzyme combination. The enzyme combination comprises endonuclease activity, DNA N-glycosylase activity, AP lyase activity, polymerase activity, 3′-diesterase activity, polynucleotide kinase activity, and DNA ligase activity. In some embodiments, the degraded DNA comprises a covalent modification such as, for example, a deamination, or an intra-strand or inter-strand cross linkage. In some embodiments, the degraded DNA comprises a nick, which can be either a single-stranded break or a double-stranded break. In some embodiments, the degraded DNA comprises an abasic site. In some embodiments, the endonuclease activity is provided by endonuclease VIII, endonuclease IV, or is provided by a combination of endonuclease IV and endonuclease VIII. In some embodiments, the DNA N-glycosylase activity is provided by endonuclease VIII, uracil DNA glycosylase, or 8-oxoguanine DNA glycosylase. In some embodiments, the AP lyase activity is provided by endonuclease VIII, or 8-oxoguanine DNA glycosylase. In some embodiments, the polymerase activity is provided by DNA Polymerase I, Large (Klenow) Fragment. In some embodiments, the 3′-diesterase activity is provided by endonuclease IV. In some embodiments, the polynucleotide kinase activity is provided by T4 polynucleotide kinase. In some embodiments, the DNA ligase activity is provided by T4 DNA ligase. In some embodiments, the degraded DNA comprises a degraded base such as, for example, urea, 5,6-dihydroxythymine, thymine glycol, 5-hydroxy-5-methylhydantoin, uracil glycol, 6-hydroxy-5,6-dihydrothymine, methyltartronylurea, 7,8-dihydro-8-oxoguanine (8-oxoguanine), 8-oxoadenine, fapy-guanine, methyl-fapy-guanine, fapy-adenine, aflatoxin B1-fapy-guanine, 5-hydroxy-cytosine, or 5-hydroxy-uracil.
The present invention also provides methods for amplification of degraded DNA in vitro. The degraded DNA is contacted with an enzyme combination comprising endonuclease activity, DNA N-glycosylase activity, AP lyase activity, polymerase activity, 3′-diesterase activity, polynucleotide kinase activity, and DNA ligase activity to produce repaired DNA. The repaired DNA is purified from the enzyme combination. The repaired DNA is amplified by methods such as for example, polymerase chain reaction, helicase-dependent amplification, LATE-PCR assymmetric amplification, single primer isothermal linear amplification, or multiple displacement amplification. In some embodiments, the polymerase used to effect the multiple displacement amplification is irradiated with an effective amount of ultraviolet light to destroy contaminating DNA in the preparation of the polymerase. In some embodiments, the polymerase is Φ29 polymerase.
The present invention also provides kits comprising an endonuclease, a DNA N-glycosylase, an AP lyase, a DNA polymerase, a 3′-diesterase, a polynucleotide kinase, and a DNA ligase. In some embodiments, the kits also comprise a mixture of random primers, a mixture of deoxynucleotide triphosphates, and/or a highly processive strand displacing polymerase.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 indicates that irradiated human mitochondrial DNA does not amplify in the whole genome amplification reaction. Lane (a): positive control—1 ng of non-irradiated mitochondrial DNA); Lane (b): size ladder; Lane (c): 10 ng of irradiated DNA treated with the heat denaturation step described in Example 2; Lane (d): 10 ng irradiated DNA without the heat denaturation step; Lanes (e and f): negative controls amplified with and without the heat denaturation step.
FIG. 2 indicates that irradiated DNA performs poorly in PCR reactions targeting the HV2 region of human mitochondrial DNA. Lanes 1-8 have 1 ng, 500 pg, 100 pg, 25 pg, 12 pg, 6 pg, 3 pg and 300 fg of irradiated mitochondrial DNA template added to the HV2 PCR reactions. Lane 9 is a positive control and lane 10 is a negative control.
FIG. 3 indicates improved performance of irradiated DNA in human mitochondrial DNA HV2 region PCR following treatment with high concentrations of T4 DNA ligase and 6 other enzymes for 2 hours at 37° C. followed by 12 hours at 16° C. (lane 94) The protocol used for lane 94 is described in detail in Example 1. Even number lanes had 2 ng of irradiated DNA used in the repair reaction and odd numbered lanes had 400 pg of irradiated DNA used in the repair reactions. 5% of the repaired DNA was subsequently used in the HV2 PCR reaction. Lanes 88 and 89 have repair cocktail with low ligase concentration (2 units) and were incubated at 37° C. for 2 hours. Lanes 90 & 91 had low ligase and were incubated at 37° C. for 12 hours. Lanes 92 & 93 had low ligase and were incubated for two hours at 37° C. followed by 12 hours at 16° C. and lanes 94 & 95 had repair cocktail made using high concentration of ligase and were incubated at 37° C. for two hours followed by 16° C. for 12 hours.
FIG. 4 demonstrates that commercially obtained Φ29 polymerase used for whole genome amplification reactions is contaminated with DNA which can be destroyed by irradiation with ultraviolet light. The left half of the gel shows the effect of irradiating the enzyme preparation for 10 sec, 30 sec, 2 min. and 4 min. prior to amplification of a 1 ng sample of human DNA. The gel indicates that the extent of amplification is essentially equivalent for each of the four reactions. In contrast, the right half of the gel indicates that contaminating DNA is amplified in the absence of expressly added human DNA template, but that this contaminating DNA fails to amplify to the same extent when the Φ29 polymerase preparation is exposed to ultraviolet light for 2 to 4 minutes.



