Restriction enzymes sequence

Restriction enzymes, sequence-dependent cleavage of DNA by, 12 497—498 Restriction fragment linked polymorphism (RFLP), 12 500 procedure, 12 103-104 Restriction sites, as genetic markers, 12 500... 

Restriction enzyme Sequence recognized Fragment end-structure after cleavage... 

The mam use of PCR is to amplify or make hundreds of thousands—even mil lions—of copies of a portion of the polynucleotide sequence m a sample of DNA Sup pose for example we wish to copy a 500 base pair region of a DNA that contains a total of 1 million base pairs We would begin as described m Section 28 14 by cleaving the DNA into smaller fragments using restriction enzymes then use PCR to make copies of the desired fragment... 

Workers in the early 1970s recognized that restriction enzymes provided tools not only for DNA mapping but also for constmction of new DNA species not found in nature. A collection of recombinant DNA species consisting of many passenger sequences joined to identical vector molecules is called a hbrary. Individual recombinant DNAs are isolated from single clones of the Hbrary for detailed analysis and manipulation. 

After a desired clone is obtained and mapped with restriction enzymes, further analysis usually depends on the deterrnination of its nucleotide sequence. The nucleotide sequence of a new gene often provides clues to its function and the stmcture of the gene product. Additionally, the DNA sequence of a gene provides a guidepost for further manipulation of the sequence, for example, lea ding to the production of a recombinant protein in bacteria. 

Figure 7.8 Sequence-specific recognition sites in the major groove of DNA for three restriction enzymes—Eco RI, Bal I, and Sma I. The DNA sequences that are recognized by these enzymes ate represented by tbe color code defined in Figure 7.7.

Type II restriction enzymes have received widespread application in the cloning and sequencing of DNA molecules. Their hydrolytic activity is not ATP-depen-dent, and they do not modify DNA by methylation or other means. Most importantly, they cut DNA within or near particular nucleotide sequences that they specifically recognize. These recognition sequences are typically four or six nucleotides in length and have a twofold axis of symmetry. For example, E. coU has a restriction enzyme, coRI, that recognizes the hexanucleotide sequence GAATTC ... 

This polylinker contains restriction sites for BamHl, EeoRL, Pst, Sal, Smal, Sphl, and Xbal. Indicate the location of each restriction site in this sequence. (See Table 11.5 of restriction enzymes for their cleavage sites.)... 

The first step in DNA sequencing is to cleave the enormous chain at known points to produce smaller, more manageable pieces, a task accomplished by the use of restriction endonucleases. Each different restriction enzyme, of which more than 3500 are known and approximately 200 are commercially available, cleaves a DNA molecule at a point in the chain where a specific base sequence occurs. For example, the restriction enzyme Alul cleaves between G and C in the four-base sequence AG-CT. Note that the sequence is a palindrome, meaning that the sequence (5 )-AGCT-(3 ) is the same as its complement (3 )-TCGA-(5 ) when both are read in the same 5 — 3 direction. The same is true for other restriction endonucleases. 

Alec Jeffryes, an English geneticist, discovered in the 1980s how to apply this principle to forensics. To do this, it is necessary to locate that portion of the DNA molecule in which the base sequence differs significantly from one individual to another. That part of the molecule is cut out by a "restrictive enzyme" in much the same way that trypsin splits a protein molecule into fragments. The DNA sample obtained in this way from a suspect can be compared with that derived from blood, hair, semen, saliva, and so on, found at the scene of a violent crime. 

Nielsen P.E., Egholm M., Berg R. H., Buchardt O. Sequence specific inhibition of DNA restriction enzyme cleavage by PNA. Nucleic Acids Res. 1993 21 197-200. 

Restriction enzymes are named after the bacterium from which they are isolated. For example, EcoRI is from Escherichia coli, and BamEII is from Bacillus amyloliquefaciens (Table 40-2). The first three letters in the restriction enzyme name consist of the first letter of the genus (E) and the first two letters of the species (co). These may be followed by a strain designation (R) and a roman numeral (I) to indicate the order of discov-ery (eg, EcoRI, EcoRIE). Each enzyme recognizes and cleaves a specific double-stranded DNA sequence that is 4—7 bp long. These DNA cuts result in blunt ends (eg,... 

Bacterial plasmids are small, circular, duplex DNA molecules whose natural function is to confer antibiotic resistance to the host cell. Plasmids have several properties that make them extremely useful as cloning vectors. They exist as single or multiple copies within the bacterium and replicate independently from the bacterial DNA. The complete DNA sequence of many plasmids is known hence, the precise location of restriction enzyme... 

A similar analysis could be made for a number of other diseases. Point mutations are usually defined by sequencing the gene in question, though occasionally, if the mutation destroys or creates a restriction enzyme site, the technique of restriction fragment analysis can be used to pinpoint the lesion. Deletions or insertions of DNA larger than 50 bp can often be detected by the Southern blotting procedure. 

Restriction enzyme An endodeoxynuclease that causes cleavage of both strands of DNA at highly specific sites dictated by the base sequence. 

Sequencing the amplicon is the most conclusive confirmatory technique. The main consideration is that the DNA must be appropriately purified to achieve unambiguous sequencing data. However, sequencing requires expensive laboratory equipment that may not be available in all labs. Sequencing does not depend upon the specificity of a probe, or restriction enzyme, but gives a direct identification of the amplicon of interest. 

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