Techniques Of Restriction Mapping
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A description of restriction endonuclease cleavage sites within a piece of DNA is referred to as a restriction map. Such a map is usually generated as the first step in characterizing an unknown DNA, and a prerequisite to manipulating it for other purposes. Restriction enzymes that cleave DNA infrequently (e.g. those with 6 bp recognition sites) are relatively inexpensive are used to produce at a map (Chakraborty, Pandey, et.al., 2006). Restriction sites are specific recognition sites where enzymes known as endonucleases cleave the DNA. e.g. EcoRI cuts at GAATTC (Gale, 2003). When discovered in archaea and bacteria, these enzymes were part of the defense mechanism of such organisms, limiting the foreign DNAs to act upon the cell. Theses enzymes will defend cells by digesting invading DNA into small, non-functional pieces. Thus this is where the name "restriction enzyme" comes from; the function of the enzyme, i.e. the ability of the enzyme to restricting access to the cell (Carroll, Griffiths, et.al., 2008). Restriction maps show the relative location of a selection of restriction sites along linear or circular DNA.
Restriction mapping involves a series of restriction enzymes digesting the DNA and then separating the resultant fragments by agarose gel electrophoresis. The patterns of fragments that are produced by restriction enzyme digestion determine the distance between restriction enzyme sites; this is how information about the structure of an unknown piece of DNA can be obtained (Champness & Snyder, 2007).
Techniques of Restriction Mapping.
There are several methods for restriction mapping; the most straightforward being the digesting of samples of the plasmid with a set of individual and pairs of those enzymes; these digests are then "run out" on an agarose gel to determine sizes of the fragments generated.
Consider to illustrate these ideas, a plasmid that contains a 3000 base pair (bp) fragment of unknown DNA. Immediately flanking the unknown DNA within the vector are unique recognition sites for the enzymes Kpn I and BamH I. Then, consider digestions with Kpn I and BamH I separately. In essence, single digests are used to determine which fragments are in the unknown DNA, and double digests to order and orient the fragments correctly (Chakraborty, Pandey, et.al., 2006).
If a DNA fragment is labeled with a radioisotope on one end only, this can directly reveal where the cleavage sites are located as by partially digesting the fragment with restriction enzymes, labeled fragments are generated (Chakraborty, Pandey, et.al., 2006).
If the sequence is known, any number of computer programmes for example ââ‚¬Å“Mapperââ‚¬ can be used to build up a map. It is simply a matter of feeding the sequence into the programme which will then search the sequence for dozens of restriction enzyme recognition sites and build you a map (Chakraborty, Pandey, et.al., 2006).
Uses and Applications of Restriction Mapping:
Restriction map information is important for many techniques used to manipulate DNA; one application being the cutting large pieces of DNA into smaller fragments for allowing it to be sequenced. Another application is to use restriction mapping to compare DNA fragments without having any information of their nucleotide sequence (Gale, 2003).
Restriction mapping has contributed immensely towards our knowledge of vectors and plasmids (OUP, 1995). It has also contributed heavily to our ability to genetically engineer organisms and recombinant DNA technology where an organism's genes are manipulated indirectly; examples of this include the generation of synthetic human insulin using modified bacteria and the production of erythropoietin in hamster ovary cells, amongst many more (Banting, 1929).
Industries like medicine, agriculture etc. also use this technique for the production of several medically useful substances like the hepatitis-B vaccine, human interferon and human growth hormone. Identifying the sequences with restriction mapping has allowed for plants to produce their own pesticides ant to perform nitrogen fixation by genetically adjusting the plant species. Bacteria capable of biodegrading oil have been produced using this technique for the use in oil-spill cleanups. The technique of restriction mapping has its applications in the field of gene knock out experiments in mice as well as identification of gene before introduction into a foreign organism to make transgenic pigs and cats. Similarly, we have been able to express several medicinal proteins in bacterial systems using restriction techniques; the most famous examples are insulin(Banting 1929).
Is restriction mapping still useful?
The process of restriction mapping is simple and easy. It can be carried out in 1-2 days. The advancements in the field of computing have enabled automated softwares to virtually analyze the sequence by identifying the restriction sites. Restriction mapping is a helpful tool for experiments where sequencing can be out of budget or not necessary. It can be used to determine whether a gene has been cloned into the plasmid. It is a much better technique for relatively short segments of DNA.
Technologies That Succeeded Restriction Mapping.
One major method that has replaced restriction mapping is the High-Throughput Sequencing and Genotyping, which is to facilitate the unraveling of genetic information across the large and diverse collection of animals, plants and microbes. This has been very useful in cases where DNA does not contain any known restriction sites, or DNA, which contains sites for enzymes, which are not commercially available. It is also advisable to send the sample for sequencing when the sample is very small (Mitchelson, 2007).
Restriction fragment length polymorphisms (RFLP) are variations in DNA fragment-banding patterns (from different individuals of a species ) of electrophoresed restriction digests of DNA (Appa Rao, Mohan, et.al., 1994).
Random amplification of polymorphic DNA (RAPD) is a molecular marker technique using PCR with arbitrary primers for amplifying anonymous stretches of DNA (Chang & Meyerowitz, 1991).
Southern blotting is a method of detection of specific DNA sequences in DNA samples. A southern blot combines the transfer of electrophoresis-separated DNA fragments to filter membranes and subsequent fragment detection by probe hybridization (Bignon, Roux-Dosseto, et.al., 1990).
With regards to in vitro enzymatic amplification of DNA, the polymerase chain reaction (PCR) has developed into one of the most promising methods allowing widespread applications in DNA cloning, sequencing and mutagenesis related studies (Appa Rao, Mohan, et.al., 1994).
Restriction mapping is a technology to identify the unknown genes without sequencing. It has enabled to industry of biotechnology to its riches now. Though there are many techniques that replaced restriction mapping, it is still in use for low-cost academic exercises and other experiments. It has contributed heavily to our knowledge of DNA manipulation studies. It is an essential tool in comparing DNA fragments together. Many of the technologies like, RFLP, RAPD, PCR, HTGS have replaced restriction mapping nowadays though it would be immature to underestimate the value of restriction mapping.