This lecture explains about different types of restriction endonuclease digestion. [ Ссылка ]
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This video talks about the properties of restriction endonuclease and the use of them in recombinant DNA technology.
A restriction enzyme (or restriction endonuclease) is an enzyme that cuts DNA at or near specific recognition nucleotide sequences known as restriction sites.[1][2][3] Restriction enzymes are commonly classified into three types, which differ in their structure and whether they cut their DNA substrate at their recognition site, or if the recognition and cleavage sites are separate from one another. To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone (i.e. each strand) of the DNA double helix.
These enzymes are found in bacteria and archaea and provide a defense mechanism against invading viruses.[4][5] Inside a prokaryote, the restriction enzymes selectively cut up foreign DNA in a process called restriction; while host DNA is protected by a modification enzyme (a methylase) that modifies the prokaryotic DNA and blocks cleavage. Together, these two processes form the restriction modification system.[6]
Over 3000 restriction enzymes have been studied in detail, and more than 600 of these are available commercially.[7] These enzymes are routinely used for DNA modification and manipulation in laboratories, and are a vital tool in molecular cloning.[8][9][10]
They are used to assist insertion of genes into plasmid vectors during gene cloning and protein expression experiments. For optimal use, plasmids that are commonly used for gene cloning are modified to include a short polylinker sequence (called the multiple cloning site, or MCS) rich in restriction enzyme recognition sequences. This allows flexibility when inserting gene fragments into the plasmid vector; restriction sites contained naturally within genes influence the choice of endonuclease for digesting the DNA since it is necessary to avoid restriction of wanted DNA while intentionally cutting the ends of the DNA. To clone a gene fragment into a vector, both plasmid DNA and gene insert are typically cut with the same restriction enzymes, and then glued together with the assistance of an enzyme known as a DNA ligase.[48][49]
Restriction enzymes can also be used to distinguish gene alleles by specifically recognizing single base changes in DNA known as single nucleotide polymorphisms (SNPs).[50][51] This is only possible if a SNP alters the restriction site present in the allele. In this method, the restriction enzyme can be used to genotype a DNA sample without the need for expensive gene sequencing. The sample is first digested with the restriction enzyme to generate DNA fragments, and then the different sized fragments separated by gel electrophoresis. In general, alleles with correct restriction sites will generate two visible bands of DNA on the gel, and those with altered restriction sites will not be cut and will generate only a single band. The number of bands reveals the sample subject's genotype, an example of restriction mapping.[citation needed]
In a similar manner, restriction enzymes are used to digest genomic DNA for gene analysis by Southern blot. This technique allows researchers to identify how many copies (or paralogues) of a gene are present in the genome of one individual, or how many gene mutations (polymorphisms) have occurred within a population. The latter example is called restriction fragment length polymorphism (RFLP).[52] Source of the article published in description is Wikipedia. I am sharing their material. © by original content developers of Wikipedia.
Link- [ Ссылка ] PPT source: The biology of life by Neil A. Campbell, Jane B. Reece, Lawrence G. Mitchell, Copyright Pearson/Benjamin Cummings
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