More discrete sequence alterations rely heavily on the use of the PCR, which allows rapid gene amplification and analysis. Moreover, PCR makes it possible to perform genetic testing and mutational analysis with small amounts of DNA extracted from leukocytes or even from single cells, buccal cells, or hair roots. Screening for point mutations can be performed by numerous methods (Table 62-9); most are based on the recognition of mismatches between nucleic acid duplexes, electrophoretic separation of single- or double-stranded DNA, or sequencing of DNA fragments amplified by PCR. DNA sequencing can be performed directly on PCR products or on. | Chapter 062. Principles of Human Genetics Part 31 More discrete sequence alterations rely heavily on the use of the PCR which allows rapid gene amplification and analysis. Moreover PCR makes it possible to perform genetic testing and mutational analysis with small amounts of DNA extracted from leukocytes or even from single cells buccal cells or hair roots. Screening for point mutations can be performed by numerous methods Table 62-9 most are based on the recognition of mismatches between nucleic acid duplexes electrophoretic separation of single- or double-stranded DNA or sequencing of DNA fragments amplified by PCR. DNA sequencing can be performed directly on PCR products or on fragments cloned into plasmid vectors amplified in bacterial host cells. RT-PCR may be useful to detect absent or reduced levels of mRNA expression due to a mutated allele. Protein truncation tests PTT can be used to detect the broad array of mutations that result in premature termination of a polypeptide during its synthesis. The isolated cDNA is transcribed and translated in vitro and the proteins are analyzed by gel electrophoresis. Comparison of electrophoretic mobility with the wild-type protein allows detection of truncated mutants. The majority of traditional diagnostic methods are gel-based. Novel technologies for the analysis of mutations genotyping large-scale sequencing and mRNA expression profiles are in rapid development. DNA chip technologies allow hybridization of DNA or RNA to hundreds of thousands of probes simultaneously. Microarrays are being used clinically for mutational analysis of several human disease genes as well as for the identification of viral sequence variations. Together with the knowledge gained from the HGP these technologies provide the foundation to expand from a focus on single genes to analyses at the scale of the genome. Faster and cheaper sequencing technologies are under development and it has been anticipated that sequencing the whole genome of an
