Direct amplification of single-stranded DNA for pyrosequencing using linear-after-the-exponential (LATE)–PCR

Jesse J Salk; J. Aquiles Sanchez; Kenneth E Pierce; John E Rice; Kevin C Soares; Lawrence J Wangh

doi:10.1016/j.ab.2006.02.012

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Direct amplification of single-stranded DNA for pyrosequencing using linear-after-the-exponential (LATE)–PCR

Journal article

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Direct amplification of single-stranded DNA for pyrosequencing using linear-after-the-exponential (LATE)–PCR

Jesse J Salk, J. Aquiles Sanchez, Kenneth E Pierce, John E Rice, Kevin C Soares and Lawrence J Wangh

Analytical biochemistry, Vol.353(1), pp.124-132

2006

DOI: https://doi.org/10.1016/j.ab.2006.02.012

PMCID: PMC1533996

PMID: 16540077

Abstract

Asymmetric PCR

IVS 110

LATE–PCR

Pyrosequencing

Single-cell

Single-nucleotide polymorphism

Single-stranded DNA

β-Thalassemia

Pyrosequencing is a highly effective method for quantitatively genotyping short genetic sequences, but it currently is hampered by a labor-intensive sample preparation process designed to isolate single-stranded DNA from double-stranded products generated by conventional PCR. Here linear-after-the-exponential (LATE)–PCR is introduced as an efficient and potentially automatable method of directly amplifying single-stranded DNA for pyrosequencing, thereby eliminating the need for solid-phase sample preparation and reducing the risk of laboratory contamination. These improvements are illustrated for single-nucleotide polymorphism genotyping applications, including an integrated single-cell-through-sequencing assay to detect a mutation at the globin IVS 110 site that frequently is responsible for β-thalassemia.

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Title: Direct amplification of single-stranded DNA for pyrosequencing using linear-after-the-exponential (LATE)–PCR
Creators: Jesse J Salk - Department of Biology, Brandeis University, Waltham, MA 02454, USA
J. Aquiles Sanchez - Department of Biology, Brandeis University, Waltham, MA 02454, USA
Kenneth E Pierce - Department of Biology, Brandeis University, Waltham, MA 02454, USA
John E Rice - Department of Biology, Brandeis University, Waltham, MA 02454, USA
Kevin C Soares - Department of Biology, Brandeis University, Waltham, MA 02454, USA
Lawrence J Wangh - Department of Biology, Brandeis University, Waltham, MA 02454, USA
Publication Details: Analytical biochemistry, Vol.353(1), pp.124-132
Publisher: Elsevier Inc
Identifiers: 9924029017401921
Academic Unit: Department of Biology
Language: English
Resource Type: Journal article

Direct amplification of single-stranded DNA for pyrosequencing using linear-after-the-exponential (LATE)–PCR

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