Types of molecular markers principles methods and uses
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Principle
The restriction enzymes recognize and cut DNA at specific sites called restriction sites. Polymorphism can be detected if there is any variation in any two individuals at the restriction sites of any particular restriction enzyme. Such polymorphism can be visualized as variation in length after cutting the DNA with the enzyme and carrying out Southern blotting.
Method
RFLP analysis involves digestion of DNA by restriction enzyme followed by separation of the fragments using agarose gel electrophoresis and detection by Southern hybridization using a labeled probe.
As discussed before, DNA sequence may be altered in different individuals due to mutations. Even single nucleotide changes can alter the restriction sites (new restriction sites can be created and the older ones can be disrupted). Therefore, the patterns of restriction digestion can be different in different individuals, resulting in detection of polymorphism. Also, large insertions and deletions (also called Indels) can occur which cause length polymorphism.
Figure: An example of pattern of southern hybridization generated after the RFLP process, in case where a single probe is used for DNA of three different plants. First lane shows that the individual is homozygous for the larger size allele. Third lane shows individual homozygous for the smaller allele and the second lane shows the heterozygous condition.
Principle
The restriction enzymes recognize and cut DNA at specific sites called restriction sites. Polymorphism can be detected if there is any variation in any two individuals at the restriction sites of any particular restriction enzyme. Such polymorphism can be visualized as variation in length after cutting the DNA with the enzyme and carrying out Southern blotting.
Method
RFLP analysis involves digestion of DNA by restriction enzyme followed by separation of the fragments using agarose gel electrophoresis and detection by Southern hybridization using a labeled probe.
As discussed before, DNA sequence may be altered in different individuals due to mutations. Even single nucleotide changes can alter the restriction sites (new restriction sites can be created and the older ones can be disrupted). Therefore, the patterns of restriction digestion can be different in different individuals, resulting in detection of polymorphism. Also, large insertions and deletions (also called Indels) can occur which cause length polymorphism.
Figure: An example of pattern of southern hybridization generated after the RFLP process, in case where a single probe is used for DNA of three different plants. First lane shows that the individual is homozygous for the larger size allele. Third lane shows individual homozygous for the smaller allele and the second lane shows the heterozygous condition.
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