Libraries of Genomic DNA

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. 

The otherwise impossible task of analyzing the 4 to 8 x 109 base pairs of human genomic DNA can be simplified. Many of the 23 human chromosomes can be physically separated from each other, their DNA 

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Yeast (Saeeharomyees eerevisiae) has a genome size of 1.21 X 10 bp. If a genomic library of yeast DNA was constructed in a bacteriophage A vector capable of carrying 16-kbp inserts, how many indi-... 

Figure 2.3 Metagenomic cloning experiments. Isolation of genomic DNA directly from environments (soil, plants, mixed environments or thermal-vent worms are the examples Illustrated here) can recover DNA fragments which could encode for enzymes. The DNA fragments can be ligated to plasmids or DNA linkers, and then subjected to functional screening (expression cloning) and/or sequence analysis. Amplification by PCR can sometimes be used to yield libraries enriched with clones containing selected sequence motifs relating to families of enzymes...

Over the last decades, as the human genome was sequenced, scientists have assembled a vast library of small DNA-sequence differences that are precisely located within the genome. The marker sequences were used for the reassembly of the sequenced pieces of the 3 billion base pairs. Some of these markers are found within a gene that has changed in people with an inherited condition. Other markers are just that—small sequence differences that may not by themselves contribute to an inherited condition. Depending on their location, they may or may not be inherited by family members with a known inherited condition. These small sequence differences are sometimes just the substitution of one nucleotide for another for example, a G instead of a C. In each person s DNA, there are millions of these single nucleotide polymorphisms, inherited differences among individuals (called SNPs), in about 1 out of every 1,200 bases. 

Elements Found by Random Isolation of Clones or by Hybridization of Genomic DNA to Genomic Library... 

In a variation of genomic libraries, clone libraries of complementary DNA (cDNA) molecules, called cDNA libraries, are produced from mRNA molecules by reverse transcription. This technique can be used to evaluate the transcriptome of certain cell types under... 

Goman, M., Langsley, G., Hyde, J. E., Yankovsky, N. K., Zolg, J. W., and Scaife, J. G. (1982). The establishment of genomic DNA libraries for the human malaria parasite Plasmodium falciparum and identification of individual clones by hybridisation. Mol. Biochem. Parasitol. 5,391-400. 

The approach taken by the private Celera group is somewhat different. They bypass the use of BAG libraries of long DNA pieces. Instead they break up the entire genome info fragments only 2000 to 10,000 nt long and sequence each of these, in chunks of up to about 700 bases (the resolution limit of the instruments). If we have 3 billion bases to worry about, this means a lot of pieces to sort out and assemble in the proper order (actually, only a portion of those are sorted, but it is still a lot). Powerful computers do the sorting to put the puzzle pieces together. 

Figure 1.2-6. Construction of jumping (a and b) and linking libraries (b). B represents BamHI, and N denotes Notl sites. Digestion of genomic DNA with BamHI is a first step in construction of a linking library.

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