Detect Known Mutations

There is a vast array of techniques that are currently being used to look for mutations for clinical molecular diagnostic use. The following overview of methods describes some of the most common techniques to detect recurrent mutations used in many diagnostic laboratories. As detection of unique mutations is not of relevance to pharmacogenetic applications, this topic will not be discussed in this chapter. 

The basis of the allele-specific oligonucleotide (ASO) assay is that DNA duplexes which contain a mismatch are destabilized and have a lower melting temperature than correctly paired duplexes. To test for mutations using ASO, two probes, one containing the normal sequence and one containing the mutant sequence, are produced and hybridized to the patient s DNA. For each normal and mutant probe, conditions can be found where the probe will hybridize to only its perfectly matched duplex. If the patient sample contains only normal sequence, only the normal probe will hybridize. In a heterozygous sample, both the mutant and normal probes will hybridize, and in a homozygous mutant sample only the mutant probe will hybridize. 

An advantage of the ASO method is that it can be used to simultaneously test samples for several different mutations by the use of multiple probes bound to a solid matrix. In practice, the success of this method relies on precisely establishing conditions for optimal oligonucleotide hybridization in order to ensure specific probe hybridization, and so multiplex ASO assays can be difficult to develop. Molecular diagnostic kits for use in genetic disorders based on ASO methods are available (14). 

There have been improvements to the ASO assay, specifically by development of a multiplex allele-specific diagnostic assay (MASDA) in which the ASO technique is adapted to a solid support and multiple regions are probed simultaneously. This has been achieved by altering probe 

The Allele-Specific Amplification Assay (ASA) assay is based on the fact that Taq polymerase will not initiate amplification from a primer that has a mismatch at the 3 ends. Two primers are designed so that the 3 base of the primer corresponds to the site of the genetic mutation to be tested, with either the normal or the mutant sequence at the 3 base positions. An unknown sample can then be tested for the presence of the mutation by using both the normal and the mutant primers in PCR with a common reverse primer. If the sample contains only normal sequence, a PCR product will only be produced when the normal primer is used, and similarly when the sample contains mutant sequence a product will only result from use of the mutant primer. Like the PCR-restriction enzyme method discussed, the ASA approach has also been applied to the detection of mutations in the CYP2D6 gene (16). 

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At this point, if the results have suggested a specific mitochondrial disorder (e.g., MELAS), then mtDNA isolated from blood cells can be tested for any known mutations associated with the suspected disorder. Unfortunately, many disease-causing mutations are not detectable in mtDNA isolated from blood cells (because of the rapid turnover in these cells, defective mitochondria are often lost). Therefore, inconclusive results may warrant further testing. 

Tian H, Brody LC, Fan S, Huang Z, Landers JP. Capillary and microchip electrophoresis for rapid detection of known mutations by combining allele-specific DNA amplification with heteroduplex analysis. Clin Chem 2001 47 173-185. 

Fig. 4. The detection of mutations is shown for two viruses, HRV 14 and tobacco mosaic virus. The 83 Da mass difference for the HRV14 mutant helps identify the mutation as Cysl99Tyr as the only possible mutation. For the tobacco mosaic virus, a mutation at Asp77Arg is identified similarly by comparison of mass difference with the known sequence for the tryptic fragment...

The dideoxy fingerprinting (ddF) is described by Sarkar et al. (92) to be a hybrid between dideoxy sequencing and SSCP. The method is rapid, large and small regions can be amplified and screened. In the initial publication, Sarkar et al. detected 84 out of 84 known mutations. The frequency of false positivity has been described to be low—in the order of 5% (92,97). The ddF technique has been explored on 73 primary breast cancers (97). Sarkar et al. claimed that this technique detected 100% of the gene mutations, but compared with SSCP, ddF technique requires 50% more effort (97). 

Schaeffeler E, Eichelbaum M, Brink-mann U, Penger A, Asante-Poku S, et al. 2001. Application of DHPLC to detect known and novel mutations in the MDR1 gene in different ethnicities. Pharmacol. Toxicol. 89(Suppl. I) 105 (Abstr.)... 

RFLP is frequently used to detect known point mutations or single nucleotide polymorphisms (SNP).i" It can also be used to separate two amplified sequences that are highly similar in nucleotide composition. Figure... 

There are a number of techniques available to detect point mutations and small deletions and insertions. In practice, the choice of method depends on mutation type, location (a known hot spot versus randomly distributed mutations), required sensitivity, specimen type, and test volume. 

Real-time PCR-based detection methods are also frequently used in clinical molecular laboratories. They may be even more preferable because they are fast and run in a closed PCR system, which reduces the risk of contamination. To detect mutations, two probes complementary to wild-type sequences are designed so that one of the probes spans the known mutation site, such as codons 12 and 13 in the KRAS gene. If no mutation... 

To detect point mutations with multiple hot spots, such as mutations in the TP53 gene, DNA sequencing and SSCP analysis are most commonly used. All exons known to harbor mutations are hrst amplihed in several PCR reactions then they are subjected to DNA sequencing and analyzed for the presence of a mutation. Alternatively, the SSCP analysis can be employed, and only those PCR products that show abnormal migration patterns in the SSCP gel (Fig. 2.10) can be selected for DNA sequencing. 

The fundamental step in the analysis of nucleic acids is the amplification of the target sequence by the polymerase chain reaction (PCR). The amplified DNA is subsequently analyzed by post-PCR steps to detect pathological relevant mutations. If known mutations, like specific codon within an oncogene, have to be detected. 

Thomas, G., et al., Capillary and microelectrophoretic separations of ligase detection reaction products produced from low-abundant point mutations in genomic DNA, Electrophoresis, 25,1668, 2004. BraziU, S.A. and Kuhr, W.G., A single base extension technique for the analysis of known mutations utilizing capillary gel electrophoresis with electrochemical detection. Anal Chem, 74, 3421, 2002. 

This test uses specially constructed bacteria to detect reverse mutations. Each tester strain has enhanced sensitivity and selectivity for the classes of compounds it will pick up. Therefore, several tester strains are generally used to maximize the possibility of detecting a mutagenic compound. It is well known now that many compounds, for example polycyclic aromatic hydrocarbons, must undergo some kind of enzymatic or chemical modification before becoming reactive. Therefore, in many cases an enzyme preparation (commonly called S9) is added to the chemical in order to ensure conversion to a chemically-reactive species. 

Capillary and microchip electrophoresis for rapid detection of known mutations by combining aUele-specific DNA... 

Bottema, C.D.K. and S.S. Sommer, Per Amplification of Specific Alleles - Rapid Detection of Known Mutations and Polymorphisms. Mutation Research, 1993. 2 1) p. 93-102. 

While many diseases have long been known to result from alterations in an individual s DNA, tools for the detection of genetic mutations have only recently become widely available. These techniques rely upon the catalytic efficiency and specificity of enzyme catalysts. For example, the polymerase chain reaction (PCR) relies upon the ability of enzymes to serve as catalytic amplifiers to analyze the DNA present in biologic and forensic samples. In the PCR technique, a thermostable DNA polymerase, directed by appropriate oligonucleotide primers, produces thousands of copies of a sample of DNA that was present initially at levels too low for direct detection. 

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