Reagents and kit compositions for single-cell whole genome amplification

Abstract

The present invention provides reagents, kits, and methods for single-cell whole genome amplification using Phi 29 DNA polymerase.

1 - 21 . (canceled) 
     
     
         22 . A method of producing an amplified product, the method comprising:
 (a) preparing a reaction mixture comprising:
 (i) genomic DNA from a single cell; 
 (ii) random primers of 40 nucleotides or fewer in length, comprising from 5′ to 3′: a tag region of at least from 1 to 20 nucleotides in length having a defined sequence, and a random region of from 6 to 20 nucleotides in length at which random nucleotides are incorporated at each position, wherein at least the 3′nucleotide of the primer has a phosphorothioate linkage; 
 (iii) a Phi29 DNA polymerase; 
 (iv) a buffering agent to provide a pH of from about 7.2 to about 7.7; 
   (b) incubating the reaction mixture at a temperature in the range from about 35° C. to about 40° C. for a time sufficient to produce an amplified product.   
     
     
         23 . A reaction mixture for amplification of genomic DNA from a single cell comprising: (i) genomic DNA from a single cell; (ii) random primers of 40 nucleotides or fewer in length, comprising from 5′ to 3′: a tag region of at least from 1 to 20 nucleotides in length having a defined sequence, and a random region of from 6 to 20 nucleotides in length at which random nucleotides are incorporated at each position, wherein at least the of the primer has a phosphorothioate linkage; (iii) a Phi29 DNA polymerase; and (iv) a buffering agent to provide a pH of from about 7.2 to about 7.7.