Identification of polymorphisms by genomic denaturing gradient gel electrophoresis: application to the proximal region of human chromosome 21

Abstract

Genomic Denaturing Gradient Gel Electrophoresis (gDGGE) provides an alternative to the standard method of restriction fragment length polymorphism (RFLP) analysis for identifying polymorphic sequence variation in genomic DNA. For gDGGE, genomic DNA is cleaved by restriction enzymes, separated in a polyacrylamide gel containing a gradient of DNA denaturants, and then transferred by electroblotting to nylon membranes. Unlike other applications of DGGE, gDGGE is not limited by the size of the probe and does not require probe sequence information. gDGGE can be used in conjunction with any unique DNA probe. Here we use gDGGE with probes from the proximal region of the long arm of human chromosome 21 to identify polymorphic DNA sequence variation in this segment of the chromosome. Our screening panel consisted of DNA from nine individuals, which was cleaved with five restriction enzymes and submitted to electrophoresis in two denaturing gradient conditions. We detected at least one potential polymorphism for nine of eleven probes that were tested. Two polymorphisms, one at D21S4 and one at D21S90, were characterized in detail. Our study demonstrates that gDGGE is a fast and efficient method for identifying polymorphisms that are useful for genetic linkage analysis.

Detection of the common alpha‐1‐antitrypsin variants by denaturing gradient gel electrophoresis

“…DGGE also requires the synthesis of long GC-clamped primers and the purchase of some dedicated equipment. Recent developments of DGGE, however, have widened its applicability for the identification of DNA polymorphisms in amplified and unamplified genomic DNA (Abrams et al 1990;Sheffield et al 1990;Burmeister et al 1991). Constant denaturing gel electrophoresis (CDGE) may provide an opportunity for even better resolution of alleles within a single melting region and the analysis of higher melting domains (Hovig et al 1991).…”

Technologies for Detection of DNA Damage and Mutations

“…Moreover, since gDGGE is not PCR-based, heteroduplex formation is not feasible and one has to rely entirely on the resolution of two doublestranded DNA molecules differing by one base change. Nevertheless, gDGGE has been successfully applied for the identification of polymorphic sequence variations in human chromosome 21 (Burmeister et al, 1991), and to screen for mutations in Drosophila (Doerig et al, 1988;Curtis et aI., 1989;Gray et aI., 1991). have developed a modified protocol to generate heteroduplex molecules between a GC-clamped radiolabeled DNA probe and genomic DNA restriction fragments which are then analyzed by DGGE.…”

Mutation detection by denaturing gradient gel electrophoresis (DGGE)

“…In genomic DGGE (gDGGE) genomic DNA is digested with a restriction enzyme, electrophoresed through a denaturing gradient gel, transferred to nylon filters, and hybridized to a unique DNA probe (Borresen et al, 1988). Clear advantages of gDGGE over its parental protocol are that (1) it is not limited to any specific target sequence nor to its length (any available unique probe of any length can be used), (2) it does not require sequence information, and (3) of polymorphic sequence variations in human chromosome 21 (Burmeister et al, 1991), and to screen for mutations in Drosophila (Doerig et al, 1988;Curtis et al, 1989;Gray et al, 1991).…”