Figure 2225

Immunological Screening of a DNA Library

Bacteria carrying a library are grown on agar, transferred to a membrane and lysed. Released proteins are bound to the membrane. This figure shows only one attached protein, but in reality, a large number of different proteins will be present. These proteins include both those from the library as well as many bacterial proteins. The membrane is incubated with a primary antibody that only binds the protein of interest. Any non-specifically bound antibody is washed away. Finally a second antibody that binds the primary antibody and that also carries a detection system is added.

are often longer than the exons and the overall length of the gene is therefore much larger than the coding sequence. This creates two problems. First, cloning large segments of DNA is technically difficult; plasmids with large inserts are often unstable and transform poorly. Secondly, bacteria cannot process RNA to remove the introns and so eukaryotic genes containing introns cannot be expressed in bacterial cells. Using a DNA copy of mRNA, known as complementary DNA or cDNA, solves both problems since the mRNA has already been processed so that all the introns are removed.

To make a cDNA library, the mRNA must be isolated and used as a template. The library generated therefore reflects only those genes expressed in the particular tissue under the chosen conditions. First, total RNA is extracted from a particular cell culture, tissue or specific embryonic stage. The messenger RNA from eukaryotic cells is normally isolated from the total RNA by taking advantage of its poly(A) tail. Since adenine base pairs to thymine or uracil, columns containing oligo(U) or oligo(dT)

complementary DNA (cDNA) DNA copy of a gene that lacks introns and therefore consists solely of the coding sequence. Made by reverse transcription of mRNA

oligo(dT) Stretch of single-stranded DNA consisting solely of dT or deoxythymidine residues oligo(U) Stretch of single-stranded RNA consisting solely of U or uridine residues

aDD ANTIBODY SPECIFIC FOR TARGET PROTEIN

Add second antibody that

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