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Restriction sites can be used for multiple applications in molecular biology such as identifying restriction fragment length polymorphisms . Restriction sites are also important consideration to be aware of when designing plasmids .
A pUC19 cloning vector showing the multiple cloning site sequence with restriction enzyme sites. A multiple cloning site (MCS), also called a polylinker, is a short segment of DNA which contains many (up to ~20) restriction sites—a standard feature of engineered plasmids. [1]
A restriction map is a map of known restriction sites within a sequence of DNA.Restriction mapping requires the use of restriction enzymes.In molecular biology, restriction maps are used as a reference to engineer plasmids or other relatively short pieces of DNA, and sometimes for longer genomic DNA.
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 recognition sequences. This allows flexibility when inserting gene fragments into the plasmid vector; restriction sites contained naturally within genes influence ...
The restriction sites in the MCS are first cleaved by restriction enzymes, then a PCR-amplified target gene also digested with the same enzymes is ligated into the vectors using DNA ligase. The target DNA sequence can be inserted into the vector in a specific direction if so desired. The restriction sites may be further used for sub-cloning ...
Plasmids are the most-commonly used bacterial cloning vectors. [39] These cloning vectors contain a site that allows DNA fragments to be inserted, for example a multiple cloning site or polylinker which has several commonly used restriction sites to which DNA fragments may be ligated.
pUC19 encodes of the N-terminal fragment of β-galactosidase gene of E. coli, also referred to as the α-peptide.[4] [3] The multiple cloning site (MCS), which contains many restriction sites, is split into codons 6-7 of the lacZ gene. [4]
A restriction fragment is a DNA fragment resulting from the cutting of a DNA strand by a restriction enzyme (restriction endonucleases), a process called restriction. [1] Each restriction enzyme is highly specific, recognising a particular short DNA sequence, or restriction site, and cutting both DNA strands at specific points within this site.
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