Analysis of ligase chain reaction products amplified in a silicon-glass chip using capillary electrophoresis

J Chromatogr A. 1996 Apr 26;732(1):151-8. doi: 10.1016/0021-9673(95)01257-5.

Abstract

Ligase chain reaction (LCR) is a useful molecular technique for detecting known point mutations. We report the first example of the use of a disposable silicon-glass micro-chip for LCR and the first application of capillary electrophoresis (CE) to analyze samples amplified by LCR in a chip. Silicon-glass chips were manufactured using conventional photolithography and anodic bonding. The chips provide three distinct advantages for LCR: excellent thermal conductivity, a micro reaction volume ( < 10 microliters), and reproducible, low-cost manufacturing. Investigation and quantitation of amplification efficiency of LCR in a chip or in a tube requires an analytical technique that is faster and more convenient than the conventional slab gel methods. Slab gel electrophoresis uses relatively large amounts of sample and is labor-intensive and time-consuming, and thus is unsuitable for the separation and detection of LCR products. In contrast CE requires sample volume (original LCR products) of less than 1 microliter and is therefore well-suited to analysis of the micro-volume reaction mixture from chips. We combined CE with a sensitive laser induced fluorescence (LIF) detection system for the rapid separation and quantitative detection of LCR products amplified from the lacI gene in a silicon-glass chip. Comparative studies were made with LCR between tubes and silicon-glass chips. CE-LIF analysis is ideally suited to examination of micro-LCR amplification with high throughput. The technologies may find medical uses in disease diagnosis and research.

MeSH terms

  • Base Sequence
  • Electrophoresis, Capillary / instrumentation*
  • Ligases / metabolism*
  • Molecular Sequence Data
  • Oligonucleotide Probes
  • Polymerase Chain Reaction / methods*
  • Silicon

Substances

  • Oligonucleotide Probes
  • Ligases
  • Silicon