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
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