TY - JOUR
T1 - Denaturing gradient gel electrophoresis (DGGE) increases resolution and informativity of Alu-directed inter-repeat PCR
AU - Van Orsouw, Nathalie J.
AU - Li, Daizong
AU - Vijg, Jan
N1 - Funding Information:
We thank Dr Charles Buys and Dr Charis Eng for critically reading the manuscript and CEPH (Centre d’Etude du Polymorphisme Humain, France) for providing us with DNA of pedigrees. This work was supported by a research grant from Toyobo Co. Ltd., Osaka, Japan.
PY - 1997/4
Y1 - 1997/4
N2 - 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.
AB - 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.
KW - CEPH polymorphisms
KW - DGGE
KW - Inter-Alu PCR
KW - Sequence variation
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U2 - 10.1006/mcpr.1996.0089
DO - 10.1006/mcpr.1996.0089
M3 - Article
C2 - 9160323
AN - SCOPUS:0031127272
SN - 0890-8508
VL - 11
SP - 95
EP - 101
JO - Molecular and Cellular Probes
JF - Molecular and Cellular Probes
IS - 2
ER -