Fast pcr for str genotyping

Abstract

Disclosed is a method of amplifying a nucleic acid sequence, wherein the method comprises subjecting a reaction mixture to at least one amplification cycle, wherein the reaction mixture comprises a double-stranded nucleic acid and at least two primers capable of annealing to complementary strands of the double-stranded nucleic acid and amplifying at least one short tandem repeat (STR) using a Family A DNA polymerase in a Fast PCR protocol having a two-step amplification cycle in 25 seconds or less. Also disclosed are real-time PCR methods using the two-step protocol and kits for STR profiling using the Fast PCR protocol.

What is claimed is: 
     
         1 . A kit for detection of an STR profile comprising:
 a pair of primers configured to amplify at least one short tandem repeat (STR) locus;   a master mix composition for forming a reaction mixture with a sample;   an indicator probe; and   a Family A DNA polymerase provided so as to be present at a concentration of greater than 0.2 units/μl and up to 0.6 units/μl within the reaction mixture when formed.   
     
     
         2 . The kit of  claim 1 , wherein the Family A polymerase comprises a polymerase from  Thermus aquaticus, Thermus thermophilus  HB-8,  Thermus thermophilus  HB-27,  Thermus flavus, Thermus maritime ; large fragment,  Thermotoga naepolitana, Thermococcus gorgonarius, Thermococcus litoralis, Thermococcus aggregans , or  Thermomicrobium roseum.    
     
     
         3 . The kit of  claim 2 , wherein the Family A polymerase comprises Taq DNA polymerase. 
     
     
         4 . The kit of  claim 1 , wherein the Family A polymerase is antibody-bound. 
     
     
         5 . The kit of  claim 1 , wherein the pair of primers is configured to amplify a region of the amelogenin locus. 
     
     
         6 . The kit of  claim 1 , wherein the indicator probe is included in the master mix composition. 
     
     
         7 . The kit of  claim 1 , wherein the indicator probe is a 5′-nuclease probe comprising a signal moiety capable of producing a detectable signal upon cleavage of the indicator probe. 
     
     
         8 . The kit of  claim 1 , wherein the Family A DNA polymerase is provided so as to be present at a concentration of 0.3 units/μl to 0.6 units/μl within the reaction mixture when formed. 
     
     
         9 . The kit of  claim 8 , wherein the Family A DNA polymerase is provided so as to be present at a concentration of 0.3 units/μl to 0.5 units/μl within the reaction mixture when formed. 
     
     
         10 . The kit of  claim 1 , further comprising an indicator molecule configured to indicate an amount of nucleic acid in the reaction mixture. 
     
     
         11 . The kit of  claim 1 , wherein the indicator probe is a is a hairpin probe comprising a signal moiety capable of producing a detectable signal. 
     
     
         12 . The kit of  claim 1 , wherein the master mix composition comprises a divalent cation. 
     
     
         13 . The kit of  claim 1 , further comprising an allelic ladder. 
     
     
         14 . The kit of  claim 1 , further comprising an electrophoresis loading solution. 
     
     
         15 . The kit of  claim 1 , wherein the master mix composition comprises deoxyribonucleotide triphosphates (dNTPs). 
     
     
         16 . The kit of  claim 1 , wherein the kit comprises multiple pairs of primers each configured to amplify a different STR locus. 
     
     
         17 . The kit of  claim 16 , wherein each primer is included so as to be present at a concentration of 0.1 μM or more within the reaction mixture when formed. 
     
     
         18 . A kit for detection of an STR profile comprising:
 multiple pairs of primers each configured to amplify a different short tandem repeat (STR) locus;   a master mix composition for forming a reaction mixture with a sample;   an indicator probe comprising a signal moiety capable of producing a detectable signal upon cleavage of the indicator probe; and   an antibody-bund Taq polymerase provided so as to be present at a concentration of greater than 0.2 units/μl and up to 0.6 units/μl within the reaction mixture when formed.   
     
     
         19 . The kit of  claim 18 , wherein the Taq polymerase is provided so as to be present at a concentration of 0.3 units/μl to 0.6 units/μl within the reaction mixture when formed. 
     
     
         20 . A method of amplifying a nucleic acid sequence, comprising:
 subjecting a reaction mixture to at least one amplification cycle, wherein the reaction mixture comprises
 a nucleic acid, 
 a pair of primers configured to amplify at least one short tandem repeat (STR), and 
 a Family A DNA polymerase included at a concentration of 0.3 units/μl to 0.6 units/μl; 
   wherein the time to complete one amplification cycle is 25 seconds or less.