US 2007/0190540 A1 · Methods for the amplification, quantitation and identification of nucleic acids

Abstract

The invention relates to improved methods of amplifying and optionally quantifying and/or identifying a plurality of selected nucleic acid molecules from a pool of nucleic acid molecules. A first round of multiplex amplification used where the amplification reaction is allowed to proceed to a point prior to that at which significant competition between amplicons for reaction components has occurred. This is the followed by a second round of amplification that typically includes a fluorescent reporter to allow for each of the selected nucleic acid sequences to be quantified. The methods are useful for the amplification and quantification of nucleic acids from a variety of sources, such as gene expression products, whereby many such products may be amplified and quantified from very limited samples and from degraded archival samples.

Claims37

Dependent claims

A method of amplifying a plurality of selected nucleic acid molecules from a pool of nucleic acid molecules comprising:

(a) amplifying a plurality of selected nucleic acid molecules in a first round multiplex amplification reaction including a plurality of outer primer pairs each pair being specific for a selected nucleic acid sequence wherein the amplification reaction is allowed to proceed to a point prior to that at which significant competition between amplicons for reaction components has occurred;
and
(b) further amplifying said selected nucleic acid molecules in a plurality of second round amplification reactions, each including a portion of the completed multiplex reaction as a template and at least one pair of inner primers each pair being specific for one of said selected nucleic acid sequences such that each second round reaction further amplifies a subset of said plurality of selected nucleic acid molecules respectively.

A method of estimating the number of selected nucleic acid molecules from a pool of nucleic acid molecules comprising:

(a) amplifying a plurality of selected nucleic acid molecules in a first round multiplex amplification reaction including a plurality of outer primer pairs each pair being specific for a selected nucleic acid sequence wherein the amplification reaction is allowed to proceed to a point prior to that at which significant competition between amplicons for reaction components has occurred;
(b) further amplifying said selected nucleic acid molecules in a plurality of second round amplification reactions, each including a detectible reporter, a portion of the completed multiplex reaction as a template and at least one pair of inner primers each pair being specific for one of said selected nucleic acid sequences whereby each second round reaction further amplifies a subset of the plurality of selected nucleic acid molecules respectively;
and
(c) monitoring each second round amplification reaction by means of said detectible reporter such that the number of selected nucleic acid molecules of each selected sequence is estimated.

1 more element

A method of estimating the number of selected nucleic acid molecules from a pool of nucleic acid molecules comprising:

(a) amplifying a plurality of selected nucleic acid molecules in a first round multiplex amplification reaction including a plurality of outer primer pairs each pair being specific for a selected nucleic acid sequence wherein the amplification reaction is allowed to proceed to a point prior to that at which significant competition between amplicons for reaction components has occurred;
and
(b) further amplifying said selected nucleic acid molecules in a plurality of second round amplification reactions, each including a detectible reporter, a portion of the completed multiplex reaction as a template and at least one pair of primers each pair comprising an inner primer and one of said outer primers and being specific for one of said selected nucleic acid sequences such that each second round reaction further amplifies a subset of said plurality of selected nucleic acid molecules respectively;
and
(c) monitoring each second round amplification reaction by means of said detectible reporter such that the number of selected nucleic acid molecules of each selected sequence is estimated.

2 more elements

Dependencies are inferred from the claim text.

Methods for the amplification, quantitation and identification of nucleic acids

Life of this patent

Methods for the amplification, quantitation and identification of nucleic acids

Life of this patent

Abstract

Claims37