Screening Methods For Detection Of Unknown Point Mutations
Back to genes, promoters, DNA, & genetics page
Rapid detection of new mutations and substitutions in large number of samples is quite important for clinical diagnostics, population genetics, and molecular epidemiology. There are three major approaches being used:
Each these methods must be confirmed by DNA sequencing. In general, target sequences are amplified by PCR before analysis. Since polymerases errors are critical, positive results should be confirmed by alternative techniques.
1. Differences in electrophoretic mobilities of wild type and mutant nucleic acids
1) Denaturing Gradient Gel Electrophoresis* (DGGE) and Temperature Gradient Gel Electrophoresis (TGGE). Double-stranded DNA (ds DNA) is electrophoresed through a gradient of increasing concentration of denaturing agent (urea or/and formamide) or of increasing temperature. With increasing concentration of denaturant or temperature, domains in the DNA dissociate according to their melting temperature (Tm). Dissociation of strands results in a decrease in electrophoretic mobility. A 1-bp difference between two ds DNA homoduplexes can change the Tm by 1oC or more. Base mismatches in heteroduplexes lead to a significant destabilization of domains, resulting in differences of Tm between homoduplex and heteroduplex of up to 6 oC. For this reason, heteroduplexes between wild type and mutant fragments are generally used for the analysis of point mutations. Radioactive labeling, ethidium bromide, or silver stain are used for detection.
2) Single-Strand Conformational Polymorphism* (SSCP). Under certain conditions, single-stranded (ss) nucleic acids form secondary structure in solution. The secondary structure depends on the base composition and may be altered by a single nucleotide substitution, causing differences in electrophoretic mobility under nondenaturing conditions. Fragments for detection are labeled radioactively, or silver staining is used.
3) Heteroduplex Analysis (HET). Heteroduplexes are generated by heat denaturation and reannealing of a mixture of wild type and mutant DNA molecules. In nondenaturing polyacrylamide gels, homoduplexes and heteroduplexes exhibit distinct electrophoretic mobilities. They are detected by ethidium bromide or silver staining.
Cleavage of heteroduplexes
1) RNase A Cleavage Method. Under defined conditions, mismatches within RNA:DNA heteroduplexes are cleaved by RNase A. After cleavage, labeled fragments are analyzed by DGGE (Denaturing Gradient Gel Electrophoresis).
2) Chemical Cleavage Method (CCM). Mispaired nucleotides within heteroduplexes are modified by chemical agents by using Maxam-Gilbert sequencing chemistry. Hydroxylamine reacts with mispaired cytosine residues, osmium tetroxide with mispaired thymidine residues. DNA:DNA or DNA:RNA heteroduplexes are cleaved by piperidine at the sites of chemical modification. Radioactively labeled fragments are used. Silver staining or fluorescence labeling are also applied.
3) Enzyme Mismatch Cleavage (EMC). Heteroduplexes generated by heat denaturation of PCR products of polymorphic DNA or wild-type and mutant alleles, respectively, are incubated and cleaved by either the bacteriophage T4 endonuclease YII or T7 endonuclease I. DNA fragments are analyzed by electrophoresis. Radioactively labeled primers are used for PCR. Silver staining should be feasible.
4) Cleavage Fragments Length Polymorphism (CFLP). When single strands of DNA refold after denaturation, sequence- dependent secondary structures consisting of folded, hairpin-like configurations are formed. The cleavase I endonuclease cleaves just 5' of the hairpin loop at the junction between ss and ds DNA, generating a collection of fragments that is unique to that strand of DNA. Changes of the sequence (e.g. point mutations, insertions, & deletions) of that strand will alter the secondary structure formed and the CFLP pattern detected. CFLP patterns are resolved on short , denaturing PAGE . Detection of fragments proceeds through labeling of one of the PCR primers.
Association of mismatch binding proteins with mismatches in heteroduplexes
1) Mutation Detection by Mismatch Binding Proteins. Mutations are detected by binding of the MutS protein, a component of Escherichia coli DNA mismatch repair system (or human hMSH2 and GTBP proteins), to ds DNA molecules containing mismatched bases. Heteroduplexes are generated by heat denaturation and subsequent reannealing of wild type and mutant alleles. DNA duplices are incubated with MutS protein, and mutations are detected by mobility shift assay. A simple assay is based on the fact that MutS binding protects heteroduplex from exonuclease degradation.
2) Protein Translation Test (PTT). The PTT is based on a combination of PCR, transcription and translation. The test selectively detects translation-termination mutations, which are revealed on the protein level by SDS-PAGE. A T7 promoter and an eucaryotic translation initiation sequence is linked to a PCR primer. PCR products are used as templates in coupled transcription-translation reactions in the presence of radioactively labeled amino acids. The size of translation products is analyzed by gel electrophoresis. Stop codons generated by point or frameshift mutations lead to a premature stop of translation and to a reduced size of the translated proteins.
Back to genes, promoters, DNA, & genetics page
Go to: dictionary | cDNAs | antibodies | DNA &
genes | logic
& exptl design |
GPIN home Page