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Molecular diagnostics

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

University of Debrecen

Chapter 13. 13. The methodology of molecular diagnostic procedures II.

Chapter 13. 13. The methodology of molecular diagnostic procedures II.

Table of Contents

In diagnostics, the molecular situation will be the basis of the chosen methodology. Mutation screening methods are used when the location of the possible mutation is not known in the gene. These methods are based on the PCR and use the physical properties of the DNA. Previously mainly SSCP, single strand conformational polymorphism, DGGE, denaturing gradient gel electrophoresis, and HA, heteroduplex analysis were used.

When an SSCP analysis is performed, first the PCR reaction is carried out. After the completion of the PCR, the sample is heated then quickly cooled. The quick cooling step does not allow the complementer DNA strands to find each other, instead, intramolecular stabilization will occur. The resulting fragments are then separated by non-denaturing electrophoresis and the difference in the conformation that based on the sequence can be detected by the difference in mobility. The theory behind the heteroduplex analysis is very similar, though, in that case the cooling step is slow, which is why the complementer strands are able to find each other. In the course of DGGE, the denaturation is performed during a gradient gel electrophoresis.

Later, the development of the suitable instrumentation has made some automation possible (dHLPC, Figure 13.1.). This method is based on the presence of both possible alleles, wild type allele and mutant allele. The PCR is followed by denaturation and a rehybridization step. In the presence of a mutation, two types of complexes can be formed:

  • - homoduplexes, where the wild type sense strand is hybridized with its wild type antisense strand, and the same occurs with PCR products possessing a mutation,

  • - heteroduplexes, where the wild type sense strand is hybridized with the mutant antisense strand, and the mutant sense strand is hybridized with the wild type antisense strand.

After the formation of the above-mentioned complexes, the next step is running the samples on an HPLC machine under partially denaturing conditions. The mobility of the heteroduplexes and homoduplexes are different, therefore the number of peaks indicate the presence of mutation. This method, similarly to the other mutation screening methods, does not provide information about the nature and location of the mutation, but only shows its presence within the PCR product. Therefore, the samples showing aberrant mobility need to be analyzed further using other methods, like DNA sequencing in order to determine the consequence of the detected alteration (i.e., harmless polymorphism or synonymous mutation, or pathogenic that causes the disease).

Figure 13.1. Figure 13.1. Mutation screening methods: denaturing HPLC (dHPLC)

Figure 13.1. Mutation screening methods: denaturing HPLC (dHPLC)

There are situations, where no hot spot is known in the disease-causing gene, but it is established that the disease is mainly caused by truncating mutations rather than missense changes. In such a case, protein truncation test (PTT) might be help in the establishment of the molecular genetic background of the disease.

PTT can be used for the analysis of mutations which affect the splice site, causing nonsense or frameshift. It is based on in vitro transcription and translation. The starting material of PTT is usually mRNA or a large exon of genomic DNA. mRNA is reverse transcribed into cDNA. Using modified primers, start codon is incorporated into the sample and the in vitro transcription and translation are performed. The resulting protein products are separated using SDS-PAGE and then detected with a specific method (i.e., by using antibodies). The special nature of the method is its advantage and drawback at the same time: it is capable of the analysis of mutations that replace the original stop codon (either by truncating the expressed protein, or by generating longer product by mutation of the original stop codon) only, the most common missense mutations will not alter the length of the expressed protein. Lanes 3, 8, 11 are mutations leading to truncated protein, while lane 5 represents a mutation that has removed the original stop codon. As the in vitro system does not contain the relevant components of nonsense-mediated mRNA decay, PTT will not answer the question whether or not the mutated product will reach its protein level at all. PTT has not become a popular screening method because it is complicated and labor-intensive.

Figure 13.2. Figure 13.2. Evaluation of the pathogenicity of mutations: protein truncation test (PTT)

Figure 13.2. Evaluation of the pathogenicity of mutations: protein truncation test (PTT)