cDNA Library Construction Protocol

• Isolation of mRNA. First of all, it involves the isolation of total mRNA from a cell type or tissue of interest.
• Synthesis of the first strand of cDNA.
• The second strand of cDNA generation.
• Incorporation of cDNA into a vector.
• Cloning of cDNAs.

Screening a cDNA or Genomic Library

1. immobilize members of the library onto a nylon membrane and denature them so that they are single-stranded.
2. prepare a radiolabelled probe and denature it to make it single-stranded.
3. hybridize the probe to the library of clones.
4. wash the excess probe and expose an X-ray film.

The traditional method for screening a cDNA library for clones representing a gene of interest is hybridization of labeled gene-specific DNA probes to colonies or plaques transferred to a nylon filter [reviewed in (17)]. This procedure can generate cDNAs that are as complete as the starting gene model.

cDNA libraries are commonly used when reproducing eukaryotic genomes, as the amount of information is reduced to remove the large numbers of non-coding regions from the library. cDNA libraries are used to express eukaryotic genes in prokaryotes.

cDNA is often used to clone eukaryotic genes in prokaryotes. When scientists want to express a specific protein in a cell that does not normally express that protein (i.e., heterologous expression), they will transfer the cDNA that codes for the protein to the recipient cell.

The cDNA is made from mRNA with the use of a special enzyme called reverse transcriptase, originally isolated from retroviruses. Using an mRNA molecule as a template, reverse transcriptase synthesizes a single-stranded DNA molecule that can then be used as a template for double-stranded DNA synthesis.

A genomic library is a set of DNA clones that ideally contains the entire DNA content of a genome from which the library was derived.

two kinds