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Updated: Aug 10, 2026

Pyrosequencing: A Simple Method for Accurate Genotyping
Published on: January 8, 2008
Direct amplification of single-stranded DNA for pyrosequencing using linear-after-the-exponential (LATE)-PCR
Jesse J Salk1, J Aquiles Sanchez, Kenneth E Pierce
1Department of Biology, Brandeis University, Waltham, MA 02454, USA. jsalk@fhcrc.org
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.
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