Restriction digest
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A restriction digest is a procedure used in
The resulting digested DNA is very often selectively amplified using
Restriction site
A given
After restriction digest, DNA can then be analysed using agarose gel electrophoresis. In gel electrophoresis, a sample of DNA is first "loaded" onto a slab of agarose gel (literally pipetted into small wells at one end of the slab). The gel is then subjected to an electric field, which draws the negatively charged DNA across it. The molecules travel at different rates (and therefore end up at different distances) depending on their net charge (more highly charged particles travel further), and size (smaller particles travel further). Since none of the four nucleotide bases carry any charge, net charge becomes insignificant and size is the main factor affecting rate of diffusion through the gel. Net charge in DNA is produced by the sugar-phosphate backbone. This is in contrast to proteins, in which there is no "backbone", and net charge is generated by different combinations and numbers of charged amino acids.
Possible uses
Restriction digest is most commonly used as part of the process of the molecular cloning of DNA fragment into a vector (such as a cloning vector or an expression vector). The vector typically contains a multiple cloning site where many restriction site may be found, and a foreign piece of DNA may be inserted into the vector by first cutting the restriction sites in the vector as well the DNA fragment, followed by ligation of the DNA fragment into the vector.
Restriction digests are also necessary for performing any of the following analytical techniques:
- RFLP – Restriction fragment length polymorphism
- AFLP – Amplified fragment length polymorphism
- STRP – Short tandem repeat polymorphism
Various restriction enzymes
There are numerous types of restriction enzymes, each of which will cut DNA differently. Most commonly used restriction enzymes are Type II restriction endonuclease (See article on Restriction enzymes for examples). There are some that cut a three base pair sequence while others can cut four, six, and even eight. Each enzyme has distinct properties that determine how efficiently it can cut and under what conditions. Most manufacturers that produce such enzymes will often provide a specific buffer solution that contains the unique mix of cations and other components that aid the enzyme in cutting as efficiently as possible. Different restriction enzymes may also have different optimal temperatures under which they function.
Note that for efficient digest of DNA, the restriction site should not be located at the very end of a DNA fragment. The restriction enzymes may require a minimum number of base pairs between the restriction site and the end of the DNA for the enzyme to work efficiently.[2] This number may vary between enzymes, but for most commonly used restriction enzymes around 6–10 base pair is sufficient.
See also
- Agarose gel electrophoresis
- DNA sequencing
- Genetic fingerprinting
- PCR
- Restriction fragment length polymorphism
References
- ISBN 0-7637-0913-1
- ^ "Cleavage Close to the End of DNA Fragments". New England Biolabs Inc.
External links
- New England Biolabs – Producer of restriction enzymes. This site contains highly detailed information on numerous enzymes, their optimal temperatures, and recognition sequences.
- REBASE