US 2010/0304444 A1Published application

Method of amplification

AssigneeMonoquant Pty Ltd.

InventorsAlexander Alan Morley, Michael Julian Brisco

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Abstract

The present invention relates generally to a method of amplifying a nucleic acid region of interest and, more particularly, to a method of amplifying a nucleic acid region of interest via a nested single tube PCR. The method of the invention is designed to provide a means to selectively inactivate the functionality of the outer primer or primers and to maintain amplification efficiency throughout the reaction. The development of a means to achieve efficient amplification by the outer primer followed by efficient amplification with the inner primers, in the context of a single tube nested PCR, is useful in a range of applications including, but not limited to, the diagnosis and/or monitoring of disease conditions which are characterized by specific gene sequences and the characterization or analysis of specific gene regions of interest. Still further, the method of the present invention enables quantification to be performed and not just simple detection.

Claims20

Dependent claims

A method of amplifying a nucleic region of interest, said method comprising:

(i) contacting a nucleic acid sample with:
(a) a first forward primer directed to said region of interest and a first reverse primer directed to said region of interest;
and
(b) a second forward primer directed to said region of interest wherein said second forward primer is directed to a nucleic acid sequence located 3′ to the sequence to which said first forward primer is directed and which second forward primer has a Tm lower than the Tm of said first forward primer;
and
a second reverse primer directed to said region of interest wherein said second reverse primer is directed to a nucleic acid sequence located 5′ to the sequence to which said first reverse primer is directed and which second reverse primer has a Tm lower than the Tm of said first reverse primer;
and
(c) one or more antisense oligonucleotides directed to said first primer, which antisense oligonucleotides have a Tm lower than said first primer;
and
wherein the molecules of parts (b) and (c) can be added to the reaction before, during or after the amplification of step (ii) but before the amplification of step (iii).
(ii) amplifying the nucleic acid sample of step (i) at an annealing temperature which enables hybridization of said first primers but which does not enable hybridization of said second primers and said antisense oligonucleotides;
and
(iii) amplifying the nucleic acid sample of step (ii) at an annealing temperature which enables hybridization of said second primers and said antisense oligonucleotides.

10 more elements

Dependencies are inferred from the claim text.