Plasmid DNA sequence

Confirm the plasmid DNA sequence using a pTD 1-161-179 primer (5 -GCAGATTGTACTGAGAGTG-3 ) for N-terminal sequencing and a pTD 1-412-394 primer (5 -ACAACGCACAGAATCTAGC-3 ) for C-terminal sequencing. The annealing temperature of these primers is 50°C see Note 7 and 8). 

Rodriguez H, Loechler EL. 1993. Mutational specificity of the (+) - anti-diol epoxide of benzo [a] pyrine in a supF gene of an Escherichia coli plasmid DNA sequence context influences hotspots, mutagenic specificity and the extent of SOS enhancements of mutagenesis. Carcinogenesis 14(3) 373-383. 

Culp P, Nusslein-Volhard C, Hopkins N (1991) High-frequency germ-line transmission of plasmid DNA sequences injectedinto fertilized zebrafish eggs. Proc Natl Acad Sci USA 88 7953-7957. 

This transformation usually results in hundreds to thousands of E. coli clones. It is usually only necessary to pick one or two E. coli clones because the DNA was derived from a single yeast clone, and thus, most colonies will contain the same plasmid DNA sequence. 

As the second educt (B), the plasmid ONA with complementary sticky ends is prepared separately. In the first step the isolated plasmid DNA is cut open by a special type of enzyme called restriction endonuclease. It scans along the thread of DNA and recognizes short nucleotide sequences, e.g., CTGCAG, which ate cleaved at a specific site, e.g., between A and G. Some 50 of such enzymes are known and many are commercially available. The ends are then again extended witfa he aid of a terminal transferase by a short sequence of identical nucleotides complementary to the sticky ends of educt (A). 

Plasmid Vectors for Facile Introduction of Passenger DNA and Selection of Recombinants. The map of a commonly used plasmid vector, pUC19 (7), is shown in Figure 2. Three parts of the vector are key to its utility. The origin sequence, oh, allows the repHcation of plasmid DNA in high copy number relative to the chromosome. A gene, amp, encoding the enzyme beta-lactamase, which hydrolyzes penicillin compounds, allows... 

FIGURE 13.2 Foreign DNA sequences can be inserted into plasmid vectors by opening the circnlar plasmid with a restriction endonnclease. The ends of the linearized plasmid DNA are then joined with the ends of a foreign sequence, reclosing the circle to create a chimeric plasmid. 

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... 

Cosmid A plasmid into which the DNA sequences from bacteriophage lambda that are necessary for the packaging of DNA (cos sites) have been inserted this permits the plasmid DNA to be packaged in vitro. 

In gene therapy, production of missing or poorly expressed protein is induced by transferring the coding sequence to the cells in plasmid DNA [223]. In principle, the sequence of any protein can be transferred to the cells for protein production. Regulatory elements of DNA, such as cell specific and inducible promotors, can be... 

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