DNA, phages

Figure 5.26 Integration of lambda DNA into the host. Integration always occurs at a specific site on the host DNA, involving a specific attachment site (att) on the phage. Some of the host genes near the attachment site are given. A sitespecific enzyme (integrase) is involved, and specific pairing of the complementary ends results in integration of phage DNA.

Bustamante C, Marko JF, Siggia ED et al (1994) Entropic elasticity of X-phage DNA. Science 265 1599-1600... 

Amplified electrochemical detection of DNA in monolayer assemblies was accomplished by the conjugation of bioelectrocatalytic transformations to the DNA recognition events. This was exemplified with the amplified electrochemical analysis of M13 phage DNA (Fig. 12.20a).75 A capturing nucleic acid, (20), complementary to... 

Tudek, B., Boiteux, S. Laval. J. (1992) Biological properties of imidazole ring-opened N7-methylguanine in MI3mpl8 phage DNA. Nucleic Acids Res., 20, 3079-3084... 

The first site-specific recombination system studied in vitro was that encoded by bacteriophage A. When A phage DNA enters an E. coli cell, a complex series of regulatory events commits the DNA to one of two fates. 

About 45% of the sequence of the RNA polymerase encoded by phage T7, which transcribes RNA from the phage DNA, is also similar to that of the Klenow fragment. Sequences of these DNA polymerases are distantly related to those of reverse transcriptases.279280 The 136-kDa polymerase y functions in mitochondria but is encoded in a nuclear gene. It is the only DNA polymerase that is inhibited by antiviral nucleotide analogs such as AZT (Box 28-C).280a b... 

Standard curve obtained by electrophoresis of X phage DNA fragments from EcoRI cleavage. 

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. 

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. 

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