DNA using restriction endonucleases

The first step in this procedure is to produce restriction fragments of DNA using restriction endonucleases. 

Kaplan N, Risko K (1981) An improved method for estimating sequence divergence of DNA using restriction endonuclease mappings. J Mol Evol 17 156-162... 

Loureiro, 2000 Deak, 2002 Capece et al., 2003). Each requires initial digestion of the harvested DNA using restriction endonucleases, enzymes that cleave DNA at specific nucleotide sequences unique for that enzyme. The DNA digest is the amplified at specific or randomly selected regions by polymerase chain reaction (PCR). Fragments are subsequently separated electrophoretically and their patterns compared against those of other isolates or databases. 

The detection of restriction fi agment length polymorphisms (RFLPs) facilitates prenatal detection of hereditary disorders such as sickle cell trait, beta-thalassemia, infant phenylketonuria, and Huntington s disease. Detection of RFLPs involves cleavage of double-stranded DNA by restriction endonucleases, which can detect subtle alterations in DNA that affect their recognized sites. Chapter 40 provides further details concerning the use of PCR and restriction enzymes for diagnosis. 

PUTTING YOUR DNA INTO A VECTOR Vectors are specialized pieces of DNA used to move other pieces of DNA around. Modern vectors are usually either bacterial plasmids or viral genomes. The act of isolating your DNA in the first place usually involves putting it into a vector and then selecting the vector that has your DNA in it. DNA pieces (called inserts when they are placed in a vector) are usually placed in vectors using restriction endonucleases. The vector is cut with two restriction enzymes of different specificity (Fig. 6-3). This removes a... 

FIGURE 3.13 Insertion of a foreign DNA sequence into a plasmid cloning vehicle, using restriction endonucleases and ligase. 

DNA isolated from any type of cell can be fragmented using restriction endonucleases. These are... 

Mundy LM, Sahm DF, Gilmore MS (2000) Relationships between enterococcal virulence and antimicrobial resistance. Chn Microbiol Rev 13 513-522 Murray BE (1990) The life and times of the Enterococcus. Chn Microbiol Rev 3 46-65 Murray BE, Singh KV, Heath JD, Sharma BR, Weinstock GM (1990) Comparison of genomic DNAs of different enterococcal isolates using restriction endonucleases with infrequent recognition sites. J Clin Microbiol 28 2059-2063... 

The most often used restriction endonucleases for staphylococci are HinAlll, Clal (Tenover et al. 1994), EcoRI (Blumberg et al. 1992), EcoRV or Kpnl (Blanc et al. 1994). The fragments containing specific sequences are then detected using a labelled piece of homologous DNA as a probe. The most often used is DNA complementary to 16S and 23S rRNA isolated from Escherichia coli and commercially available for ribotyping (Falkinham 1994 Pfaller and Hollis 2004). 

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