Method, compositions and kit for detection of microorganisms and bi-directional sequencing of nucleic acid polymers
First Claim
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.
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Accused Products
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.
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Citations
12 Claims
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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. - View Dependent Claims (2)
- 50 to 1;
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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. - View Dependent Claims (4, 5, 6, 7, 8, 9, 10, 11, 12)
- and
Specification