Use of taq polymerase mutant enzymes for nucleic acid amplification in the presence of pcr inhibitors

Abstract

The present invention generally relates to detection of a target nucleic acid in standard PCR, real-time PCR, RT PCR, and real-time RT PCR. One aspect of the invention provides mutant DNA polymerase enzymes that are resistant to PCR inhibitors, such as dye, blood, and soil. Another aspect of the invention provides for methods of real-time PCR assays using mutant DNA polymerase enzymes resistant to PCR inhibitors with samples containing dye, blood, and/or soil. Another aspect of the invention provides for methods of standard PCR assays using mutant DNA polymerase enzymes resistant to PCR inhibitors with samples containing blood and/or soil.

1 . A method of amplifying a target nucleic acid in a real-time polymerase chain reaction (PCR) comprising:
 forming an assay mixture comprising:
 a sample comprising a target nucleic acid; 
 primers specific for the target nucleic acid; 
 a buffer; 
 at least one dye; and 
 at least one polymerase, wherein the at least one polymerase is a dye-resistant polymerase; and 
   amplifying the target nucleic acid in the assay mixture in a real-time PCR.   
     
     
         2 . The method of  claim 1  wherein the target nucleic acid is a DNA molecule. 
     
     
         3 . The method of  claim 1  wherein:
 the target nucleic acid is an RNA molecule; 
 the real-time PCR is a real-time reverse transcriptase (RT) PCR; and 
 the assay mixture further comprises a reverse-transcriptase. 
 
     
     
         4 . The method of  claim 1  wherein the at least one polymerase is (i) a dye-resistant and blood-resistant polymerase; (ii) a dye-resistant and soil-resistant polymerase; or (iii) a dye-resistant, blood-resistant, and soil-resistant polymerase. 
     
     
         5 . The method of  claim 1  wherein the at least one polymerase has a polypeptide sequence comprising SEQ ID NO: 1 or at least 95% identical to SEQ ID NO: 1 and dye-resistant polymerase activity. 
     
     
         6 . The method of  claim 5  wherein the at least one polymerase having a polypeptide sequence at least 95% identical to SEQ ID NO: 1 comprises at least one amino acid substitution at an amino acid residue position selected from the group consisting of positions 626, 707, and 708. 
     
     
         7 . The method of  claim 1  wherein the at least one polymerase has a polypeptide sequence comprising SEQ ID NO: 2 or at least 95% identical to SEQ ID NO: 1 and dye-resistant polymerase activity. 
     
     
         8 . The method of  claim 7  wherein the at least one polymerase having a polypeptide sequence at least 95% identical to SEQ ID NO: 2 comprises at least one amino acid substitution at an amino acid residue position selected from the group consisting of 626, 707, and 708. 
     
     
         9 . The method of  claim 1  wherein the sample further comprises whole blood or a blood component. 
     
     
         10 . The method of  claim 9  wherein the blood component is selected from the group consisting of blood plasma and blood serum. 
     
     
         11 . The method of  claim 9  wherein the whole blood or blood component is at least about 1% up to about 25% of a total volume of the PCR assay mixture. 
     
     
         12 . The method of  claim 1  wherein the sample further comprises soil or soil extract. 
     
     
         13 . The method of  claim 12  wherein the soil or soil extract is at least about 1% up to about 90% of a total volume of the PCR assay mixture. 
     
     
         14 . The method of  claim 12  wherein the soil or soil extract comprises a humic acid and the soil or soil extract is present in the assay mixture at a soil or soil extract equivalent amount that provides up to about 25 ng of humic acid per 50 uL reaction volume. 
     
     
         15 . The method of  claim 1  wherein the at least one dye is a fluorescent dye selected from the group consisting of SYBR Green, Ethidium Bromide, PICO, TOTO, YOYO or LC Green. 
     
     
         16 . The method of  claim 1  wherein the dye is present in the PCR assay mixture at least about 0.5× up to about 256×, where × is a manufacturer unit for concentration for use in PCR. 
     
     
         17 . The method of  claim 1  wherein the assay mixture further comprises a PCR enhancer. 
     
     
         18 . An isolated polypeptide comprising
 a polypeptide sequence of SEQ ID NO: 2;   a polypeptide sequence at least 95% identical to SEQ ID NO: 2 and having dye-, soil-, or blood-resistant polymerase activity, or a combination thereof;   a polypeptide sequence at least 95% identical to SEQ ID NO: 2 comprising at least one amino acid substitution at an amino acid residue position selected from the group consisting of 626, 707, and 708, and having dye-, soil-, or blood-resistant polymerase activity, or a combination thereof.   
     
     
         20 . A method of amplifying a target nucleic acid in a polymerase chain reaction (PCR) comprising:
 forming an assay mixture comprising:
 a sample comprising a target nucleic acid;
 wherein the sample optionally contains:
 whole blood or a blood component selected from blood plasma or blood serum; or 
 soil or soil extract; 
 
 
 primers specific for the target nucleic acid; 
 a buffer; 
 at least one polymerase comprising the isolated polypeptide of claim  19 ; and 
   amplifying the target nucleic acid in the assay mixture in a PCR.