Cloning of DNA fragments

Directional cloning of DNA fragments at a large distance from an initial probe ... 

The cosmid is another variation of the plasmid and viral DNA vectors that has been developed to allow the cloning of DNA fragments that range from 40 to 50 kb. As the linear bacteriophage A DNA is... 

Multiple cloning sites (MCS). These are fragments of DNA that contain a number of unique different restriction sites, enabling the use of a choice of restriction enzymes for the cloning of DNA fragments. 

Fig. 24.1 Simplified diagram of the plasmid pUCl 8. lacZ represents the insertional inactivation marker coding for fl-galactosidase activity. A multiple cloning site (MCS) is present within the LacZ gene to enable the cloning of DNA fragments. Ori represents the origin of replication which, in this case, works in Escherichia coli. Finally, Ampr represents an ampicillin resistance marker.

Kunkel LM, Monaco AP, Middlesworth W, Ochs H, Latt SA. Specific cloning of DNA fragments absent from the DNA of a male patient with an X-chromosome deletion. Proc Natl Acad Sci USA 1985 82 4778-82. 

Collins, F.S. and Weissman, S.M. (1984) Directional cloning of DNA fragments at a large distance from an initial probe a circularization method. Proc. Natl. Acad. Sci. U.S.A., 21, 6812 -6816. 

To increase the amount of DNA covered in each step, three different strategies have been used One approach, the use of vector-host systems likely to allow the direct cloning of DNA fragments larger than those accommodated in cosmid, has led to the recent development of artificial yeast cloning vectors (13). 

An economical alternative to the cloning of DNA fragments obtained in Subheading 3.4, step 4 and subsequent sequencing of individual clones is to sequence the PGR products directly... 

L Cloning of DNA fragments. When using EcoRI (or other restriction enzyme) linkers to insert cDNA or DNA fragments into the EcoRI site of a vector (plasmid, phage A vectors), any potential internal EcoRI sites must first be protected by methylation. The linkers are then attached to the DNA by blunt-end ligation and are cleaved by EcoRI to open cohesive ends. The cohesive-ended DNAs are ligated to the vector and are transfected into a bacterial host (refer to [9] below). 

Figure 40-2. Results of restriction endonuclease digestion. Digestion with a restriction endonuclease can result in the formation of DNA fragments with sticky, or cohesive, ends (A) or blunt ends (B).This is an important consideration in devising cloning strategies.

Shotgunning Cloning of a complete set of DNA fragments from a particular genome. 

You just isolated a novel recombinant clone and purified the desired insert (a 10,000 bp linear duplex DNA) from the vector. Now you wish to map the recognition sequences for restriction endonucleases A and B. You cleave the DNA with these enzymes and fractionate the digestion products according to size by agarose gel electrophoresis. Comparison of the pattern of DNA fragments with marker DNAs of known sizes yields the following results ... 

Describe a procedure for cloning a DNA fragment into the BamHl site of pBR322. 

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