Page 214 - Concise Pathology for Exam Preparation ( PDFDrive )

8 Genetic and Paediatric Disorders 199

Northern Hybridization (Blotting)

• This is a sensitive and quantitative method for mRNA detection.
• RNA is denatured with a variety of reagents. To obtain a high-quality RNA yield, it is
important to avoid contamination and to inhibit RNAases during processing of tissue.
• Although RNA is single stranded, denaturation is required for effective separation on
agarose gel.
• Denaturation is typically performed by using formamide with subsequent separation by
electrophoresis on a formaldehyde gel.
• Separated RNA on the gel will be transferred to filter paper by capillary action and
hybridized in a similar way, as Southern method, to a complementary target molecule.
• These are hybridized with radiolabelled probes.

Dot Blot Hybridization

This is a useful technique for quantitative measurement of target DNA sequences where
the size of the target is known or is unnecessary. A membrane blotted with known DNA
sequences will be hybridized with a test sample for the detection of a specific sequence.
Western Blotting

In this technique, electrophoretically separated components are transferred from a gel onto
a solid support and probed with antibodies specific to certain epitopes on target protein.

In situ Hybridization

• The technique is ideal for visualization and localization of specific nucleic acid
sequences in cells. A tissue sample or cell preparation mounted on a slide can be used
as a target for probe hybridization.
• In situ DNA: The target for this technique is nuclear DNA and the probe sequences
include centromeric, whole chromosome and specific gene.
• In situ mRNA: The target in this method is the mRNA transcript in the cytoplasm. The
probes are manufactured in vitro by inserting a cloned DNA fragment of interest into a
vector near a regulatory sequence (promoters) that direct RNA polymerase to transcribe
sequence to RNA.
• A sense or antisense RNA probe can be generated depending on the orientation of
inserted fragment. Each affected individual has an affected parent unless the condition
has arisen from a new mutation in the germ cells forming that individual.
• The applications include gene localization and determination of translocation, ploidy
and gene amplification.

Q. Write briefly on polymerase chain reaction (PCR).
Ans. PCR is an in vitro technique of nucleic acid synthesis that allows for rapid, sensitive
and specific replication of nucleic acid for the detection and isolation of a targeted
sequence.
• The method involves exponential amplification of DNA, and is based on the function of
DNA polymerase enzyme to create a new complementary DNA strand from a template
strand.
• If RNA is used as a substrate, it is first reverse transcribed to obtain cDNA and then
amplified by PCR.
• The technique requires a pair of oligonucleotide primers that complement the opposite
ends of each strand of a target sequence and are aligned for DNA synthesis to proceed
only in the region between the primers.
• A reaction mixture for PCR amplifications typically contains a DNA sample, a pair
of primers, thermostable Taq DNA polymerase enzyme and 4-deoxynucleotide
triphosphates (dNTPs) in a buffered solution.

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