METHODS AND COMPOSITIONS FOR MULTIPLE DISPLACEMENT AMPLIFICATION OF NUCLEIC ACIDS
Disclosed are methods for multiple displacement amplification of a nucleic acid sequence in a sample. The nucleic acid is contacted with a reaction mixture that includes a set of oligonucleotide primers and a plurality of polymerase enzymes. The reaction mixture is subjected to conditions under which the nucleic acid sequence is amplified to produce an amplification product in a multiple displacement amplification reaction. Also disclosed are kits containing a set of oligonucleotide primers with random sequences having lengths of 6 to 8 nucleobases. At least some of the individual members of the primers have one or more ribose modifications that stabilize or lock the ribose ring in a 3′-endo conformation. At least some of the primers have one or more universal nucleobases.
1 . A method for multiple displacement amplification of a nucleic acid sequence in a sample comprising the steps of:
contacting said nucleic acid with a reaction mixture, wherein said reaction mixture includes a set of oligonucleotide primers and a plurality of polymerase enzymes; and
subjecting said reaction mixture to conditions under which said nucleic acid sequence is amplified to produce an amplified product in a multiple displacement reaction.
2 . The method of claim 1 wherein said sample is a forensic sample comprising human DNA.
3 . The method of claim 1 wherein said plurality of polymerase enzymes have 5′→3′ DNA polymerase activity, 3′→5′ exonuclease activity, and 5′→3′ excision repair activity.
4 . The method of claim 1 wherein members of said set of oligonucleotide primers have random sequences.
5 . The method of claim 4 wherein said oligonucleotide primers have lengths of 6, 7 or 8 nucleotide residues.
6 . The method of claim 4 wherein said oligonucleotide primers include at least one universal nucleobase.
7 . The method of claim 6 wherein said universal nucleobase is inosine.
8 . The method of claim 6 wherein said universal nucleobase is located at the 3′-terminal end of said oligonucleotide primers.
9 . The method of claim 1 wherein said set of oligonucleotide primers includes primers having one or more modifications of the ribose ring that favor a 3′-endo conformation of said ribose ring or lock said ribose ring in said 3′-endo conformation.
10 . The method of claim 9 wherein said modifications comprise a 2′ to 4′ ribose bridge.
11 . The method of claim 10 wherein said modifications are located at the 2nd and 5th positions of said primers.
12 . The method of claim 1 wherein said one or more enzymes include phi29 polymerase and pol I polymerase.
13 . The method of claim 1 wherein said one or more enzymes further includes pyrophosphatase.
14 . The method of claim 1 including the proviso that bovine serum albumin is not included in said reaction mixture.
15 . The method of claim 1 wherein said reaction mixture comprises betaine and trehalose.
16 . The method of claim 1 wherein said conditions comprise thermal cycling of said reaction mixture.
17 . The method of claim 1 wherein said multiple displacement amplification results in whole genome amplification.
18 . The method of claim 1 wherein said multiple displacement amplification reaction produces said amplification product at a constant ratio relative to production of other amplification products of other nucleic acids present in said sample.
19 . The method of claim 1 wherein the total quantity of said nucleic acid sequence is between about 2 picograms to about 1000 picograms.