Format

Send to

Choose Destination
See comment in PubMed Commons below
Biotechniques. 2012 Aug 14;2012. doi: 10.2144/000113913.

Improving the limit of detection for Sanger sequencing: A comparison of methodologies for KRAS variant detection.

Author information

1
Life Technologies, Foster City, CA, USA.

Abstract

Fluorescent dye terminator Sanger sequencing (FTSS), with detection by automated capillary electrophoresis (CE), has long been regarded as the gold standard for variant detection. However, software analysis and base-calling algorithms used to detect mutations were largely optimized for resequencing applications in which different alleles were expected as heterozygous mixtures of 50%. Increasingly, the requirements for variant detection are an analytic sensitivity for minor alleles of <20%, in particular, when assessing the mutational status of heterogeneous tumor samples. Here, we describe a simple modification to the FTSS workflow that improves the limit of detection of cell-line gDNA mixtures from 50%-20% to 5% for G>A transitions and from 50%-5% to 5% for G>C and G>T transversions. In addition, we use two different sample types to compare the limit of detection of sequence variants in codons 12 and 13 of the KRAS gene between Sanger sequencing and other methodologies including shifted termination assay (STA) detection, single-base extension (SBE), pyrosequencing (PS), high- resolution melt (HRM), and real-time PCR (qPCR).

PMID:
23083368
DOI:
10.2144/000113913
Free full text
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Informa Healthcare, USA
    Loading ...
    Support Center