Considerations in the application of selected molecular biology techniques in the clinical laboratory: preanalytical...

As a prerequisite to the utilization of molecular biology techniques in the clinical laboratory one must be aware of preanalytical pitfalls associated with these techniques. Procedures for the identification of restriction fragment length polymorphism (RFLP) resulting from variation in the number of tandem repeats (VNTR) of a short DNA segment, or even simple tandem repeats (STRs) such as dinucleotide repeats require scrutiny to understand both the strengths and limitations of these techniques. In terms of minimizing analytical pitfalls particular attention should be given to the composition of DNA probes. Many variables affect PCR (polymerase chain reaction). These include cycling temperatures chosen for denaturation of synthesized DNA strands, annealing and primer extension steps, the type of thermocyclers used to achieve temperature cycling, requirements for primers, to name just a few. Particular attention should be given for trouble shooting PCR products. A multiplicity of kits are currently available for the isolation of DNA and RNA. These kits while simplifying the isolation procedure could in some instances introduce a preanalytical variable depending on the type of detergent used in cell lysis which may impact on the amplification of DNA by techniques such as the polymerase chain reaction (PCR). The anticoagulants used for blood collection could also affect digestion with restriction enzymes and amplification reactions, such as been reported with heparin under certain conditions. Residual red blood cells can inhibit taq polymerase enzyme used in PCR amplification. The type of tissue fixative and the duration of fixation can affect the efficiency of amplification reactions. Finally, contamination from the working environment should be controlled to minimize both preanalytical and postanalytical error.