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the nucleic acid starting material. Since the genome of an organism is fixed, chromo-

somal DNA may be isolated from almost any cell type in order to prepare genomic

libraries. In contrast, however, cDNA libraries only represent the mRNA being pro-

duced from a specific cell type at a particular time. Thus, it is important to consider

carefully the cell or tissue type from which the mRNA is to be derived in the

construction of cDNA libraries.

There are a variety of cloning vectors available, many based on naturally occurring

molecules such as bacterial plasmids or bacteria-infecting viruses. The choice of vector

depends on whether a genomic library or cDNA library is constructed. The various

types of vectors are explained in more detail in Section 6.3.

6.2.4 Genomic DNA libraries

Genomic libraries are constructed by isolating the complete chromosomal DNA from a

cell, then digesting it into fragments of the desired average length with restriction

endonucleases. This can be achieved by partial restriction digestion using an enzyme

that recognises tetranucleotide sequences. Complete digestion with such an enzyme

would produce a large number of very short fragments, but if the enzyme is allowed to

cleave only a few of its potential restriction sites before the reaction is stopped, each

DNA molecule will be cut into relatively large fragments. Average fragment size will

depend on the relative concentrations of DNA and restriction enzyme, and in particu-

lar, on the conditions and duration of incubation (Fig. 6.4). It is also possible to

produce fragments of DNA by physical shearing although the ends of the fragments

Genome library

Extract chromosomal DNA

Digest chromosomal DNA with

restriction endonuclease

Insert each DNA fragment into vector

(recombinant DNA)

Transform bacteria

Clone each recombinant

cDNA library

Extract mRNA

Produce cDNA

Insert each cDNA into vector

(recombinant DNA)

Transform bacteria

Clone each recombinant

Fig. 6.3Comparison of the general steps involved in the construction of genomic and complementary DNA

(cDNA) libraries.

199 6.2 Constructing gene libraries