Methods and compositions for multiple displacement amplification of nucleic acids

Abstract

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.

What is claimed is: 
     
         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.