Explain how genomic libraries are constructed and the two methods that are used

Genomic DNA libraries are usually prepared by isolating total DNA from an organism, digesting the DNA with a restriction endonuclease, and inserting the restriction fragments into an appropriate cloning vector. Two different procedures are used to insert the DNA fragments into the cloning vector. If the restriction enzyme that is used makes staggered cuts in DNA, producing complementary single-stranded ends, the restriction fragments can be ligated directly into vector DNA molecules cut with the same enzyme. An advantage of this procedure is that the foreign DNA inserts can be precisely excised from the vector DNA by cleavage with the restriction endonuclease used to prepare the genomic DNA fragments for cloning. If the restriction enzyme cuts both strands of DNA at the same position, producing blunt ends, complementary single-stranded tails must be added to the DNA fragments in vitro. This is accomplished by using the enzyme terminal transferase to add nucleotides to the 3 termini of the DNA strands after the 5 ends are cut back with phage exonuclease. Usually, poly(A) tails are added to the cleaved vector DNA, and poly(T) tails are added to the genomic DNA fragments, or vice versa. Then, the T-tailed genomic DNA fragments are inserted into the A-tailed vector DNA molecules with DNA ligase. Since the T and A tails will not always be the same length, the E. coli enzymes exonuclease III and DNA polymerase I are used to cut back overhangs and fill in gaps, respectively. DNA ligase will only seal nicks between adjacent nucleotides; it will not add nucleotides if gaps are present. Once the genomic DNA fragments are ligated into vector DNA, the recombinant DNA molecules must be introduced into host cells for amplification by replication in vivo. This step usually involves transforming antibioticsensitive recipient cells under conditions where a single recombinant DNA molecule is introduced per cell (for most cells). When E. coli is used, the bacteria must first be made permeable to DNA by treatment with chemicals or a short pulse of electricity. Transformed cells are then selected by growing the cells under conditions where the selectable marker gene of the vector is essential for growth. A good genomic DNA library contains essentially all of the DNA sequences in the genome of interest. For large genomes, complete libraries contain hundreds of thousands of different recombinant clones.