DESCRIPTION OF EMBODIMENTS
The present invention is directed to, inter alia, methods for repairing degraded DNA in vitro comprising contacting the degraded DNA with an enzyme combination. The enzyme combination comprises endonuclease activity, DNA N-glycosylase activity, AP lyase activity, polymerase activity, 3′-diesterase activity, polynucleotide kinase activity, and DNA ligase activity, or any combination thereof. The degraded DNA can be contacted with any combination or all of the foregoing enzymes in a single cocktail or a series of cocktails, or can be contacted sequentially in any order.
As defined herein, the term “in vitro” refers to DNA isolated from a cell or tissue.
As defined herein, the term “AP site” refers to an apurinic or apyrimidinic site (i.e., a nucleobase within a segment of DNA whose purine or pyrimidine base has been removed).
In some embodiments, the endonuclease activity is provided by one or more enzymes such as, for example, endonuclease VIII or endonuclease IV, or a combination thereof. Other suitable enzymes having endonuclease activity are known to those skilled in the art and can also be used in the methods described herein.
In some embodiments, the DNA N-glycosylase activity is provided by one or more enzymes such as, for example, endonuclease VIII, uracil DNA glycosylase, or 8-oxoguanine DNA glycosylase, or any combination thereof. Other suitable enzymes having DNA N-glycosylase activity are known to those skilled in the art and can also be used in the methods described herein.
In some embodiments, the AP lyase activity is provided by one or more enzymes such as, for example, endonuclease VIII or 8-oxoguanine DNA glycosylase, or a combination thereof. Other suitable enzymes having AP lyase activity are known to those skilled in the art and can also be used in the methods described herein.
In some embodiments, the polymerase activity is provided by one or more enzymes such as, for example, DNA Polymerase I, Large (Klenow) Fragment. Other suitable enzymes having polymerase activity are known to those skilled in the art and can also be used in the methods described herein.
In some embodiments, the 3′-diesterase activity is provided by one or more enzymes such as, for example, endonuclease IV. Other suitable enzymes having 3′-diesterase activity are known to those skilled in the art and can also be used in the methods described herein.
In some embodiments, the polynucleotide kinase activity is provided by one or more enzymes such as, for example, T4 polynucleotide kinase. Other suitable enzymes having polynucleotide kinase activity are known to those skilled in the art and can also be used in the methods described herein.
In some embodiments, the DNA ligase activity is provided by one or more enzymes such as, for example, T4 DNA ligase. Other suitable enzymes having DNA ligase activity are known to those skilled in the art and can also be used in the methods described herein.
In some embodiments, the degraded DNA comprises a degraded base such as, for example, modified purine and pyrimidine bases including, but not limited to, urea, 5,6-dihydroxythymine, thymine glycol, 5-hydroxy-5-methylhydantoin, uracil glycol, 6-hydroxy-5,6-dihydrothymine, methyltartronylurea, 7,8-dihydro-8-oxoguanine (8-oxoguanine), 8-oxoadenine, fapy-guanine, methyl-fapy-guanine, fapy-adenine, aflatoxin B1-fapy-guanine, 5-hydroxy-cytosine, or 5-hydroxy-uracil.
In some embodiments, the degraded DNA comprises other classes of DNA damage including, but not limited to, a backbone nick, an abasic (apurinic or apyrimidinic) site, or an inter- or intra-strand cross linkage.
The present invention is also directed to methods for amplification of degraded DNA comprising repairing the degraded DNA by contacting the degraded DNA with an enzyme combination that contains endonuclease activity, DNA N-glycosylase activity, AP lyase activity, polymerase activity, 3′-diesterase activity, polynucleotide kinase activity, and DNA ligase activity. The repaired DNA is purified from the enzyme combination and the DNA is amplified by amplification methods including, but not limited to, PCR, multiple displacement amplification, rolling circle amplification, helicase-dependent amplification, LATE-PCR assymmetric amplification, and single primer isothermal linear amplification.
