Though at low statistical probability, errors may be misinterpreted as mutations when analyses are performed with low numbers of starting templates (200 bp, have been reported for RNA-SSCP ( 13).
Depending on the method of choice, polymerase errors may contribute reasonably to unspecific background, limiting the level of detection, particularly in situations where few mutated alleles are analyzed in a great excess of wild-type alleles (for theoretical considerations see ref. The error rate of Taq polymerase is in the range of 10 −4 to 10 −5 per nucleotide and is strongly affected by the reaction conditions (e.g., concentrations of magnesium chloride and dNTPs, pH, and temperature). At present, Taq polymerase is widely used for amplification. In general, target sequences are amplified by PCR before analysis. Recently, a new principle that depends on the association of mismatch binding proteins with mismatches in heteroduplices has been described. The second group of methods is based on the cleavage of heteroduplices. One set of methods relies on the differences in electrophoretic mobilities of wild-type and mutant nucleic acids. Screening Methodsĭisregarding direct sequencing of PCR products, two different approaches for the detection of unknown point mutations can be distinguished. For detailed information the reader is referred to some review articles ( 1)( 2). We do not intend to present an in-depth review. Special attention will be paid to performance and quality assessment. For this reason, mutations assumed from the results of screening methods must be confirmed by DNA sequencing. Though DNA sequencing techniques will not be covered, we stress that DNA sequencing is considered the gold standard and remains the definitive procedure for the detection of mutations so far. Screening methods for unknown mutations as well as methods for the detection of known mutations are included.
In general, PCR is either used for the generation of DNA fragments, or is part of the detection method. Here, different methods for the detection of point mutations and small deletions or insertions will be discussed on the basis of the above criteria (for simplification, we shall refer to point mutations only in the text, though in general, small deletions or insertions are detected equally well by the methods described). For the appropriate choice of any one of these methods, several criteria must be considered:ġ) What type of nucleic acid is analyzed (DNA or RNA)?Ģ) What kind of specimen is analyzed (e.g., peripheral blood, bone marrow, tissues, secretions, excretions)?ģ) Are the mutations to be detected known before analysis?Ĥ) How large is the number of potential mutations to be detected?ĥ) Need each of the potential mutations be detected?Ħ) What is the ratio between wild-type and mutant alleles?ħ) How reliable is the method to be used, and how far can it be standardized?ĩ) Is the test suited for routine diagnosis?ġ0) What kind of quality assessment can be achieved? Indexing terms: alleles, electrophoresis, gene insertions, gene deletions, polymerase chain reactionĪ variety of methods for the detection of point mutations as well as small deletions or insertions has been described.
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