Methods, compositions, and kits for amplifying and sequencing polynucleotides
First Claim
1. A method of amplifying and sequencing at least a region of a polynucleotide, said method comprising the steps of:
- a) incubating the polynucleotide with a composition comprising;
at least one amplification primer;
a processive-amplification polymerase having strand displacement activity;
a sequencing primer;
a sequencing polymerase;
deoxynucleoside triphosphates suitable for template-dependent primer extension, wherein the deoxynucleoside triphosphates suitable for template-dependent primer extension are present throughout the method; and
four different terminating nucleotides, each of which terminates template-dependent primer extension at a different template nucleotide,wherein a first annealing temperature of a duplex formed between the polynucleotide and the at least one amplification primer is at least 5°
C. lower than a second annealing temperature of a duplex formed between the sequencing primer and an amplification product;
b) maintaining a first incubation temperature isothermally at a temperature suitable for amplifying the polynucleotide with the amplification polymerase;
c) elevating the incubation temperature to two or more temperatures suitable for forming a plurality of differently-sized extended sequencing primers with the sequencing polymerase, wherein the two or more temperatures are above the first incubation temperature; and
d) determining a sequence of the polynucleotide by evaluating the plurality of differently-sized extended sequencing primers.
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Accused Products
Abstract
In one aspect, there are provided methods of amplifying and sequencing a polynucleotide. In some embodiments, the method includes (a) amplifying the polynucleotide with at least one amplification primer, a processive amplification polymerase, a sequencing primer, a sequencing polymerase, deoxynucleoside triphosphates suitable for template-dependent primer extension, and one or more terminating nucleotides, the incubation being carried out at a first temperature suitable for amplifying the polynucleotide with the processive amplification polymerase; (b) incubating the product of step (a) at a second temperature suitable for forming a plurality of differently-sized extended sequencing primers with the sequencing polymerase; (c) evaluating the extended sequencing primers in order to determine the sequence of the polynucleotide. The reactions at the first and second temperatures can be carried out in a single reaction vessel. In other aspects, compositions and kits for carrying out the methods are also provided.
14 Citations
25 Claims
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1. A method of amplifying and sequencing at least a region of a polynucleotide, said method comprising the steps of:
- a) incubating the polynucleotide with a composition comprising;
at least one amplification primer; a processive-amplification polymerase having strand displacement activity; a sequencing primer; a sequencing polymerase; deoxynucleoside triphosphates suitable for template-dependent primer extension, wherein the deoxynucleoside triphosphates suitable for template-dependent primer extension are present throughout the method; and four different terminating nucleotides, each of which terminates template-dependent primer extension at a different template nucleotide, wherein a first annealing temperature of a duplex formed between the polynucleotide and the at least one amplification primer is at least 5°
C. lower than a second annealing temperature of a duplex formed between the sequencing primer and an amplification product;b) maintaining a first incubation temperature isothermally at a temperature suitable for amplifying the polynucleotide with the amplification polymerase; c) elevating the incubation temperature to two or more temperatures suitable for forming a plurality of differently-sized extended sequencing primers with the sequencing polymerase, wherein the two or more temperatures are above the first incubation temperature; and d) determining a sequence of the polynucleotide by evaluating the plurality of differently-sized extended sequencing primers. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25)
- a) incubating the polynucleotide with a composition comprising;
Specification