Molecular biology

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Molecular biology


Molecular techniques now play a central role in the diagnosis and management of blood disorders, particularly haematological malignancies. This is a rapidly changing field and the following is a summary of some of the most commonly used and newest technologies and applications.



Selected techniques used in the analysis of DNA



Polymerase chain reaction (PCR)


The object of PCR is to amplify a preselected sequence of DNA many times over. This amplification greatly facilitates subsequent analysis of the DNA sequence for point mutations and polymorphisms, and often allows direct analysis of the product by gel electrophoresis without the use of probes.


The method can only be briefly described here. Essentially two specific oligonucleotide primers are added to the DNA. These have sequences matching the regions flanking the region of interest. A DNA polymerase is added and the mixture heated, causing the DNA to dissociate into two single strands. Following cooling the single strands bind to the oligonucleotides which are in excess. The oligonucleotide then acts as a primer for DNA polymerase and is extended to form a new double-stranded molecule. With each repeat of the cycle the amount of DNA is doubled. Generally about 30 cycles are used and amplification of approximately 106 can be achieved.



Fluorescence in situ hybridisation (FISH)


FISH describes the hybridisation of specific DNA or RNA sequences in situ to cellular targets attached to microscope slides. The most popular probes are chromosome-specific DNA sequences which generate a brilliant signal in both metaphase and interphase nuclei. The technique is particularly useful in the demonstration of chromosomal monosomies or trisomies but chromosome translocations (Fig 50.1), deletions and amplification of specific genes can also be detected. The results of FISH may be further improved by image processing.




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Jun 12, 2016 | Posted by in HEMATOLOGY | Comments Off on Molecular biology

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