Nature of the mismatch

The presence of a mismatch, e.g., a C>T within a double-stranded fragment, will generate four species: a wild-type homoduplex (composed of C:G), a mutant homoduplex (T:A), and two heteroduplexes, one containing a C:A mismatch and the other a T:G. Highsmith et al.[5] designed a DNA ''toolbox,'' a set of molecules of varying length and GC content containing all possible mismatches at a particular location, to determine which factor had the greatest effect on the ability to detect a single-base substitution. They reported that the factor with the greatest effect was the nature of the nucleotide change; the order of detection being G:G/C:C> A:C/T:G> A:A/T:T. They concluded that this hierarchy of resolution was in agreement with the measured thermodynamic stability of oligonu-cleotides with different base-pair mismatches.[20-22] It was presumed that the larger the distortion due to several unstacked bases, the greater the differential migration in the gel compared with fully complementary strands.

In an analysis of all mutations recorded in the Human Gene Mutation Database (HGMD), Krawczak et al.[23] reported that 60% were transitions. One reason for the predilection toward transitions in any set of alterations is the relatively high incidence of CG>TG and CG>CA substitutions caused by deamination of methylcytosine.

Methylated cytosines most commonly occur in CpG dinucleotides and are susceptible to deamination favoring the formation of thymine (CG>TG). CG>CA substitutions occur if, following deamination of a methyl-cytosine in the antisense strand, there is a miscorrection of G>A in the sense strand. In an update to the study of Hoskins et al.[16] (unpublished) these types of mutation accounted for 21% of substitutions and 33% of transitions consistent with Krawczak et al.'s findings. The majority of the remaining transitions were A>G substitutions, with five of seven adenines preceded by another adenine. The local DNA sequence environment may therefore influence the sensitivity of techniques such as MCHA.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

Get My Free Ebook


Post a comment