dA:dT Joints,Terminal Transferase Method

dA:dT Joints,Terminal Transferase Method

dA:dT Joints,Terminal Transferase Method - Berg and his associates employed the terminal transferase procedure to insert SV40 DNA into E. coli DNA through the lambda phage vector. SV40 and lambda phage DNA are closed circular molecules.

The enzyme EcoRI endonuclease was first used to cleave the loops and produce linear DNA. The linear molecules were then treated with lambda exonuclease, which cuts off nucleotides at the 5' ends

This results in linear DNA with projecting single stranded 3' termini. In the presence of cobalt ions terminal transferase can add nucleotides directly to the 3' termini without prior removal of a portion of the complementary strand. By adding A TP, a chain of adenine (A) nucleotides, 50-200 nucleotides long, was formed at the 3' ends of one DNA species by the enzyme terminal transferase.

Similarly, by adding TTP a chain of thymine (T) nucleotides was formed on the other DNA species. The poly(A) and the poly(T) terminus DNA species were now mixed together. The ends of the two DNA species became linked (annealed) by complementary dA: dT pairing through hydrogen bonding.

The gaps at the ends of the 5' strands were filled in with nucleotides under the catalytic action of exonuclease III and DNA polymerase I. The nicks in the strands were finally sealed with. DNA ligase By joining together the DNA of the two viruses, SV40 and lambda phage, Berg and his associates became the first to combine genetic material from two different organisms.

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