Research Method

DNA sample Plasmids

1| A DNA sample and plasmids are cleaved with the same restriction endonuclease.

2| Fragments and plasmids are mixed and spliced with DNA ligase.

3Ï A mixture of plasmids, all with different fragments inserted, results.

4| Bacteria take up the plasmids and are grown in a nutrient medium that selects for recombinant clones.

5} Colonies containing clones of each fragment of the original DNA are separated and maintained as a pure culture. Each such culture is a "volume' in the gene library.

16.7 Constructing a Gene Library Human chromosomes are broken up into fragments of DNA using restriction enzymes.The fragments are inserted into vectors (plasmids are shown here) and taken up by host bacterial cells, each of which then harbors a single fragment of the human DNA.The information in the resulting bacterial cultures and sets of colonies constitutes a gene library.

produces a colony of recombinant cells, each of which harbors many copies of the same fragment of human DNA.

Using plasmids, which are able to insert up to 10,000 base pairs of foreign DNA into a bacterium, about 200,000 separate fragments are required to make a library of the human genome. By using phage X, which can carry four times as much DNA as a plasmid, the number of volumes can be reduced to about 250,000. Although this seems like a large number, a single growth plate can hold up to 80,000 phage colonies, or plaques, and is easily screened for the presence of a particular DNA sequence by denaturing the phage DNA and applying a particular probe.

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Essentials of Human Physiology

Essentials of Human Physiology

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