Patent ID: 8486627

Claim:
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.