Restriction digest

A restriction digest is a procedure used in molecular biology to prepare DNA for analysis or other processing. It is also known as DNA fragmentation. It uses a number of restriction enzymes to selectively cleave strands of DNA into shorter fragments, or to isolate short fragments of interest. The resulting digested DNA is very often selectively amplified using PCR, making it more suitable for analytical techniques such as agarose gel electrophoresis, and chromatography. It is used in genetic fingerprinting, and RFLP analysis.

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A given restriction enzyme cuts DNA segments within a specific nucleotide sequence. These recognition sequences are typically four, six, eight, ten, or twelve nucleotides long. Because there are only so many ways to arrange the four nucleotides which compose DNA (Adenine, Thymine, Guanine and Cytosine) into a four- to twelve-nucleotide sequence, recognition sequences tend to occur by chance in any long sequence. Restriction enzymes specific to hundreds of distinct sequences have been identified and synthesized for sale to laboratories, and as a result, several potential "restriction sites" appear in almost any gene or locus of interest on any chromosome. Furthermore, almost all artificial plasmids include an (often entirely synthetic) polylinker (also called "multiple cloning site") that contains dozens of restriction enzyme recognition sequences within a very short segment of DNA. This allows the insertion of almost any specific fragment of DNA into plasmid vectors, which can be efficiently "cloned" by insertion into replicating bacterial cells.

After restriction digest, DNA can then be analysed using gel electrophoresis. In gel electrophoresis, a sample of DNA is first "loaded" onto a slab of agarose gel (litterally pipetted into small wells at one end of the slab). The gel is then subjected to an electric current, which draws the negatively charged DNA across it. In gel electrophoresis, molecules travel at different rates (and therefore 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 digests are necessary for performing any of the following analytical techniques:

• RFLP - Restriction Fragment Length Polymorphism
• AFLP - Amplified Fragment Length Polymorphism
• RAPD - Randomly Amplified Polymorphic DNA
• STRP - Simple Tandem Repeat Polymorphism

Various Restriction Enzymes

There are numerous types of restriction enzymes, each of which will cut DNA differently. (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 produces 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 also have different optimal temperatures under which they function.

See also

• DNA sequencing
• Genetic fingerprinting
• PCR
• Restriction fragment length polymorphism
• Agarose gel electrophoresis