A DNA library is a collection of cloned restriction fragments of the DNA of an organism. Two kinds of libraries are commonly used: genomic libraries and complementary DNA (cDNA) libraries. Genomic libraries ideally contain a copy of every DNA nucleotide sequence in the genome. In contrast, cDNA libraries contain those DNA sequences that only appear as processed messenger RNA (mRNA) molecules, and these differ according to cell type and environmental conditions. [Note: cDNA lacks introns and the control regions of the genes, whereas these are present in genomic DNA.] 1.
Genomic DNA libraries: A genomic library is created by digestion of the total DNA of an organism with a restriction endonuclease and subsequent ligation to an appropriate vector. The recombinant DNA molecules replicate within host bacteria. Thus, the amplified DNA fragments collectively represent the entire genome of the organism and are called a genomic library. Regardless of the restriction enzyme used, the chances are good that the gene of interest contains more than one restriction site recognized by that enzyme. If this is the case, and if the digestion is allowed to go to completion, the gene of interest is fragmented (that is, it is not contained in any one clone in the library). To avoid this usually undesirable result, a partial digestion is performed in which either the amount or the time of action of the enzyme is limited. This results in cleavage occurring at only a fraction of the restriction sites on any one DNA molecule, thus producing fragments of ~20 kb. Enzymes that cut very frequently (that is, those that recognize 4-bp sequences) are generally used for this purpose so that the result is an almost random collection of fragments. This insures a high degree of probability that the gene of interest is contained, intact, in some fragment.
2.
Complementary DNA libraries: If a protein-coding gene of interest is expressed at a high level in a particular tissue, the mRNA transcribed from that gene is likely also present at high concentrations in the cells of that tissue. For example, reticulocyte mRNA is composed largely of molecules that code for the α-globin and β-globin chains of hemoglobin
A (HbA). This mRNA can be used as a template to make a cDNA molecule using the enzyme reverse transcriptase (Fig. 34.7). Therefore, the resulting cDNA is a double-stranded copy of mRNA. [Note: The template mRNA is isolated from transfer RNA and ribosomal RNA by the presence of its poly-A tail.] cDNA can be amplified by biologic cloning or by PCR. It can be used as a probe to locate the gene that encodes the original mRNA (or fragments of the gene) in mixtures containing many unrelated DNA fragments. If the mRNA used as a template is a mixture of many different size species, the resulting cDNA is heterogeneous. These mixtures can be cloned to form a cDNA library. Because cDNA has no introns, it can be cloned into an expression vector for the synthesis of eukaryotic proteins by bacteria (Fig. 34.8). These special plasmids contain a bacterial promoter for transcription of the cDNA and a Shine-Dalgarno (SD) sequence (see p. 454) that allows the bacterial ribosome to initiate translation of the resulting mRNA molecule. The cDNA is inserted downstream of the promoter and within a gene for a protein that is expressed in the bacterium (for example, lacZ; see p. 466), such that the mRNA produced contains an SD sequence, a few codons for the bacterial protein, and all the codons for the eukaryotic protein. This allows for more efficient expression and results in the production of a fusion protein. [Note: Therapeutic human insulin is made in bacteria through this technology. However, the extensive co-and posttranslational modifications required for most other human proteins (for example, blood clotting factors) necessitates the use of eukaryotic, even mammalian, hosts.]
C. Sequencing cloned DNA fragments