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, a sequencing primer, a sequencing polymerase, deoxynucleoside triphosphates suitable for template-dependent primer extension, and four different terminating nucleotides, each of which terminates template-dependent primer extension at a different template nucleotide, the incubating 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) determining a sequence of said polynucleotide by evaluating said plurality of differently-sized extended sequencing primers.
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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.
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Citations
27 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:
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a) incubating the polynucleotide with a composition comprising;
at least one amplification primer, a processive amplification polymerase, a sequencing primer, a sequencing polymerase, deoxynucleoside triphosphates suitable for template-dependent primer extension, and four different terminating nucleotides, each of which terminates template-dependent primer extension at a different template nucleotide, the incubating 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) determining a sequence of said polynucleotide by evaluating said 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)
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26. A method of amplifying and sequencing at least a region of a polynucleotide, said method comprising the steps of:
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a) incubating the polynucleotide with a composition comprising;
at least one amplification primer, a processive amplification polymerase, a sequencing primer, a sequencing polymerase, deoxynucleoside triphosphates suitable for template-dependent primer extension, a terminating nucleotide, the incubating being carried out at a first temperature suitable for amplifying the polynucleotide with the processive amplification polymerase, wherein said sequencing primer is labeled with a detectable label;
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, wherein step (a) and step (b) are repeated in four separate incubations in the presence of a terminating nucleotide suitable for terminating primer extension at A, C, G/I, or T/U, respectively;
c) determining a sequence of said polynucleotide by evaluating said plurality of differently-sized extended sequencing primers.
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27. A method of amplifying and sequencing a polynucleotide, said method comprising the steps of:
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a) amplifying at least a region of a polynucleotide with a processive polymerase and at least one amplification primer at a first temperature in the presence of a sequencing primer, a sequencing polymerase, nucleoside triphosphates suitable for template-dependent primer extension, four different terminating nucleotides, the amplifying being carried out at a first temperature suitable for amplifying the polynucleotide with the processive amplification polymerase;
b) forming at a second temperature a plurality of differently-sized extended and labeled sequencing primers, wherein said second temperature is suitable for forming a plurality of differently-sized extended sequencing primers with the sequencing polymerase;
c) determining a sequence of said polynucleotide by evaluating said plurality of differently-sized extended sequencing primers.
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Specification