Mutation detection using ligase chain reaction in passivated silicon-glass microchips and microchip capillary electrophoresis

• Published in: BioTechniques Vol. 37; no. 3; pp. 392 - 398
• Main Authors: Lou, Xing Jian, Panaro, Nicholas J, Wilding, Peter, Fortina, Paolo, Kricka, Larry J
• Format: Journal Article
• Language: English
• Published: Natick, MA Future Science Ltd 01.09.2004; Eaton; Taylor & Francis Group
• Subjects: Biological and medical sciences; Diverse techniques; DNA Mutational Analysis - methods; DNA Primers - analysis; Electrophoresis, Capillary; Fundamental and applied biological sciences. Psychology; Glass; Humans; Ligase Chain Reaction - instrumentation; Ligase Chain Reaction - methods; Lymphocytes; Molecular and cellular biology; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; Povidone - chemistry; Silicon; Silicon Dioxide - chemistry; Surface Properties; Enzyme; Capillary electrophoresis; Ligases; Glass; Silicon; Mutation; Detection
• ISSN: 0736-6205; 1940-9818
• DOI: 10.2144/04373ST03

The ligase chain reaction (LCR) following PCR is one of the most sensitive and specific methods for detecting mutations, especially single nucleotide polymorphisms (SNPs). Performing LCR in microchips remains a challenge because of the inhibitory effect of the internal surfaces of silicon-glass microchips. We have tested a dynamic polymer-based surface passivation method for LCR conducted in oxide-coated silicon-glass microchips. The combination of polyvinylpyrrolidone 40 (PVP-40) at 0.75% (w/v) with an excess of the ligase produced successful LCR in the silicon-glass microchips, with yields of ligated primers comparable to reactions performed in conventional reaction tubes. Ligated primers were detected and quantified simply and conveniently using microchip capillary electrophoresis.