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

ABSTRACT: 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 beta-thalassemia.

SUBMITTER: Salk JJ

PROVIDER: S-EPMC1533996 | biostudies-literature | 2006 Jun

REPOSITORIES: biostudies-literature

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

Salk Jesse J JJ Sanchez J Aquiles JA Pierce Kenneth E KE Rice John E JE Soares Kevin C KC Wangh Lawrence J LJ

Analytical biochemistry 20060228 1

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 sampl ...[more]

PMID: 16540077

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