DNA Libraries

Yeast artificial chromosome vectors act like real chromosomes in yeast and can store much longer DNA fragments, some over 150 kilobases in size, big enough for several genes along with their regulatory sequences. However, YAC vectors are difficult to manipulate, are prone to spontaneous rearrangement, and have been supplanted by bacterial artificial chromosome (BAC) vectors.

BAC vectors are derived from the F plasmid of Escherichia coli. This plasmid behaves like a chromosome and not like a typical plasmid. BACs can store very large DNA fragments—in excess of three hundred kilobases in some cases, although typical fragments are about half that size. The unique features of BAC vectors are very well suited to creating and maintaining DNA libraries. For example, once a BAC vector enters a cell, it will exclude all other BAC vectors, which means that a given E. coli clone will contain only one unique library fragment. Furthermore, E. coli cells are relatively easy to grow and store, and DNA purification from the bacterium is straightforward. BAC libraries played a key role in the massive sequencing efforts that made up the Human Genome Project.

Many of the large-format DNA libraries (YACs, BACs) are used exclusively to store genomic DNA for sequencing projects. Larger fragments permit A cDNA (complementary DNA) library begins with all the RNA expressed by a cell under a specific condition. The RNA is reverse transcribed into DNA, and then each DNA copy is inserted into a separate plasmid. These are taken up by E.coli bacteria, one to a cell, which are then grown in culture. At that point, the DNA can be purified for analysis or further cloning, "expressed sequence tags" for each can be sequenced, or the proteins can be re-expressed by the bacteria to yield enough protein for further study. easier assembly of finished DNA sequence and require the maintenance of fewer clones, which is particularly important when sequencing large genomes. DNA sequencing, however, is only one application of DNA libraries.

DNA libraries are created by generating a set of DNA fragments of the desired size and then attaching those fragments to the appropriate vector sequence. For genomic DNA, the fragments are normally generated by either enzymatic digestion or simple mechanical shearing of all the DNA of the genome, including noncoding sequences. Fragments are then enzymatically attached to the vector sequences, in a reaction known as ligation. The collected fragments, now attached to vector sequences, are then moved into the appropriate host organism for growth and evaluation. Conditions are chosen so that only one fragment enters each organism, which can then be grown up into a colony whose individuals all carry the same fragment.