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Molecular diagnostics

Dr. István Balogh, Dr. János Kappelmayer, Dr. József Tőzsér (2011)

University of Debrecen

Chapter 14. 14. Methodology of the molecular diagnostic procedures III

Chapter 14. 14. Methodology of the molecular diagnostic procedures III

Table of Contents

It has been shown before that although unknown mutations can be investigated in some cases, most of the molecular testing is performed in order to find known gene variants, like mutational hot spots, founder mutations, molecular risk factors or mutations/polymorphisms of pharmacogenetic importance. Traditionally, the most widely used method for the detection of point mutations is the PCR followed by restriction digestion (PCR-RFLP, restriction fragment length polymorphism). As the bacterial restriction endonucleases recognize a very specific sequence motif, they are very useful fotr the detection of mutations affecting restriction sites. There are a few hundred such enzyme. During the testing, the last step is an electrophoresis on agarose or acrylamide to separate the fragments according to their size. The number and size distribution of the restriction fragments are the function of the genotype.

Allele-specific PCR method can be used, when the site of the mutation is known, as a mutation detection method. Allele-specific PCR is based on the fact, that Taq polymerase enzyme needs perfectly hybridized primer (on its 3' end) for its function. In the case of 3' incomplementarity, Taq enzyme is unable to use the oligonucleotide as primer. The system contains a common oligonucleotide (right side of Figure 14.1) and contains primers specific to the possible alleles (one is shown on the left-hand side of the picture). Allele-specific PCR reactions are separated physically. With careful optimization, allele-specific amplification is possible. In the upper part of the picture a perfect hybridization is shown resulting in PCR product. In the lower panel, no product is formed due to the 3' end incomplementarity. Using several pairs of primers, multiplexing is possible. The method is capable of showing all possible genotypes, i.e., wild type, heterozygous, homozygous.

Allele-specific PCR has been popular under many names, like ASO-PCR, ARMS. There are commercially available mutation detection diagnostic kits that are based on this principle.

Figure 14.1. Figure 14.1. Allele-specific PCR

Figure 14.1. Allele-specific PCR

Although the allele specific PCR has significant advantages compared to PCR-RFLP as it can be multiplexed, it still requires electrophoretic separation. There have been many efforts to develop a methodology that does not involve electrophoresis during allele discrimination.

Allele specific oligonucleotide hybridization is a general method distinguishing between alleles that differ by even a single nucleotide substitution (Figure 14.2.). The method is based on the difference in the melting temperature between perfectly matched and mismatched probe-template hybrids. In this procedure the allele specific oligonucleotides (ASOs) are immobilized on a nitrocellulose or nylon membrane and hybridized with a labelled PCR product spanning the variant nucleotide site. The discrimination between the two alleles is based on the fact that in a specific hybridization temperature the perfectly matched hybrid is more stable than the mismatched one. After hybridization and washing the detection is mainly colorimetric: for example if the PCR product is labelled with biotin, the detection is based on the streptavidin- alkaline phosphatase conjugate (or horseradish peroxidase) enzyme reaction with a substrate. By using two ASOs, it is possible to determine each combination: wild type, heterozygous, homozygous. The advantage of this method that multiplexing is possible (at least 5-10 different mutations are detectable at the same time) and it does not involve a gel electrophoretic step, though many washing steps are needed and high stringency conditions are required. There are commercially available mutation detection diagnostic kits that are based on this principle.

Figure 14.2. Figure 14.2. Mutation detection using allele-specific oligonucleotide hybridization

Figure 14.2. Mutation detection using allele-specific oligonucleotide hybridization

One of the most inmportant use of the allele specific oligonucleotide hybridization is shown in Figure 14.3.

Mutations in the CFTR gene which cause cystic fibrosis (CF) are very heterogeneous. To date, more than 1600 CF-causing mutations have been described of which approximately 30 are more common. There are mutation testing panels to analyze the most common alterations and the available diagnostic kits include these mutations. The figure shows such an assay. The test system, which is highly multiplexed, is performed using two membane strips. Mutations are represented both with their wild type and mutated alleles. The left-hand side of the picture shows the CFTR19 and right side shows the CFTR17+Tn kit. Using both strips, many different mutations can be tested simultaneously. Sample 1 has been shown to be wild type for the analyzed mutations while sample 2 is homozygous mutant for p.F508del, the far most common CF-causing mutation as judged by the respective signals: on the CFTR19 strip only the M.F508del allele-specific probe provides signal, the W.F508del = W.I507del probe does not. Sample 3 has two mutations. M.G542X could be detected on the CFTR19 strip, and M.R117H gives a signal on the CFTR17+Tn strip. As in both cases the wild type probe also gave a signal, the genotype of the sample is compound heterozygous. Sample 4 was similar to sample 3. The detected alterations were p.S1251N and 394delTT. Using this – or similar – diagnostic approach, 80-95% of the pathogenic mutations causing CF can be detected, depending on the tested population.

Figure 14.3. Figure 14.3. Mutation detection using allele-specific oligonucleotide hybridization for the most common mutations causing cystic fibrosis.

Figure 14.3. Mutation detection using allele-specific oligonucleotide hybridization for the most common mutations causing cystic fibrosis.