Medical Genetics - V. M. Zaporozhan 2005

Methods for Diagnosing Hereditary Diseases
Molecular Genetic Methods (DNA Diagnostic Methods)
Modification of PCR

There are numerous modifications of the PCR method designed for rapid genome scanning and the detection of known Gene Mutations.

Multiplex (multi-primer) PCR relies on the simultaneous Amplification of several exons of the target gene or multiple fragments of different genes within a single reaction (see Fig. 10.8). This approach enables rapid screening for the most frequent mutations in a gene or the simultaneous detection of mutations across different genes.

Allele-specific amplification is based on The Use of different primer pairs, one complementary to the normal DNA sequence and the other to the mutant sequence. Consequently, this reaction yields two distinct types of PCR products—normal and mutant—which differ in their Structure.

The method of site-directed amplification of modified DNA involves the use of a specific mismatch primer in the PCR, which differs from the template sequence by a single nucleotide. The incorporation of such a primer into the mutant PCR product creates an artificial restriction site for a specific restriction endonuclease, thereby enabling DNA Diagnostics via restriction analysis.

Specialized PCR techniques have been developed to study not only DNA but also RNA. This is crucial, for example, when investigating RNA Viruses (such as Retroviruses, notably HIV, hepatitis C virus, Influenza A, etc.) and analyzing the expression of various genes within an Organism. Such Procedures typically comprise two stages: reverse METABOLISM/31.html">Transcription and subsequent PCR using the resulting cDNA as a template.

Real-time PCR. One of the most promising and rapidly advancing areas in DNA technology is real-time PCR (Real-time PCR). This method eliminates The Need for post-amplification Electrophoresis in agarose or polyacrylamide gels. The presence or absence of amplification products can be detected in two ways: by measuring the fluorescence of the amplification products or by analyzing the thermal Denaturation (melting) curves during the annealing step, which depend on the presence or absence of the target DNA sequence.

The application of real-time PCR significantly saves time and takes advantage of a "closed-tube system," as there is no need to open the reaction tubes after amplification. This prevents the contamination of PCR products and the occurrence of false-positive results. Real-time PCR is particularly promising for the Diagnosis of viral and bacterial infections.