Heteroduplex analysis

Heteroduplex is double-stranded DNA in which the two DNA strands do not show perfect base complementarity. When DNA is denatured, the two strands are separated. On renaturation or annealing, complementary DNA strands reassociate and form a homoduplex. However, when there is a mutation in one of the strands then a heteroduplex is formed. The electrophoretic mobility of heteroduplex in polyacrylamide gel is less than that of homoduplex, and they can be detected as an extra slow moving bands. Resolution is better in specially formulated gel matrices (Hydro-link, MDE, etc.). In fragments of under 200 bp insertions, deletions and most single base substitutions are detected. Heteroduplex analysis is mostly used combined with single confirmation analysis (SSCA) on a single gel (Fig.1). Several mutations in a gene can also be combined to form a multiplex heteroduplex analysis. Such a method has been successfully been reported in early-onset breast and /or ovarian cancer¹, Duchenne muscular dystrophy² and neurofibromatosis type 1 (NF1)³ and adenomatous polyposis coli (FAP)⁴.

This concept of heteroduplex formation also forms the basis of mutation screening when the normal (probe) and patient's DNA is mixed, denatured and annealed. Most, but not all of these methods, rely on detecting mismatched bases formed when complementary strands of a mutation and the wild type allele are allowed to hybridize to form a heteroduplex. This is likely to occur naturally when PCR products from a heterozygous person is denatured then cooled to allow single mutant strands to base -pair with complementary strands from the wild type allele. There are many ways to identify the mismatches e.g. Chemical Cleavage Mismatch Detection (CCMD), Enzymatic Cleavage of Mismatches (ECM), Degrading Gradient Gel Electrophoresis (DGGE), Protein Truncation Test (PTT).

REFERENCES:

1.Gayther, S. et al. Rapid detection of regionally clustered germ-line BRCA1 mutations by multiplex heteroduplex analysis. (1996) Am. J. Hum. Genet. 58: 451-456

2. Prior, TW. et al. Heteroduplex analysis of the dystrophin gene: application to point mutation and carrier detection. (1994) Am. J. Med. Genet. 50: 68-73.

3. Abernathy, CR. et al. NF1 mutation analysis using a combined heteroduplex /SSCP approach. (1997) Human Mut. 9: 548-554.

4. Cama, A. et al. Multiplex PCR analysis and genotype-phenotype correlations of frequent APC mutations. (1995) Human Mut. 5: 144-152.

Prepared by: M. S. Enayat October 1997