Mutation scanning methods

Gasser, R.B. (1997) Mutation scanning methods for the analysis of parasite genes. InternationalJournal for Parasitobgy 27, 1449-1463. 

The principal factor influencing the sensitivity of this technique to base changes is the combination of the mismatched bases. Greater mobility differences (relative to the homoduplex) occur in the order of G G/C C > A C/T G A G/T C > A A/T T. This and other mutation-scanning methods are useful when a wide variety of sequence alterations might be present. Particularly when most of the samples tested are wild-type ("normal )) it is more economical to scan for the presence of mutations before performing specific genotyping or DNA sequence analysis. 

Highsmith WE Jr, Nataraj AJ, Jin Q, O Connor JM, Ei-Nabi SH, Kusukawa N, Garner MM, Use of DNA toolbox for the characterization of mutation scanning methods. II evaluation of single-strand conformation polymorphism analysis. Electrophoresis 1999 20 1195-203. 

These techniques can be broadly split into two groups, the first of which can be represented by pooling methods, where deconvolution is obtained via various chemical steps, run in parallel or after the library synthesis. Pooling methods normally require multiple synthesis of many library members, including inactive individuals, in different pool formats. They are not single bead methods, so they are independent from analytical methods for structure determination. This group includes iterative deconvolution, recursive deconvolution, subtractive deconvolution, positional scanning and mutational... 

Iterative deconvolution Recursive deconvolution Subtractive deconvolution Positional scanning/indexing Mutational surf other methods... 

We recently developed a systematic method that uses the intrinsic tryptophan residue (Trp or W) as a local optical probe [49, 50]. Using site-directed mutagenesis, tryptophan can be mutated into different positions one at a time to scan protein surfaces. With femtosecond temporal and single-residue spatial resolution, the fluorescence Stokes shift of the local excited Trp can be followed in real time, and thus, the location, dynamics, and functional roles of protein-water interactions can be studied directly. With MD simulations, the solvation by water and protein (residues) is differentiated carefully to determine the hydration dynamics. Here, we focus our own work and review our recent systematic studies on hydration dynamics and protein-water fluctuations in a series of biological systems using the powerful intrinsic tryptophan as a local optical probe, and thus reveal the dynamic role of hydrating water molecules around proteins, which is a longstanding unresolved problem and a topic central to protein science. 

The diagnosis of P-thalassemia minor, with appropriate indices in the CBC, is dependent on the finding of a raised Hb A.2 concentration (>3.5%). Iron deplete individuals should become iron replete before a definitive diagnosis of P-thalassemia is made as the Hb A2 may be falsely low. HPLC is the preferred method for this quantification since densitometric scanning of the Hb A2 band on an alkaline electrophoresis gel is not recommended because of poor precision and accuracy. In 30% to 40% of all cases of P-thaiassemia minor, the Hb F will also be raised (>1.0%). The lifespan of the RBC may be reduced, and diabetics may show a lower Hb Ai compared with normal individuals with equivalent glycemic control. The P-thalassemia mutation may be identified either by Southern blot using mutation specific probes or by gap-PCR. 

Selected Methods Error-prone PCR Mutator strains Chemical mutagens Combinatorial cassette mutagenesis Recursive ensemble mutagenesis Scanning saturation mutagenesis... 

The effect of mutations on Drosophila development. Scanning electron micrographs of the eye from lef a wild-type fly, (middle) a fly carrying a dominant developmental mutation produced by recombinant DNA methods, and (right) a fly carrying a suppresor mutation that partially reverses the effect of the dominant mutation. [Courtesy of llaria Rebay, Whitehead Institute, MIT]... 

ITERATIVE DECONVOLUTION RECURSIVE DECONVOLUTION SUBTRACTIVE DECONVOLUTION POSITIONAL SCANNING/INDEXING MUTATIONAL SURF OTHER METHODS... 

Table 3.4 Methods for scanning a gene for point mutations...

TABLE 15.5 Common methods used to scan genes for mutations... 

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