Genomic Library

Constructing Genomic Libraries using Plasmids

This is the process of cloning human DNA fragments by inserting them between two EcoRI digested sites of a plasmid cloning vector. After the human DNA fragments are inserted into the plasmid, the plasmids in turn are inserted into bacterial cells. In the end, these host bacterial cells contain their own chromosomal DNA together with plasmid DNA. As these bacteria replicate their chromosomes, they also replicate with them the plasmid cloning vectors. Plasmids are very convenient to work with because they contain one origin of replication, one of more selectable markers (such as a gene that confers resistance to antibiotics), [Page 729]and one or more restriction sites that can be cut and used for ligation of foreign DNA molecules.

Constructing Genomic Libraries Using Bacteriophage Vectors:

The human genome is partially digested with a restriction enzyme like Sau3A in a specific way so that some of the sites are cleaved and others are not. By this way, random cleavage of the sites occurs and a collection of overhanging fragments of length suitable for cloning can be obtained, which are then ligated into bacteriophage lambda “arms” prepared so that the Sau3A ends of human DNA fragments can be ligated into the vector. The recombinant lambda chromosomes are then packaged into the infectious bacteriophage, and then the library, containing 1 million or more fragments of genomic DNA, can be stored for the future isolation of many genes. A collection of several hundred thousand phages would represent the entire DNA from the human genome.

Constructing Genomic Libraries with Yeast Artificial Chromosomes (YACs):

Large fragments of human DNA (over 500 kb) are generated by partial EcoRI digestion of human genomic DNA. Individual vector arms contain telomeres at one end and EcoRI compatible overhangs on at the other end. Individual vectors also carry a different selectable marker, and one arm also contains a centromere and selectable markers at each end and a yeast chromosome at the other end. The YACs are transferred into yeast, and the selectable markers are used to select only those yeasts that contain a properly constructed YAC.

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