Methods, compositions, and kits for amplifying and sequencing polynucleotides

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, 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.