Method, compositions and kit for detection of microorganisms and bi-directional sequencing of nucleic acid polymers

Abstract

Evaluation of a sample for the presence and qualitative nature of a microorganism can be performed in a single vessel by combining a natural abundance DNA sample with a sequencing mixture containing a primer pair, a thermally stable polymerase such as ThermoSequenase™ which incorporates dideoxynucleotides into an extending nucleic acid polymer at a rate which is no less than about 0.4 times the rate of incorporation of deoxynucleotides, nucleotide triphosphate feedstocks, and a chain terminating nucleotide triphosphate. The mixture is processed through multiple thermal cycles for annealing, extension and denaturation to produce a product mixture which is analyzed by electrophoresis.

1 . A composition comprising a mixture of four deoxynucleotide triphosphates and at least one dideoxynucleotide triphosphate corresponding to one of the four deoxynucleotide triphosphates, wherein the dideoxynucleotide triphosphate is present in a mole ratio to the corresponding deoxynucleotide triphosphate of from 1:50 to 1:500, said composition further comprising a thermally stable polymerase enzyme which incorporates dideoxynucleotides into an extending nucleic acid polymer at a rate which is no less than 0.4 times the rate of incorporation of deoxynucleotides.  
     
     
         2 . The composition according to  claim 1 , wherein the mole ratio is from 1:100 to 1:300.  
     
     
         3 . A kit for detection of a target microorganism comprising, in packaged combination, 
 (a) a pair of primers which bind to the sense and antisense strands, respectively, and flank a selected region within the genome target microorganism; and    (b) a mixture of four deoxynucleotide triphosphates and at least dideoxynucleotide triphosphate corresponding to one of the four deoxynucleotide triphosphates, wherein the dideoxynucleotide triphosphate is present in a mole ratio to the corresponding deoxynucleotide triphosphate of from 1:50 to 1:1000    (c) a polymerase enzyme which incorporates dideoxynucleotides into an extending nucleic acid polymer at a rate which is no less than 0.4 times the rate of incorporation of deoxynucleotides.    
     
     
         4 . The kit according to  claim 3 , wherein the mole ratio is from 1:100 to 1:500.  
     
     
         5 . The kit according to  claim 3 , wherein at least one of the primers is labeled with a fluorescent label.  
     
     
         6 . The kit according to  claim 3 , wherein the primers are each labeled with a spectroscopically-distinct fluorescent label.  
     
     
         7 . The kit according to  claim 3 , wherein the target microorganism is  Chlamydia trachomatis.    
     
     
         8 . The kit according to  claim 7 , wherein the first and second primers are selected from the group consisting of the oligonucleotides given by Seq. ID. Nos. 1-17.  
     
     
         9 . The kit according to  claim 3 , wherein the target microorganism is human immunodeficiency virus.  
     
     
         10 . The kit according to  claim 9 , wherein the first and second primers are selected from the group consisting of the oligonucleotides given by Seq. ID. Nos. 18-20.  
     
     
         11 . The kit according to  claim 3 , wherein the target microorganism is human papilloma virus.  
     
     
         12 . The kit according to  claim 11 , wherein the first and second primers are selected from the group consisting of the oligonucleotides given by Seq. ID. Nos. 21-22.