( Clive R. Pullinger Argh...)Detection of Apolipoprotein Gene Variants by DGGE Using the DCode System

Clive R. Pullinger, Arghavan K. Shahidi, Andrea L. Verhagen and John P. Kane^*

Cardiovascular Research Institute, ^*Department of Medicine, Department of Biochemistry and Biophysics, University of California, San Francisco, California

Introduction
In a number of fields, techniques are required for searching candidate genes for mutations that underlie hereditary diseases. We have used the denaturing gradient gel electrophoresis method (DGGE)¹ using the Bio-Rad DCode system to identify a number of new, as well as known, mutations and polymorphisms in the genes for apolipoprotein A-I (apoA-I) and apolipoprotein B (apoB).

Materials And Methods
Genomic DNA was prepared from patients with dyslipidemia. Segments of the apoA-I and apoB genes were amplified from genomic DNA using a temperature cycler (Ericomp Inc., San Diego, CA). Both oligonucleotide primers had GC clamps.² Following restriction endonuclease digestion, the samples were subjected to DGGE.

The amplification reactions were performed in 50 mM Tris-HCl, pH 9 (at 25 C), 20 mM (NH₄)₂SO₄, 1.5 mM MgCl₂, 200 M of each dNTP, 1 unit of Hot Tub polymerase (Amersham Life Science Inc., Cleveland, OH), 15 pmol of each primer and a minimum of 200 ng of DNA in a total volume of 50 l. After initial denaturation at 96 C for 1 minute, PCR* was carried out for a total of 33 cycles at an annealing temperature of 62 C (apoA-I) or 58 C (apoB), in each case for 30 s. The denaturing and elongation steps were 96 C for 30 s and 72 C for 60 s. A final elongation step of 8 minutes at 72 C w
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