In some embodiments, the polymerase used to effect the multiple displacement amplification is irradiated with an effective amount of ultraviolet light to destroy contaminating DNA in the preparation of the polymerase used for multiple displacement amplification. In some embodiments, the polymerase is Φ29 polymerase.
The present invention is also directed to kits for repairing degraded DNA. In some embodiments, the kits include an enzyme combination that contains DNA N-glycosylase activity, AP lyase activity, DNA polymerase activity, a 3′-diesterase activity, polynucleotide kinase activity, and DNA ligase activity, or any combination thereof. In some embodiments, the kit comprises packaging material containing the components thereof.
The kit may also comprise a sufficient quantity of reagents for carrying out multiple displacement amplification reactions (whole genome amplification), including, for example, Φ29 polymerase, a mixture of random primers, nucleotide triphosphates, and/or a highly processive strand displacing polymerase, or any combination thereof. A kit may further include instructions pertinent for the particular embodiment of the kit, such instructions describing how to irradiate the polymerase to deactivate contaminating DNA. A kit may also comprise amplification reaction containers such as microcentrifuge tubes and the like.
The present invention also comprises compositions comprising any two or more of the following enzymes: DNA N-glycosylase activity, AP lyase activity, DNA polymerase activity, a 3′-diesterase activity, polynucleotide kinase activity, and DNA ligase activity, or any combination thereof. Representative examples of each of these enzymes are described above.
In order that the invention disclosed herein may be more efficiently understood, examples are provided below. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the invention in any manner. Throughout these examples, molecular cloning reactions, and other standard recombinant DNA techniques, were carried out according to methods described in Maniatis et al., Molecular Cloning—A Laboratory Manual, 2nd ed., Cold Spring Harbor Press (1989), using commercially available reagents, except where otherwise noted.
EXAMPLES
Example 1
Repair of Irradiated Double-Stranded DNA Prior Use in Multiple Displacement Amplification
Irradiation of DNA is used herein as a means to artificially damage DNA in order to investigate the capability of DNA repair enzyme mixtures to repair the damage. As shown in FIG. 1, human mitochondrial DNA exposed to 4 mega-rad of radiation (lanes c-f) is not amplifiable by whole genome amplification (WGA) effected by multiple displacement amplification (MDA), presumably due to the large frequency of nicks and degraded nucleotides. Likewise, the same radiation treatment results in poor PCR amplification of the HV2 region of human mitochondrial DNA relative to non-irradiated control DNA samples (FIG. 2). A significant difference in the amount of irradiated DNA needed in a PCR reaction in order to obtain a detectable PCR product was observed. This observation was fortuitous because it enabled a quick test of DNA repair formulations and conditions to quickly assess their effects on improving the performance of the irradiated DNA in PCR instead of in WGA.
For repair of irradiated DNA, the following protocol was found to be effective: a stock enzyme combination solution (herein designated RC13 enzyme mix) was prepared as shown in Table 1 and stored at −20° C. RC13 is repair cocktail formulation number 13 and its formulation is the result of over 100 different test reaction conditions and formulations. The repair assay improved the performance of irradiated DNA in both PCR and WGA reactions. The RC13 enzyme mixture contains four different enzymes that are involved with the excision of degraded nucleotides from DNA. The mixture also contains a polynucleotide kinase, a polymerase to extend and fill in gaps and a ligase to repair nicks and to concatamerize the DNA fragments. One hurdle was the identification of inhibitors from the repair cocktail that block the WGA reaction. It was found that the enzymes themselves were the source of the inhibition and that efficient amplification of the repaired DNA requires a phenol/chloroform purification step.







