Methods for sequential DNA amplification and sequencing

Patent metadata

Field	Value
Publication number	US-9745624-B2
Application number	US-201213564528-A
Country	US
Kind code	B2
Filing date	Aug 1, 2012
Priority date	Oct 18, 2004
Publication date	Aug 29, 2017
Grant date	Aug 29, 2017

Abstract

Homogenous detection during or following PCR amplification, preferably LATE-PCR, utilizing fluorescent DNA dye and indirectly excitable labeled primers and probes, improves reproducibility and quantification. Low-temperature homogeneous detection during or following non-symmetric PCR amplification, preferably LATE-PCR, utilizing fluorescent DNA dye and indirectly excitable labeled mismatch-tolerant probes permits analysis of complex targets. Sequencing sample preparation methods following LATE-PCR amplifications reduce complexity and permit “single-tube” processing.

First claim

What is claimed is:

1. A sequential DNA amplification-sequencing method comprising:
a) amplifying at least two DNA targets by LATE-PCR to generate an amplification product containing copies of at least two excess primer strands and at least two limiting primer strands;
b) processing the amplification product with a clean-up procedure consisting of diluting the amplification product by a factor of at least eighty to produce a cleaned-up amplification product;
and
c) sequencing the copies of at least one of said excess primer strands in the cleaned-up amplification product.

2. The method of claim 1 , wherein sequencing is dideoxy cycle sequencing.

3. The method of claim 1 , wherein the act of diluting is performed in two steps.

4. The method of claim 1 , wherein the LATE-PCR comprises monitoring the adequacy of production of single-stranded product, wherein the monitoring comprises determining the ratio of single-stranded product to double-stranded product.

5. The method of claim 1 , wherein the excess primer strand is sequenced using a primer having a sequence identical to a limiting primer used in the LATE-PCR.

Assignees

• Wangh Lawrence J
• Rice John E
• Sanchez J Aquiles
• Pierce Kenneth E
• Salk Jesse
• Reis Arthur H
• Hartshorn Cristina
• Univ Brandeis

Inventors

• Wangh Lawrence J
• Rice John E
• Sanchez J Aquiles
• Pierce Kenneth E
• Salk Jesse
• Reis Arthur H
• Hartshorn Cristina

Classifications

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• C12Q1/6851

  Quantitative amplification · CPC title

• C12Q2600/16

  Primer sets for multiplex assays · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q1/6844 Primary

  Nucleic acid amplification reactions · CPC title