Joining DNA Fragments

Myers, R.M., Fischer, S.G., Lerman, L.S. and Maniatis, T. (1985a) Nearly all base substitutions in DNA fragments joined to a GC-clamp can be detected by denaturing gradient gel electrophoresis. Nucleic Acids Research 13, 3131-3145. 

Name the substrates and describe the reaction catalyzed by DNA ligase. Draw the termini of the DNA fragments joined by DNA ligase. 

Fig. 3. Constmetion of a recombinant DNA by joining vector and passenger fragments where (I I I I I I) represent sticky ends. Both A and B genes represent selectable traits, so that introduction of foreign DNA in B gene leads to the loss of an identifiable function. REP represents repHcation and...

Fig. 24.3 The construction of a chimeric (or recombinant) DNA molecule hy joining together two DNA fragments produced by cleavage of different parental DNA molecules with the same restriction endonuclease.

Assuming the vector DNA and the DNA fragment to be cloned have been suitably prepared by one or another of the methods described earlier in this section, the two species are joined to one another by annealing their ends and sealing the nicks with DNA ligase (Figure 3.13). 

Cloned DNA Any DNA fragment that passively replicates in the host organism after it has been joined to a cloning vector. 

Joining two DNA fragments covalently. The enzyme DNA ligase links the cloning vector and DNA to be cloned. Composite DNA molecules comprising covalently linked segments from two or more sources are called recombinant DNAs. 

Cloning When joining two or more DNA fragments, a researcher can adjust the sequence at the junction in a variety of subtle ways, as seen in the following exercises. 

Correct answer = C. Fluoroquinolones, such as ciprofloxacin, inhibit bacterial DNA gyrase—a type II DNA topoisomerase. This enzyme catalyzes the transient breaking and rejoining of the phosphodiester bonds of the DNA backbone, to allow the removal of positive supercoils during DNA replication. The other enzyme activities mentioned are not affected. Primase synthesizes RNA primers, helicase breaks hydrogen bonds in front of the replication fork, DNA polymerase I removes RNA primers, and DNA igase joins Okazaki fragments. 

A problem with DNA fragments with cohesive ends is that they spontaneously form closed circles, a process that may compete with a desired joining to another piece of DNA. One solution to this problem is to hydrolyze off the 5 -phosphate groups with an alkaline phosphatase (Chapter 12). This prevents formation of covalently closed circles. However, pieces of DNA that retain their 5 -phosphate groups... 

As mentioned in Section B, 1, human centromeres are rich in the repetitive a-satellite DNA. By joining a-satellite DNA-containing fragments of the X-chro-mosome to cloned telomeric DNA, human minichromosomes have been created.488 These have been developed into human artificial chromosomes,489 which may be practical vehicles for gene transfer in human therapy. 

---