Nucleic acid amplification

Abstract

Disclosed are compositions and methods for amplification of nucleic acid sequences of interest. It has been discovered that amplification reactions can produce amplification products of high quality, such as low amplification bias, if performed on an amount of nucleic acid at or over a threshold amount and/or on nucleic acids at or below a threshold concentration. The threshold amount and concentration can vary depending on the nature and source of the nucleic acids to be amplified and the type of amplification reaction employed. Disclosed is a method of determining the threshold amount and/or threshold concentration of nucleic acids that can be used with nucleic acid samples of interest in amplification reactions of interest. Because amplification reactions can produce high quality amplification products, such as low bias amplification products, below the threshold amount and/or concentration of nucleic acid, such below-threshold amounts and/or concentrations can be used in amplification reactions.

1 . (canceled) 
     
     
         2 . (canceled) 
     
     
         3 . (canceled) 
     
     
         4 . A kit for amplifying a whole genome, the kit comprising
 a composition for lysis,   a composition for neutralization,   a set of primers, and   a DNA polymerase.   
     
     
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         6 . (canceled) 
     
     
         7 . The kit of  claim 4  wherein the composition for lysis comprises potassium hydroxide. 
     
     
         8 . The kit of  claim 7  wherein the composition for lysis comprises 400 mM KOH. 
     
     
         9 . The kit of  claim 8  wherein the composition for lysis comprises 400 mM KOH, 100 mM dithiothreitol, and 10 mM EDTA. 
     
     
         10 . The kit of  claim 4  wherein the composition for lysis consists of 400 mM KOH, 100 mM dithiothreitol, and 10 mM EDTA. 
     
     
         11 . The kit of  claim 26  wherein the composition for stabilization comprises Tris-HCl at pH 4.1. 
     
     
         12 . The kit of  claim 11  wherein the composition for stabilization comprises 800 mM Tris-HCl, pH 4.1. 
     
     
         13 . The kit of  claim 12  wherein the composition for stabilization consists of 800 mM Tris-HCl, pH 4.1. 
     
     
         14 . The kit of  claim 4  further comprising deoxynucleotide triphosphates. 
     
     
         15 . The kit of  claim 26  further comprising
 1M dithiotheitol,   1× Phosphase-Buffered Saline, pH 7.5, and   control DNA template,   wherein the composition for lysis comprises 400 mM KOH and 10 mM EDTA,   wherein the composition for stabilization comprises 800 mM Tris-HCl, pH 4,   wherein the set of primers comprises a reaction mix, wherein the reaction mix comprises 150 mM Tris-HCl, 200 mM KCl, 40 mM MgCl 2 , 20 mM (NH 4 ) 2 SO 4 , 4 mM deoxynucleotide triphosphates, and 0.2 mM random hexamer primers,   wherein the DNA polymerase is φ29 DNA polymerase.   
     
     
         16 . The kit of  claim 4  further comprising one or more detection probes, wherein the detection probes each comprise a complementary portion, wherein the complementary portion is complementary to a nucleic acid sequence of interest. 
     
     
         17 . The method of  claim 16  wherein the kit is designed to detect nucleic acid sequences of interest in the genome. 
     
     
         18 . The method of  claim 17  wherein the kit is designed to assess a disease, condition or predisposition of an individual based on the nucleic acid sequences of interest. 
     
     
         19 . A kit for amplifying a whole genome, the kit comprising
 a composition for lysis, wherein the composition for lysis is alkaline,   a composition for neutralization,   a set of primers, wherein the primers are 6 nucleotides long, wherein the primers each contain at least one modified nucleotide such that the primers are nuclease resistant, and   a DNA polymerase, wherein the DNA polymerase is φ29 DNA polymerase.   
     
     
         20 . (canceled) 
     
     
         21 . (canceled) 
     
     
         22 . A kit for amplifying a whole genome, the kit comprising
 a composition for stabilization,   a reaction mix comprising a set of primers, and   a DNA polymerase mix comprising a DNA polymerase.   
     
     
         23 . The kit of  claim 22  further comprising
 1M dithiotheitol,   1× Phosphase-Buffered Saline, pH 7.5, and   control DNA template,   wherein the composition for stabilization comprises 800 mM Tris-HCl, pH 4,   wherein the reaction mix comprises 150 mM Tris-HCl, 200 mM KCl, 40 mM MgCl 2 , 20 mM (NH 4 ) 2 SO 4 , 4 mM deoxynucleotide triphosphates, and 0.2 mM random hexamer primers,   wherein the DNA polymerase is φ29 DNA polymerase.   
     
     
         24 . The kit of  claim 4 , wherein the composition for neutralization is a composition for neutralization of a cell lysate. 
     
     
         25 . The kit of  claim 4 , wherein the composition for neutralization is a composition for neutralizing lysed cells. 
     
     
         26 . The kit of  claim 4 , further comprising a composition for stabilization. 
     
     
         27 . The kit of  claim 19 , wherein the composition for lysis is a composition for cell lysis, and wherein the composition for neutralization is a composition for neutralization of a cell lysate. 
     
     
         28 . The kit of  claim 19 , wherein the composition for lysis is a composition for lysing cells, and wherein the composition for neutralization is a composition for neutralizing lysed cells.