Restriction enzymes screening

Mutation screening methods involve testing for specific (previously selected) mutations in a gene. This approach is relatively inexpensive and may be useful for disorders that are caused by one or few common mutations. It is important to take the origin of the patient into consideration, since the frequency of mutations differs markedly between populations. As an example for such a method we discuss restriction enzyme analysis in more detail in this chapter. 

It is recommended to include multiple positive controls (e.g. genomic DNA with heterozygous and homozygous mutations) and a negative control (water instead of template DNA) in each series. A validation of the procedure can be performed by sequencing some of the wild-type and mutant PCR products, as described in 8.2.3.5 Mutation Screening Restriction Enzyme Digest. 

The primary structure of AspAT from Thermophilic bacillus was determined from cDNA sequence.24 Sequence information of an N-terminal portion of the native enzyme and 19 tryptic peptide fragments, recovered from HPLC, was obtained from gas phase sequencer analyses. Based on such sequence information, four oligonucleotide probes were prepared. cDNA encoding AspAT was cloned by screening restriction enzyme fragments from genomic cDNA of Thermophilic bacillus species YM-2. Amino acid sequence of Thermophilic bacillus AspAT deduced from cDNA was confirmed by the sequences made available by gas phase sequencer analysis. 

Based on the screening strategies discussed in Subheading 3.1., targeted colonies can be screened by PCR or Southern blot. Take 15 pL DNA for each restriction enzyme digestion, or, pool 5 samples of DNA to run PCR. The amount of DNA in each PCR reaction should be optimized with other reagents. 

For one patient, a new mutation was suspected but the regular screening methods (IEF and restriction enzyme analysis of most of exons 2, 3, and 4) failed to identify the mutation. ESI and MALDI MS were utilized to characterize the novel variant. 

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