Detection of human DNA polymorphisms with a simplified denaturing gradient gel electrophoresis technique.

Abstract

Single base pair differences between otherwise identical DNA molecules can result in altered melting behavior detectable by denaturing gradient gel electrophoresis. We have developed a simplified procedure for using denaturing gradient gel electrophoresis to detect base pair changes in genomic DNA. Genomic DNA is digested with restriction enzymes and hybridized in solution to labeled single-stranded probe DNA. The excess probe is then hybridized to complementary phage M13 template DNA, and the reaction mixture is electrophoresed on a denaturing gradient gel. Only the genomic DNA-probe hybrids migrate into the gel. Differences in hybrid mobility on the gel indicate base pair changes in the genomic DNA. We have used this technique to identify two polymorphic sites within a 1.2-kilobase region of human chromosome 20. This approach should greatly facilitate the identification of DNA polymorphisms useful for gene linkage studies and the diagnosis of genetic diseases.The ability to identify DNA sequence heterogeneity by restriction endonuclease analysis has revolutionized gene linkage studies and genetic disease diagnosis. However, one significant limitation of this technique is that sequence differences must be present at restriction enzyme recognition sites in order to be identified in most cases. Differences occurring outside the recognition sites cannot be detected, and even large panels of restriction enzymes recognize only a fraction of the sequence heterogeneity that is present.In contrast, denaturing gradient gel electrophoresis (DGGE) allows detection of any single-base change that occurs within the long stretches of DNA that form earlymelting domains (see ref. 1 for a review of DGGE). An analysis of predicted melting domain structure for a 35-kilobase (kb) human DNA segment suggests that up to 50-70% of all base pair substitutions should be detectable (2). In the DGGE procedure, DNA molecules are held at the temperature of incipient denaturation and electrophoresed through a polyacrylamide gel containing a gradient of increasing concentrations of urea and formamide. DNA fragments moving through the gel eventually encounter a concentration of denaturants where the early melting domain becomes unstable and abruptly melts, producing molecules that are partially double-stranded and partially disordered. These partially melted molecules migrate more slowly, if at all, through the gel. The position in the gradient at which migration becomes altered is unique and characteristic for each individual fragment. Remarkably, molecules that differ by only a single base pair in the early-melting domain can be separated on these gels because of the alteration in domain melting temperature.Recently Myers et al. reported that DGGE could be used to examine known single-base mutations in the globin genes of patients with /3-thalassemia (3). Here we show that DGGE can be used to detect previously unidentified genomic DNA polymorphisms. We also describe a simple technique for reducing probe background that improves the qu...

Angiotensin I-converting enzyme (ACE): estimation of DNA haplotypes in unrelated individuals using denaturing gradient gel blots

“…DGGE has been used previously for determining DNA sequence differences in plasmid DNA or DNA fragments amplified by PCR (PCR DGGE; Fischer and Lerman 1983;Myers et al 1985;Noll and Collins 1987;Sheffield et al 1989;Higuchi et al 1991). More recently, the DGGE method has been modified (DGGE blot technique) to detect DNA polymorphisms in genomic DNA (Gray et al 1991;Gray 1992;Krolewski et al 1992b).…”

Rationally Engineered Nucleic Acid Architectures for Biosensing Applications

“…Noll and Collins utilized denaturing gradient gel electrophoresis (DGGE) to identify DNA polymorphisms and to discriminate between two DNA molecules that differed by a single-base substitution. 507 However, this method could only detect ∼50% of all possible single-base changes for DNA fragment sizes of 50 to 1000 base pairs. Sheffield et al improved this method by using PCR to attach a 40-bp GC-rich sequence (GC-clamp) to genomic DNA fragments, achieving improved detection of single-base changes.…”

“…Gel electrophoresis can also be applied for detection of single nucleotide differences. Noll and Collins utilized denaturing gradient gel electrophoresis (DGGE) to identify DNA polymorphisms and to discriminate between two DNA molecules that differed by a single-base substitution . However, this method could only detect ∼50% of all possible single-base changes for DNA fragment sizes of 50 to 1000 base pairs.…”

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“…Although popular in medical research (e.g. Myers & Maniatis 1986;Noll & Collins 1987;Collins et al 1989), DGGE has not been widely used in population genetics studies (Norman et al 1994).…”