Methods, compositions, and kits for amplifying and sequencing polynucleotides

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.

Description

1. CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims benefit under 35 U.S.C. §119(e) to application Ser. No. 60 / 539,465, entitled “Methods, Compositions, and Kits for Amplifying and Sequencing Polynucleotides,” filed Jan. 26, 2004, the disclosure of which is incorporated herein by reference in its entirety.2. FIELD

[0002] The present disclosure relates to the field of molecular biology, and in particular, provides methods, compositions and kits for amplifying and sequencing polynucleotides. 3. INTRODUCTION

[0003] Polynucleotide amplification and sequencing are fundamental technologies in molecular biology and life sciences in general. A number of methods have been developed for amplification of polynucleotides. These include the polymerase chain reaction (PCR), ligase chain reaction (LCR), self-sustained sequence replication (3SR), polynucleotide sequence based amplification (NASBA), strand displacement amplification (SDA), amplification with Qβ replicase (Birken...

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