Unit 4
Unit 4
Unit 4
Sudha Mathpal
DNA Diagnostics
Mutational Analysis
The search for unknown mutations in genomic DNA is important for a broad spectrum of
research studies, including fundamental research on gene structure and function, the study of
genetic diseases and disorders, and species identification. In any living cell, mutations occur
when there is a failure in DNA repair.
Different Types of Mutations
Single-Strand
Strand Conformation Polymorphism
strand conformation polymorphism (SSCP) technique is based on the fact that single
Single-strand single-
sequence specific secondary structure. Sequence differences as small as a
stranded DNA has a sequence-specific
Unit-4 Dr. Sudha Mathpal
single base change can affect this secondary structure and can be detected by electrophoresis in a
nondenaturing polyacrylamide gel. Double-stranded mutant and wild-type samples are first
denatured into single strands and then loaded onto the gel. Differences in mobility of the single
strands between the control wild-type DNA and the other samples indicate a mutation. SSCP is a
widely used mutation screening method because of its simplicity. However, since experimental
conditions cannot be predicted for a particular DNA, it is important to optimize gel
electrophoresis conditions. The ability to detect single base changes rests on several factors
which optimize band resolution.
1. Fragment size — the estimated efficiency for detecting single base changes is 90–95% for
fragments less than 350 bp, but the efficiency will decrease as the length of fragment increases.
2. Gel temperature — migration differences due to a single mutation are observed at buffer
temperatures between 4–25°C. Optimal temperature must be determined empirically.
3. Gel additives — in some cases, 5–10% glycerol can be added to the gel to improve the
mobility differences in fragments. Since glycerol can reduce the mobility of single-stranded
DNA fragments at low temperatures, it is typically used with gels run near room temperature.
4. Crosslinking ratio — the acrylamide/bis ratio determines the percent of crosslinking. SSCP
gels generally use 1–2 % crosslinking. Acrylamide concentrations will vary from 5 to 10%.
5. Buffer concentration — gels are run with TBE buffer at concentrations of 0.5x or 1.0x. In
some cases, 0.5x TBE appears to give slightly better results than 1.0x TBE.
DNA fingerprinting
Satellite DNA regions are stretches of repetitive DNA which do not code for any specific
protein. These non-coding sequences form a major chunk of the DNA profile of humans. They
depict a high level of polymorphism and are the basis of DNA fingerprinting. These genes show
a high level of polymorphism in all kind of tissues as a result of which they prove to be very
Unit
Unit-4 Dr. Sudha Mathpal
useful in forensic studies. Alec Jeffreys developed this technique in which he used satellite
DNAs also called VNTRs (Variable Number of Tandem Repeats) as a probe because it showed
the high level of polymorphism.
Principle of DNA fingerprinting
The human genome consists of innumerable small noncoding sequences which are inheritable
and repeatedly present. They can be separated from the bulk DNA as satellite upon performing
density gradient centrifugation and thus known as satellite DNA. They can be categorized into
depending on the length, base composition and tandemly
either microsatellites or microsatellites depending
repetitive units. These satellite DNAs show polymorphism and this polymorphism is the basis of
DNA fingerprinting. The repeat regions can be divided into two groups based on the size of the
le number tandem repeats (VNTRs) and short tandem repeats. These repeats act as
repeat - variable
genetic markers and every individual inherits these repeats from their parents. Thus, every
individual has a particular composition of VNTRs and this is the main principle of the t DNA
fingerprinting technique.
DNA Fingerprinting Steps:
Isolating the DNA.
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Digesting the DNA with the help of restriction endonuclease enzymes.
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Separating the digested fragments as per the fragment size by the process of electrophoresis.
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Blotting the separated fragments onto synthetic membranes like nylon.
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Hybridising the fragments using labelled VNTR probes.
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Analysing the hybrid fragments using autoradiography.
Unit
Unit-4 Dr. Sudha Mathpal