Gene Cloning

Screening DNA Libraries 135
In this chapter we have considered methods for screening DNA libraries
by looking either for expression of a particular protein or by detecting the
presence of a specific DNA sequence. However, in many cases there is not
enough information to use these strategies. In many cases, for example in
the cloning of human disease genes, you may know little more than the
phenotype resulting from mutations in the gene; techniques for cloning
genes in these circumstances are discussed in the next chapter.
Q5.9. In each of the following cases decide which approach you would use
to screen the library.
(a) Remember the question at the end of Chapter 4 where a cDNA library
seemed to be the most appropriate choice when trying to clone the genes
for the enzyme rubisco from spinach. What approach would you use to
identify the genes for rubisco from a spinach leaf cDNA library?
(b) You have a clone from a wheat grain cDNA library which encodes a gene
involved in starch synthesis. You want to study the upstream regulatory
DNA sequences. How would you obtain a clone containing these
sequences?
(c) β-lactamase is a bacterial enzyme, which is capable of inactivating β-lactam
antibiotics including penicillin and hence gives rise to antibiotic resistance.
You have cloned the chromosomal ampC β-lactamase gene from
Pseudomonas aeruginosa but now you want to clone β-lactamase genes from
a range of other bacteria.
(d) You are interested in the human glutathione S-transferase (GST) which
encodes an enzyme important in detoxification in the liver.You can look up
the sequence of the gene on the human genome project database but you
need a cDNA clone for your studies. Human cDNA expression libraries
derived from a number of different tissues are commercially available, typically
containing as many as 96,000 individual clones.
(e) You have a genomic DNA clone representing a small part of the dystrophin
gene. Remember from Chapter 4 that mutations in this gene give
rise to the human sex-linked disorder muscular dystrophy.You want to find
the rest of the gene and you have both cDNA and genomic libraries.
Questions and Answers
Q5.1. Using a cosmid vector such as pHC79 how many clones would you
need to have a 99% chance of finding a particular gene in a genomic library
of (a) Saccharomyces cerevisiae and (b) human? Hint. You will need to
know the size of insert which can be cloned in pHC79 and the size of the