Denaturing gradient gel electrophoresis (DGGE) increases resolution and informativity of Alu-directed inter-repeat PCR.
By inter-repeat PCR, multiple polymorphic loci can be targeted in parallel. To improve resolution and extend the number of detectable polymorphisms, Alu-directed inter-repeat PCR products from two large pedigrees of the Centre d'Etude du Polymorphisme Humain (CEPH) were electrophoretically resolved in non-denaturing polyacrylamide gels and, separately, on the basis of sequence content by denaturing gradient gel electrophoresis (DGGE). The resolution in DGGE gels was found to be superior to that in non-denaturing gels and a higher number of fragments was detected separately. The number of polymorphic bands detected by DGGE alone, however, was lower than that after size separation. This is ascribed to the fact that because of complete melting, small polymorphic fragments can run off the gel. With three Alu-specific primers, 18 and 16 polymorphic bands per individual were detected by size separation in pedigrees 1200 and 6600, respectively. In the same two pedigrees, seven and 15 polymorphic bands, respectively, were detected by DGGE. Segregation analysis of polymorphisms in the CEPH pedigrees indicated that most polymorphisms detected by DGGE were different from those detected by size separation.
van Orsouw
N J
NJ
Department of Medicine, Beth Israel Hospital and Harvard Medical School, Harvard Institutes of Medicine, Boston, MA 02115, USA.
Li
D
D
Vijg
J
J
D004247:DNA / Q000032:analysis; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005260:Female; D006801:Humans; D008297:Male; D009691:Nucleic Acid Denaturation; D010375:Pedigree; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D016622:Silver Staining
D017931:DNA Primers; D004247:DNA