Introduction

Mutation detection typically refers to the identification of allelic variants of single or low-copy-number gene sequences. It is of tremendous value in the diagnosis and understanding of heritable diseases as well as the understanding of complex biochemical processes having no easily measurable phenotype. Direct sequencing of specific sequence regions is still the most reliable and accurate method of mutation detection, but it is exceptionally expensive and labor intensive as a method of analysis. As indicated in this chapter, PCR has greatly facilitated the development of alternative methods that provide standards of accuracy and convenience that complement direct sequencing methods.

Although there are few current methods, short of direct sequencing and specialized techniques of hybridization, that can directly pinpoint the specific identity of all single nucleotide variations in a limited segment of DNA, some of the recently developed PCR-based mutation detection assays offer highly reliable methods of identifying a single nucleotide variation in a given fragment. As a diagnostic tool, this offers the powerful advantage of allowing one to conveniently prescreen large numbers of unknown samples of which only implicated variants would need to be directly sequenced.

Individual identification through genetic typing represents a broader scope of the concept of mutation detection as a method of differentiating individuals in a closely related population of organisms. Based on the ability to easily identify repeated polymorphisms of high copy number, DNA typing methods allow questions of simple genetic linkage, paternity, evolutionary taxonomy, and population genetics to be conveniently addressed through a simple assay.

Forensic applications of genetic typing have caused a small revolution in the field of genome characterization. The need for accurate, reliable, and efficient methods of individual identification has spurred rapid growth in the identification of polymorphic sequences that are now used in numerous DNA typing methods. The rapid diversification of these techniques also reflects the growing value of being able to type genomic DNA in a wide range of organisms and their subspecies. The discovery of small, abundant, highly polymorphic repetitive sequences is the critical basis of powerful genetic typing methods, and PCR has led to dramatic innovations in this field.

The use of ever smaller polymorphic sequences combined with sensitive PCR-based sequence amplification techniques has significantly reduced the restrictions of sample quality, quantity, and sensitivity that were early obstacles to the rapid development and application of these methods. Collectively, the development of advanced mutation detection and genetic typing methods has dramatically expanded the ease and accuracy of genetic analysis without the need for direct or complete sequence knowledge of the organism being studied.