TABLE 1







Preparation of RC13 Enzyme Mix
















Amount
Final


Enzyme/Reagent
Vendor
Cat#
Units/ml
(μl)
Units/μl















Endonuclease
NEB
M0299S
10000
2
0.38


VIII


FPG
NEB
M0240S
8000
2
0.30


USER Enzyme
NEB
M5505S
1000
2
0.04


Endonuclease
Epicentre
E70100
2000
2
0.08


IV


Klenow
NEB
M0212S
5000
2
0.19


fragment


T4 Poly-
NEB
M0201S
10000
1
0.38


nucleotide


Kinase


T4 DNA ligase
NEB

2,000,000
25
657


High Conc.


50% Glycerol
—
—
—
40









RC13 buffer composed of 250 mM TRIS-HCl, pH 7.6, 50 mM MgCl2, 5 mM ATP and 5 mM dithiothreitol was used for the DNA repair reaction with the RC13 enzyme combination as follows: water was added to a DNA solution to a volume of 15 μl or 25 μl, to which was added 4 μl RC13 buffer and 1.5 μl RC13 enzyme mix for the 15 μl reaction or 6 μl RC13 buffer and 1.5 μl RC13 enzyme mix for the 25 μl reaction. The reaction mixture was then incubated in a thermocycler at 37° C. for 2 hours then at 16° C. for 12 hours. The reaction was stopped by heating to 65° C. for 15 minutes.
The repaired DNA in the resulting reaction mixture can be used directly in PCR reactions without further purification from RC13 enzyme components.
Prior to amplification of the repaired DNA by whole genome amplification, the repaired DNA is purified from the RC13 enzyme mix as follows: a standard phenol/chloroform extraction was performed in a 1.5 μl, followed by a standard chloroform extraction with final purification of the DNA sample using Micro Bio-Spin® chromatography columns (Bio-Rad, cat # 732-6223). The column is inverted sharply several times to re-suspend the settled gel and remove bubbles. The tip is snapped off and the column is placed in a 2.0 ml microcentrifuge collection tube. The column is then centrifuged at 800×g for 4 minutes, after which the collection tube is discarded. The column is placed in a clean 1.5 ml microcentrifuge tube and the DNA sample is applied to the column. The column is then centrifuged at 800×g for 4 minutes to elute the DNA sample.
The preceding method was used in an experiment which determined that high concentrations of ligase are necessary for efficient repair of irradiated DNA. In FIG. 3 the repair protocol as described above was used in lane 94 and the results indicate effective repair of the DNA.
Example 2
Whole Genome Amplification of Genomic DNA by Multiple Displacement Amplification Using Φ29 Polymerase
The following method has been found to be effective for whole genome amplification of DNA:







TABLE 2







Reaction Components for Whole Genome Amplification








Components
Final Concentration





Starting DNA
1 pg-100 ng


Water
NA


10× NEB Φ29 Rxn Buffer, Supplied
1×


with polymerase.









Modified-Random Primers (1 mM) equal
50
uM


part mix of hexamers, heptamers and octamers


Custom ordered from IDT-DNA-thiophosphate


linkages for the last two 3 prime nucleotides


DNTPs (100 mM mix) Stratagene Corp.
2
mM


Φ29 polymerase (10,000 units/ml.) -
16
Units


NEB cat # M0269L


Pyrophos-phosphatase (40 u/ml stock) -
0.008
Units


Amersham cat # E70953Z


BSA-Acetylated- Ambion Cat # 2612 (20 mg/ml)
200
ug/ml


0.1 M DTT - Invitrogen cat # Y00147
5
mM









wherein NEB buffer is 50 mM TRIS-HCl, 10 mM MgCl2, 10 mM (NH4)2SO4, 4 mM DTT, pH 7.5 at 25° C.

