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...

AI Technical Summary

Benefits of technology

[0013] The novel methods, compositions and kits provided herein are based in part on the surprising discovery by Applicants that terminating nucleotides used conventionally in polynucleotide sequencing do not substantially interfere with the amplification by processive polymerases. The amplification reaction can be carried out in the presence of reagents required for a subsequent sequencing reaction, which can be performed at an elevated temperature, thereby eliminating the need to provide separate reaction mixtures and / or vessels for carrying out both polynucleotide amplification and sequencing reactions.

Problems solved by technology

The amplification product can be viscous and difficult to accurately pipette.

However, each additional manipulative step slows down the overall analysis, increases the risk of contamination and increases the risk of contamination.

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