Reaction Restriction enzymes

Endonuclease Reaction Restriction enzyme-enriched samples... 

Molecular methods used to uncover mutations are subject to several variables. The anticoagulants used for blood collection can affect digestion with restriction enzymes and amplification reactions. The type of detergent used in cell lysis can affect amplification of DNA by inhibiting the DNA-amplifying enzyme such as the taq polymerase used in the polymerase chain reaction (116). The control of contamination is crucial in ensuring the quality of results obtained by molecular analysis (117). 

Obtain samples of selected restriction enzymes and reaction buffer (supplied with enzymes). Also obtain a DNA sample, either a viral (lambda) or bacterial plasmid. 

Restriction enzyme techniqnes are freqnently nsed in con-jnnction with the polymerase chain reaction. 

The polymerase chain reaction (PCR) is an important procedure in genetic engineering that allows any DNA segment to be replicated (amplified) without the need for restriction enzymes, vectors, or host cells (see p. 258). However, the nucleotide sequence of the segment has to be known. Two oligonucleotides (primers) are needed, which each hybridize with one of the strands at each end of the DNA segment to be amplified also needed are sufficient quantities of the four deoxyribonucleo-side triphosphates and a special heat-tolerant DNA polymerase. The primers are produced by chemical synthesis, and the polymerase is obtained from thermostable bacteria. 

Type III site-specific deoxyribonuclease [EC 3.1.21.5], also referred to as type III restriction enzyme, catalyzes the endonucleolytic cleavage of DNA to give specific, double-stranded fragments with terminal 5 -phosphates. This class of enzymes has an absolute requirement for ATP but does not hydrolyze the ATP. -Adenosylmethionine stimulates the reaction, but is not absolutely required. These enzymes recognize specific short DNA sequences and hydrolyze bonds that are a short distance away from the recognition sequence. 

Incubate the reaction mixture for 2-4 h at the temperature appropriate for the restriction enzyme used. 

For preparation of donor DNAs, 2 pg of the cDNA clones carrying the desired ORF are linearized by a restriction enzyme that can cleave the vector sequence but not ORF (teeNote 8). Prepared cDNAs are digested in 50 pL of a reaction mixture with 20 U of the restriction enzyme at the appropriate temperature for the enzyme for 1 h in a 96-well format, and the enzyme is inactivated by heating the reaction mixture whose condition is suitable for the enzyme. 

Mix 5 -12 pi PCR product or restriction enzyme digest reaction mixture with... 

Most restriction enzymes are commercially available. The enzymes are usually sold together with the appropriate reaction buffers. 

Several hundred restriction enzymes have been isolated and characterized. Nomenclature for the enzymes consists of a three-letter abbreviation representing the source (Eco — E. colt), a letter representing the strain (R), and a roman numeral designating the order of discovery. fcoRI is the first to be isolated from E. coli (strain R) and characterized. Table E15.1 lists several other restriction enzymes, their recognition sequence for cleavage, and optimum reaction conditions. 

Disposable gloves should be worn when you are handling the enzyme container. Remove the enzyme from the freezer just before you need it. Store the enzyme in an ice bucket when it is outside the freezer. The enzyme should never be stored at room temperature. Because of high cost, digestion by restriction enzymes is carried out on a microscale level. A typical reaction mixture will contain about 1 fig or less of DNA and 1 unit of enzyme in the appropriate incubation buffer. One unit is the amount of enzyme that will degrade 1 fig of A. phage DNA in 1 hour at the optimal temperature and pH. The total reaction volume is usually between 20 and 50 fiL. Incubation is most often carried out at the recommended temperature for about 1 hour. The reaction is stopped by adding EDTA solution, which complexes divalent metal ions essential for nuclease activity. 

Reaction mixtures from restriction enzyme digestion may be analyzed directly by agarose gel electrophoresis. This technique combines high resolving power and sensitive detection to allow the analysis of minute amounts of DNA fragments. 

The objective of the experiment is to evaluate the action of restriction enzymes on bacterial plasmids, A phage DNA, or viral DNA. The DNA will be incubated under the appropriate conditions with selected restriction enzymes. The reaction mixtures will be subjected to agarose gel electrophoresis in order to determine the number and molecular size of the restriction fragments. 

Approximately 2 hours are required to prepare and incubate the restriction enzyme reaction mixtures. The agarose slab gel may be prepared during the incubation period. Up to 1 to 2 hours are required to prepare the gel slab. Alternatively, precast agarose gels may be provided. The electrophoresis may be completed as a group project. Most electrophoresis chambers accommodate slab gels with 15 to 20 sample wells. 

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