A sample of DNA is diluted with nuclease-free water to a volume of 62 μl in a 200 μl PCR tube, to which is added 35.0 μl of Mix A (a mixture of 200 μl of 10×NEB, 100 μl of modified-random primer mix (1 mM) and 40 μl of 100 mM DNTP mixture), followed by vortexing and centrifuging to collect all liquid in the bottom of the tube. The mixture is then heated to 95° C. for 5 minutes and allowed to cool to 4° C. using a thermocycler. Then 3.0 μl of Mix B (a mixture of 10,000 units/ml of Φ29 polymerase, 40 units/ml pyrophos-phosphatase, 20 mg/ml BSA-acetylated (Ambion Cat. #2612) and 0.1 M DTT). The reaction mixture is then cycled through a 12 hour thermocycler program as follows:
1. 30° C. for 4 minutes
2. 15° C. for 15 seconds
3. repeat steps 1 and 2 sequentially 150 times
4. 90° C. for 3 minutes
5. maintain at 4° C.
Post amplification processing of the amplified DNA includes fragmentation of the DNA by sonication with a Misonix Sonicator 3000 equipped with a cup horn (Cat #431 A) and a cup horn stand (Cat #448), power level setting 10 with a 1 minute process time (30 sec interval on, 30 sec interval off). The PCR tube containing the WGA reaction mixture is placed in a floating foam holder in the cup horn filled 1⁄3 with water. The resulting post-amplification processed DNA is diluted 1:10 with water and can be used directly in mitochondrial DNA typing PCR reactions, such as those disclosed and claimed in U.S. patent application Ser. Nos. 10/660,998 and 10/807,019, each of which is commonly owned and incorporated herein by reference in its entirety.
Example 3
Inactivation of Contaminating DNA in Φ29 Polymerase
FIG. 4 demonstrates that commercially obtained Φ29 polymerase used for whole genome amplification reactions is contaminated with DNA which can be destroyed by irradiation with ultraviolet light. The left half of the gel shows the effect of irradiating the enzyme preparation for 10 sec, 30 sec, 2 min. and 4 min. prior to amplification of a 1 ng sample of human DNA. The gel indicates that the extent of amplification is essentially equivalent for each of the four reactions. In contrast, the right half of the gel indicates that contaminating DNA is amplified in the absence of expressly added human DNA template, but that this contaminating DNA fails to amplify to the same extent when the Φ29 polymerase preparation is exposed to ultraviolet light for 2 to 4 minutes. This experiment indicates that an added step prior to carrying out a WGA reaction wherein the Φ29 polymerase preparation is irradiated with ultraviolet light to destroy contaminating DNA is a useful addition for a whole genome amplification reaction. It is expected that this step will enable a significant lowering of the concentration (template copy number) of starting template DNA required to carry out a WGA reaction.
Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each of the patents, applications, printed publications, and other published documents mentioned or referred to in this specification are incorporated herein by reference in their entirety. Those skilled in the art will appreciate that numerous changes and modifications may be made to the embodiments of the invention and that such changes and modifications may be made without departing from the spirit of the invention. It is therefore intended that the appended claims cover all such equivalent variations as fall within the true spirit and scope of the invention.

## Claims

1. A method for repairing and amplifying degraded DNA fragments in vitro comprising:
I) contacting said degraded DNA fragments with a reaction mixture comprising:
a) a plurality of substantially purified enzymes, said enzymes having endonuclease activity, DNA N-glycosylase activity, AP lyase activity, polymerase activity, 3′-diesterase activity, polynucleotide kinase activity, and DNA ligase activity, and
b) a reaction buffer, wherein said reaction buffer further comprises ATP, wherein said contacting is under conditions such that said degraded DNA fragments are repaired, 5′ end phosphorylated, and ligated together to generate repaired concatamerized DNA;

II) purifying said repaired concatamerized DNA from said plurality of substantially purified enzymes to generate purified repaired concatamerized DNA; and
III) amplifying said purified repaired concatamerized DNA using a whole genome amplification method, wherein said whole genome amplification method employs random primers.

