Genomic DNA clones
Clones of genomic DNA, containing individual fragments of chromosomal DNA, are needed for many purposes. Some examples include:
§ to obtain detailed structures of genes,
§ to identify regulatory regions, i.e. DNA sequences needed for correct expression of the gene,
§ to map and analyze alterations to the genome, e.g. the isolate genes that when mutated cause a hereditary disease,
§ to direct alterations in the genome, e.g. by homologous recombination to replace a wild-type allele with a mutant one (to test function of the gene in mouse) or vice versa(to cure a hereditary disease, perhaps eventually in humans).
Construction of libraries of genomic DNA fragments in cloning vectors
Genomic DNA is digested with restriction enzymes (Fig. 3.20.) The more frequently an enzyme cuts (the shorter the recognition sequence), the smaller the average size of DNA fragments. Some enzymes cut very infrequently, such as NotI (8 bp recognition sequence) and can be used to generate very large fragments. Alternatively, one can do a partial digest (not all sites are cleaved) with a particular enzyme and isolate the products that are in the desired size range (e.g. 20 kb). A particularly clever way to do this is to digest partially with Sau3AI or MboI (both cut at 'GATC) and ligate these fragments into vector cut with BamHI (cuts at G'GATCC) ‑ i.e. they have the same sequence in the overhang (or sticky end). In this process one uses vectors that can accomodate large DNA fragments, such as l phage vectors, cosmids, YACs or P1 vectors.
Figure 3.20.Construction of a library of genomic DNA
Screening methods for genomic DNA clones
One method is to use complementationof a mutation in the host to select or screen for the desired gene. This works just like the situation for cDNA clones described above, and it requires that the cloned fragments be expressed in the host cell.
Far more common is to screen by hybridizationwith gene‑specific probes (Fig. 3.21). Frequently the cDNA clone is found first, and the genomic clone then isolated by hybridization screening (using the cDNA clone as a probe) against a library of genomic DNA fragments.
Figure 3.21. Screening a library of genomic DNA