Dr. István Balogh, Dr. János Kappelmayer, Dr. József Tőzsér (2011)
University of Debrecen
The large and diverse field of molecular testing includes the previously described monogenic and multifactorial diseases, the pharmacogenetic testing, but the methodology is the same in molecular oncology (pathology) and in molecular microbiology as well.
In many genetic diseases, the clinical diagnosis supported by some laboratory diagnostic test might make the direction of the further testing obvious (Figure 12.1.). If the gene is unknown, but its physical sites are known, linkage analysis can be performed using intragenic markers or markers close to the tested gene. In these cases, analysis of the members of the family for some generations is unavoidable. In the absence of such samples, the storage of the sample (biobanking) is the only solution until the genetic background of the given disease is clarified. In the case of a certain clinical diagnosis or at least suspicion (II) knowing the gene helps to a great extent the diagnostic efforts and the establishment of the genotype/phenotype associations. In those cases, the goal is to define the molecular background of the disease. If the mutations are scattered throughout the entire gene (absence of mutational hot spot), mutation screening methods can be utilized. These methods are SSCP, heteroduplex analysis, denaturating HPLC, high-resolution melting analysis. If the mutation affects large genetic structures, the use of citogenetic methods are necessary. If the mutation site is known (mutation hot spot), mutation detection methods (allele specific PCR, allele specific oligonucleotide hybridization, PCR-restriction digestion, hybridization or hydrolysis probes, DNA sequencing) can be used. Those methods are usually used in the case of mutation testing in common multifactorial diseases when diagnostic or predictive testing is performed.
The most frequently used material of the molecular genetic diagnostic procedures is the genomic DNA. In the past, DNA isolation was based on either phenol-chloroform extraction or salting-out procedures. These methods, however, are time-consuming and involve the use toxic materials, therefore, they are not ideal for a diagnostic laboratory.
The next generation of DNS isolation, the spin column purification method is based on the phenomenon that DNA/RNA binds selectively to silica in high salt concentration. Cell lysates (usually originated from EDTA- or citrate anticoagulated blood) are loaded to spin columns containing silica membrane followed by several washing steps with different buffers, and the pure DNA/RNA is eluted with low salt concentration buffer or sterile molecular biology grade water. This method is the fastest available, but it is expensive. It involves many centrifugation steps and the yield of purified DNA/RNA is relatively little. The isolated DNA can be used further if its concentration and purity are adequate. DNA yield is determined by measuring the concentration of DNA by absorbance at 260 nm (at this wavelength one OD unit is equivalent to 50 micrograms DNA). The peak absorbance of DNA is at 260 nm and that of proteins is at 280 nm, thus purity is determined by calculating the ratio of absorbance at 260 nm to absorbance at 280 nm (Abs260/280). Pure DNA has an Abs260/280 ratio of 1.7-1.9. Ratio less than 1.7 indicates protein contamination which might interfere with the downstream applications. DNA can be stored at +4°C for months, at -20°C for years, and at -70°C for at least ten years without significant degradation.
Since its development in 1985, the PCR has revolutionized the possibilities of detecting minor changes in DNA (Kary Mullis, the developer of the method, received the Nobel prize in 1993). PCR is an in vitro technique to amplify a well-defined DNA fragment. PCR uses the physical characteristics of DNA (i.e. ability for reversible de- and renaturation), and the heat stability of a special DNA polymerase enzyme (Taq polymerase). The reaction consists of cycles. Each cycle starts with denaturation at a high temperature (95 °C) to separate the strands of double-stranded DNA template. The second step is fast cooling to 50-65°C to hybridize the specific oligonucleotide probes to the template and followed by an incubation at 72°C to synthesize the new strands.
The components of standard PCR reaction:
Oligonucleotide primers. The primers determine the starting point of DNA synthesis (because the polymerase enzyme is able to initiate DNA synthesis only from a primer) and define the piece of DNA which will be amplified. It means that one of the primers will be hybridized to the sense and the other to the antisense strand. The orientation of DNA synthesis is 5'-3', therefore only the fragment between the two primers will be amplified.
Thermostable DNA polymerase enzyme. Mainly DNA polymerase in the Thermus aquaticus bacteria (living in thermal springs) (Taq polymerase) in native or recombinant form.
dNTPs. Deoxy-nucleotides in triphosphate form (dATP, dCTP, dGTP, dTTP).
Buffer (provides the necessary ionic strength and concentrations. It contains MgCl2 which is a cofactor of the Taq enzyme).
Several different types of PCR are used in genetic diagnosis:
Standard PCR: this method is very useful in detecting small variations in genetic material (point mutations, short deletions and insertions), e.g., for the detection of factor V Leiden mutation, prothrombin 20210A allele, haemochromatosis.
Multiplex PCR: this method involves paralel amplification of a different piece of gene (i.e. exons) followed by agarose gel electrophoresis. This method is very useful in certain types of genetic diseases, for example Duchenne muscular dystrophy, where the disease is caused mainly (in about 70% of the cases) by deletions of exons.
Long PCR: this method combines two enzymes: Taq polymerase and a special enzyme which has a proofreading activity to remove the incorporated nucleotides in a wrong place. With this method it is possible to amplify larger DNA fragments (maximum 40 kb). Long PCR is a useful tool, for example, in the molecular diagnosis in Friedreich ataxia.
Quantitative real-time PCR: this method is used to quantitate the copy number of a specific DNA/cDNA in the sample. In routine diagnostic procedures, the fluorescent detection is the most widely used. During the PCR amplification, the fluorescence intensity (originated from a dye intercalating to the double stranded DNA or from a dye-labelled hybridization probe specific to the amplifiable DNA) is measured in every cycle. After the completion of the PCR, the software identifies the cycle in which the PCR product fluorescence signal has just exceeded the background (crossing point, CT). If standards with known copy number of the analyzed DNA are used, the standard curve will provide information about the copy number of the sample. This method is used in the testing of the translocation t(9;22) and the measurement of the bcr/abl transcript number in the sample.