MODULE 13 : Mutation Detection Techniques
Mutation detection techniques
Allele-specific oligonucleotide hybridization
- Also known as Dot Blotting
- a method for testing known mutations.
- DNA products generated by polymerase chain reaction (PCR) are dotted in duplicate
membranes: one membrane is dotted, or hybridized, with an oligonucleotide
corresponding to the wild-type sequence, and the other to a mutant sequence.
- is based on the principle that even single nucleotide mismatches between a probe and its
target can destabilize the hybrid.
o ASO probes can be designed to be complementary and specific for the various
alleles, thus providing a simple methodology to detect any known mutation or
single nucleotide polymorphism (SNP)
- commonly used approach to analyze cloned DNA, using radioactively labeled ASO
probes to hybridize on genomic DNA that has been immobilized on a membrane after
restriction endonuclease digestion and electrophoresis
- used to detect one or more known specific mutations in population
- It makes use of nucleotide probes which are short and specific for particular DNA
sequences.
- a simpler and less expensive approach than searching for any new mutation in family
members..
Allele-specific PCR
- is an application of the polymerase chain reaction (PCR) that permits the direct detection
of any point mutation in human DNA by analyzing the PCR products in an ethidium
bromide-stained agarose or polyacrylamide gel
- Principle: Based on the modification of the primers for different alleles, in which the 3'
end of the primers is modified in such a way that one set of the primer can amplify the
normal allele and others can amplify the mutant allele
- also known as amplification refractory mutation system (ARMS) or PCR Amplification
for Specific Alleles (PASA)
- a PCR-based method used to detect known SNP
- its applications include gene mapping, genetic disease diagnosis, HLA typing, paternity
testing, and forensic science.
Microarray
- is a laboratory method used to detect the expression of thousands of genes at the same
time.
o In this method, DNA molecules attached to each slide and act as probes to
detect gene expression, which is also known as the transcriptome or the set of
messenger RNA (mRNA) transcripts expressed by a group of genes.
- Principle: It states that the complementary sequences will bind to each other. The
unknown DNA molecules are cut into fragments by restriction endonucleases;
fluorescent markers are attached to these DNA fragments
- Analysis is usually done on a blood sample.
o In the laboratory, the DNA (chromosome material) is taken from the cells in the
sample and compared to a normal DNA sample to see if there are any missing
or extra pieces of chromosome material.
- Effective tool in transcriptomics that helps us in studying and analyzing the mRNA
expression of almost every gene present in an organism.
Related links:
[Link] (ASO)
[Link] (ASO)
[Link] (Dot Blot)
[Link] (Dot Blot Technique)
[Link] (Dot Blot)
[Link] (microarray)
[Link] (Microarray)
[Link] (DNA microarray)
[Link] (DNA Microarray Technology)
[Link] (DNA microarray animation)
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