Methods for sequential DNA amplification and sequencing

• US 9,745,624 B2
• Filed: 08/01/2012
• Issued: 08/29/2017
• Est. Priority Date: 10/18/2004
• Status: Active Grant

• Current Assignee: Brandeis University
• Original Assignee: Brandeis University
• Inventors: Wangh, Lawrence J., Rice, John E., Sanchez, J. Aquiles, Pierce, Kenneth E., Salk, Jesse, Reis, Arthur H., Hartshorn, Cristina
• Primary Examiner(s): BERTAGNA, ANGELA MARIE
• Application Number:
  US13/564,528
• Publication Number:
  US 20130095479A1
• Time in Patent Office: 1,854 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2531/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2545/114 involving a quantitation step
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

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.

Claims

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.