Libraries, DNA

Typical methods usually require a five- to tenfold redundant set of clones to ensure that there is a good chance of sampling the entire target at sufficient density to allow overlap detection. 

The EST databases probably contain fragments of a majority of all genes. Thus they are an important resource for locating some part of most genes. Some of the EST databases are given in Table 15.3. 

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A DNA library is a set of cloned fragments that collectively represent the genes of a particular organism. Particular genes can be isolated from DNA libraries, much as books can be obtained from conventional libraries. The secret is knowing where and how to look. 

A gene (erstEl) encoding a thermostable esterase was isolated from Escherichia coli cells that had been transformed by DNA libraries with metagenomes from environmental samples isolated from thermal habitats. The enzyme belonged to the hormone-sensitive lipase family, could be overexpressed in E. coli, was active between 30 and 95°C, and used 4-nitrophenyl esters with chain lengths of C4-C16 (Rhee et al. 2005). 

Genomic DNA is much more complex than cDNA. Since cDNAs are synthesized in the laboratory from mRNAs using reverse transcriptase, they are no more diverse than the number of mRNAs present at the time of isolation. However, only about 2% of the mammalian genome codes for the synthesis of proteins and their mRNAs. Thus genomic DNA libraries are at least 50 times more diverse than cDNA libraries. cDNA libraries are not easier to make, however, both because of the inherent instability of mRNA compared to DNA and because reverse transcriptase prematurely terminates when copying long mRNAs. 

Figure 3.1 Overview of DNA library creation strategies. Random mutagenesis introduces mutations at positions throughout the gene sequence. Semi-rational design randomizes only the specific position(s) of interest. Gene shuffling brings existing sequence diversity from different parental DNA sequences together to form a chimeric library...

Figure 3.2 Examples of gene shuffling methods used for DNA library creation in directed evolution, (a) Homology-dependent primer-independent DNA shuffling (b) homology-dependent primer-dependent StEP (c) homology-independent SHIPREC...

Lutz, S., Ostermeier, M., Moore, G.L. et al. (2001) Creating multiple-crossover DNA libraries independent of sequence identity. Proceedings of the National Academy of Sciences of the United States of America, 98, 11248-11253. 

EPO is present in serum and (at very low concentrations) in urine, particularly of anaemic individuals. This cytokine/hormone was first purified in 1971 from the plasma of anaemic sheep, and small quantities of human EPO were later purified (in 1977) from over 2500 1 of urine collected from anaemic patients. Large-scale purification from native sources was thus impractical. The isolation (in 1985) of the human EPO gene from a genomic DNA library facilitated its transfection into CHO cells. This now facilitates large-scale commercial production of the recombinant human product (rhEPO), which has found widespread medical application. 

L3. Lichter, P., Cremer, T., Borden, J., Manuelidis, L., and Ward, D. C., Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries. Hum. Genet. 80, 224-234 (1988). 

The collection of colonies produced is referred to as a genomic DNA library. The library must be screened with a radioactive probe to identify the colony with the desired restriction fragment (see Screening DNA Libraries). 

The MnP-1 cDNA clone was used to probe a P. chiysosporium genomic DNA library in the vector XEMBL-3, and a MnP-1 genomic clone was isolated and characterized (Godfrey, B. J. Mayfield, M. B., Brown,... 

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