Recombinant cloning vectors

Figure 4.6. Recombinational cloning (RC). A. Cloning of PCR products using the attB + attP > attL + attR reaction catalyzed by Int and IHF. The result is an Entry Clone that can be used to create functional vectors. B. Conversion of an Entry Clone to a functional vector using the attL + attR > attB + attP reaction catalyzed by Int, Xis and IHF. A wide variety of functional vectors can be constructed by using a Destination Vector with the appropriate promoters and tags.

Figure 4.7. In vivo recombinational cloning in yeast. Two successive PCR reactions are performed. Each set of primers contains 5 flanking sequences that are eventually used for homologous recombination with vector sequences after transformation of yeast with the 2nd PCR fragment and the linearized vector.

Okada,T.,W.J Ramsey, J Munir, O.Wildner, and R.M. Blaese, Efficient directional cloning of recombinant adenovirus vectors using DNA-protein complex. Nucleic Acids Res, 1998.26(8) 1947-50. 

Zhang L, Sanker U, Lampe DJ, et al. The Himarl mariner transposase cloned in a recombinant adenovirus vector is functional in mammalian cells. Nucleic Acids Res 1998 26(16) 3687—3693. 

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. 

The principles that govern the delivery of recombinant DNA in clonable form to a host cell, and its subsequent amplification in the host, are well illustrated by considering three popular cloning vectors commonly used in experiments with E. coli—plasmids, bacteriophages, and bacterial artificial chromosomes—and a vector used to clone large DNA segments in yeast. 

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 ... 

Various bacterial plasmids, bacteriophages, and yeast artificial chromosomes are used as cloning vectors (Brown, 2001). At the heart of the general approach to generating and propagating a recombinant DNA molecule is a set of enzymes which synthesize, modify, cut, and join DNA molecules. 

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