Protocol

Loss of Heterozygosity: A Multiplex PCR Method to Define a Narrow Deleted Chromosomal Region of a Tumor Genome

This protocol was adapted from "Purification of High-Quality DNA," contributed by Lise Lotte Hansen and Just Justesen, Chapter 17, in PCR Primer, 2nd edition (eds. Dieffenbach and Dveksler). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.

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INTRODUCTION

In this protocol, DNA for analysis is purified using salt precipitation. The method is gentle, limits the breakage of the long chromosomal strands, and avoids the use of phenol and chloroform. It is suitable for use with cultured cells, breast tumor tissue that has been subjected to hormone receptor analysis, and blood samples. The loss of heterozygosity assay is performed using a multiplex PCR, in which one of each primer pair is labeled with a different fluorophor. Simple tandem repeats (STRs) are amplified from wild-type and tumor DNAs and analyzed by either polyacrylamide gel electrophoresis or capillary electrophoresis.

MATERIALS

Reagents

dNTP solution . . . [Full Text of this Article]

Equipment

METHOD

TROUBLESHOOTING