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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)
 
 
 
 
 

Efficient detection of point mutations on color-coded strands of target DNA.

Presently available methods for screening large genetic regions for unknown point mutations are neither flawless nor particularly efficient. We describe an approach, especially well suited to identifying mutations present in the heterozygous state, that combines several improvements in a protocol called fluorescence-assisted mismatch analysis (FAMA). Appropriate gene regions of the wild-type and the putative mutant allele are simultaneously amplified from genomic DNA by using the polymerase chain reaction, and large DNA fragments, so far up to 800 bp, are end labeled with strand-specific fluorophores. Aliquots are denatured and reannealed to form heteroduplexes and subjected to conventional cytosine- and thymine-specific modifications. Cleavages occurring on opposite strands are detected by denaturing gel electrophoresis using an automated DNA sequencer. Since the DNA fragments derived from the mutant allele are also end labeled, the number of informative mispaired bases is doubled compared to conventional searches using wild-type probes. The sensitivity of detection is also increased, because differential fluorescent end labelling allows the identification and measurement of strand-specific background cleavages at matched cytosine or thymine residues. Automatic superimposition of tracings from different subjects allows mismatch detection at sites that, because of the nature of the bases involved and of the neighboring sequence, are known to be less susceptible to cleavage. The effects of the latter parameters have been studied quantitatively on a series of point mutations found in the human C1-inhibitor gene in patients affected by hereditary angioedema. Dilution experiments have demonstrated that most mutations are detected even when the mutant chromosome is diluted 10-fold or more compared with the normal one.[1]

References

  1. Efficient detection of point mutations on color-coded strands of target DNA. Verpy, E., Biasotto, M., Meo, T., Tosi, M. Proc. Natl. Acad. Sci. U.S.A. (1994) [Pubmed]
 
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