2. The method of claim 1 wherein said whole genome amplification method comprises multiple displacement amplification.

3. The method of claim 2 wherein said covalent modification comprises a deamination.

4. The method of claim 2 wherein said covalent modification comprises an intra-strand or inter-strand cross linkage.

5. The method of claim 1 wherein said degraded DNA comprises a nick.

6. The method of claim 5 wherein said nick comprises a single-stranded break or a double-stranded break.

7. The method of claim 1 wherein said degraded DNA comprises an abasic site.

8. The method of claim 1 wherein said endonuclease activity is provided by endonuclease VIII.

9. The method of claim 1 wherein said endonuclease activity is provided by endonuclease IV.

10. The method of claim 1 wherein said endonuclease activity is provided by endonuclease IV and endonuclease VIII.

11. The method of claim 1 wherein said DNA N-glycosylase activity is provided by endonuclease VIII.

12. The method of claim 1 wherein said DNA N-glycosylase activity is provided by uracil DNA glycosylase.

13. The method of claim 1 wherein said DNA N-glycosylase activity is provided by 8-oxoguanine DNA glycosylase.

14. The method of claim 1 wherein said AP lyase activity is provided by endonuclease VIII.

15. The method of claim 1 wherein said AP lyase activity is provided by 8-oxoguanine DNA glycosylase.

16. The method of claim 1 wherein said polymerase activity is provided by DNA Polymerase I, Large (Klenow) Fragment.

17. The method of claim 1 wherein said 3′-diesterase activity is provided by endonuclease IV.

18. The method of claim 1 wherein said polynucleotide kinase activity is provided by T4 polynucleotide kinase.

19. The method of claim 1 wherein said DNA ligase activity is provided by T4 DNA ligase.

20. The method of claim 1 wherein said degraded DNA comprises a degraded base.

21. The method of claim 20 wherein the identity of said degraded base is urea, 5,6-dihydroxythymine, thymine glycol, 5-hydroxy-5-methylhydantoin, uracil glycol, 6-hydroxy-5,6-dihydrothymine, methyltartronylurea, 7,8-dihydro-8-oxoguanine (8-oxoguanine), 8-oxoadenine, fapy-guanine, methyl-fapy-guanine, fapy-adenine, aflatoxin B1-fapy-guanine, 5-hydroxy-cytosine, and 5-hydroxy-uracil.

22. The method of claim 1 wherein said degraded DNA fragments are fragments of a degraded whole genome.

23. The method of claim 1 wherein said DNA ligase activity is 657cohesive end ligation Units per microliter of enzyme combination.

24. The method of claim 1 wherein said DNA ligase activity is 39cohesive end ligation Units per microliter of reaction mixture to 66 cohesive end ligation Units per microliter of reaction mixture.

25. The method of claim 1 wherein said ATP accommodates said DNA ligase activity and accommodates the remaining enzymes in said enzyme cocktail without exhausting the ATP supply.

26. The method of claim 1 wherein said reaction mixture comprises 1.2mM to 1.3 mM ATP.

27. A method for repairing and amplifying degraded polynucleotide fragments comprising the steps of:
a) providing a substantially purified polynucleotide kinase;
b) providing a substantially purified polymerase;
c) providing a substantially purified ligase to concatamerize polynucleotide fragments;
d) providing one or more substantially purified enzymes that catalyze excision of degraded nucleotides from a polynucleotide;
e) incubating a mixture of said degraded polynucleotide fragments with said polynucleotide kinase, said polymerase, said ligase, said one or more substantially purified enzymes, and ATP under conditions such that said degraded polynucleotide fragments are repaired, 5′ end phosphorylated, and ligated together to generate repaired concatamerized nucleic acid;
f) purifying said repaired concatamerized nucleic acid from said polynucleotide kinase, said polymerase, said ligase, and said one or more substantially purified enzymes to generate purified repaired concatamerized nucleic acid; and
g) amplifying said purified repaired concatamerized nucleic acid using a whole genome amplification method, wherein said whole genome amplification method employs random primers.

28. The method of claim 27 wherein said polynucleotide kinase comprises T4 polynucleotide kinase.

29. The method of claim 27 wherein said polymerase comprises DNA Polymerase I, Large (Klenow) Fragment.

30. The method of claim 27 wherein said ligase comprises T4 DNA ligase.

31. The method of claim 27 wherein said polynucleotide fragments comprise fragments of a degraded whole genome.

32. The method of claim 27 wherein said DNA ligase concentration comprises 39 cohesive end ligation units per microliter of reaction mix to 66 cohesive end ligation units per microliter.

33. The method of claim 27 wherein said one or more enzymes that catalyze excision of degraded nucleotides comprises an endonuclease, a DNA N-glycosylase an AP lyase, a 3′-diesterase or a combination thereof.

34. The method of claim 27 wherein said whole genome amplification method comprises multiple displacement amplification.