Page 215 - Concise Pathology for Exam Preparation ( PDFDrive )

200 SECTION I General Pathology

• For the reaction to proceed, target DNA is first denatured by heating and the reaction
mixture then cooled to a certain temperature to allow the primers to anneal to the DNA
target and, lastly, extension by Taq DNA polymerase is brought about.
• Repeating these cycles leads to exponential production of the specific target.

Steps
The repeated cycles of DNA polymerase activity include the following:
1. Denaturation: Heat is typically used to break the hydrogen bonds between comple-
mentary bases of both strands.
2. Annealing: This entails the binding of the primers to the beginning sequence of one
strand and to the end of the other strand.
3. Extension: DNA polymerase and triphosphorylated deoxynucleotides are added to the
reaction including the primers to extend the complementary strand.

Types
1. Simple PCR: This entails the amplification of a single specific sequence by using a pair
of primers complementary to the flanking sequences of the target and is routinely used
for preparing more DNA targets for subsequent analyses.
2. Multiplex PCR: In this technique, a number of different primers are added to the same
reaction mixture to screen for multiple abnormalities and to avoid unnecessary testing.

Q. Write briefly on fluorescence in situ hybridization (FISH).
Ans. This is a method of identifying a chromosome or its parts by the use of specific
probes that bind to specific DNA sequences (which are complementary).
• It is more useful than traditional karyotyping, as cells can be visualized even in interphase.
• It circumvents the need for dividing cells; even those cells that are not dividing or
cannot be induced to divide can be mapped.
• The probes are attached with fluorescent dyes and are visualized under a fluorescent
microscope.

Applications
• Karyotyping of cells/chromosomes in interphase.
• Using specific complementary DNA sequence, one can look for specific regions on
a chromosome.
• Can be used for detection of numerical abnormalities, eg, aneuploidy, microdeletions
and complex translocations that are not readily visualized by karyotyping.
• Mapping/localization of newly isolated genes of clinical importance.
Chromosome painting: It is visualization of the entire chromosome using different fluorescent
dyes.
Spectral karyotyping: Using computer-generated signals the entire human genome can be
‘painted’ and visualized simultaneously.

Q. Write briefly on array comparative genomic hybridization.
Ans. Array comparative genomic hybridization, also called CMA (chromosomal micro-
array analysis) or CGH (microarray-based comparative genomic hybridization), is a
technique to detect genomic variations, at a higher resolution level than chromosome-
based CGH without prior knowledge of what these aberrations may be. DNA from a test
sample and normal reference sample are labelled differentially, using different fluorescent
dyes and hybridized to several thousand probes. The probes are derived from most of the
known genes and noncoding regions of the genome, printed on a glass slide. The ratio of
the fluorescence intensity of the test to that of the reference DNA is then calculated, to
measure the copy number changes for a particular location in the genome.

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