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Vol. 10, Issue 8, 1211-1218, August 2000

METHODS
Loss of Heterozygosity Assay for Molecular Detection of Cancer Using Energy-transfer Primers and Capillary Array Electrophoresis

Igor L. Medintz,1 Chyi-Chia Richard Lee,2 Wendy W. Wong,1 Kristin Pirkola,3 David Sidransky,2 and Richard A. Mathies1,4

1 Department of Chemistry, University of California, Berkeley, California 94720 USA; 2 Department of Otolaryngology --- Head and Neck Surgery, Division of Head and Neck Cancer Research, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 USA; 3 Molecular Dynamics, Sunnyvale, California 94086 USA

Microsatellite DNA loci are useful markers for the detection of loss of heterozygosity (LOH) and microsatellite instability (MI) associated with primary cancers. To carry out large-scale studies of LOH and MI in cancer progression, high-throughput instrumentation and assays with high accuracy and sensitivity need to be validated. DNA was extracted from 26 renal tumor and paired lymphocyte samples and amplified with two-color energy-transfer (ET) fluorescent primers specific for loci associated with cancer-induced chromosomal changes. PCR amplicons were separated on the MegaBACE-1000 96 capillary array electrophoresis (CAE) instrument and analyzed with MegaBACE Genetic Profiler v.1.0 software. Ninety-six separations were achieved in parallel in 75 minutes. Loss of heterozygosity was easily detected in tumor samples as was the gain/loss of microsatellite core repeats. Allelic ratios were determined with a precision of  ± 10% or better. Prior analysis of these samples with slab gel electrophoresis and radioisotope labeling had not detected these changes with as much sensitivity or precision. This study establishes the validity of this assay and the MegaBACE instrument for large-scale, high-throughput studies of the molecular genetic changes associated with cancer.

4 Corresponding author.

10:1211-1218 ©2000 by Cold Spring Harbor Laboratory Press  ISSN 1088-9051/00 $5.00
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