Concepts

One method of finding a gene is to create and screen a DNA library. A genomic library is created by cutting genomic DNA into overlapping fragments and cloning each fragment in a separate bacterial cell. A cDNA library is created from mRNA that is converted into cDNA and cloned in bacteria.

Screening DNA libraries Creating a genomic or cDNA library is relatively easy compared with screening the library to find clones that contain the gene of interest. The screening procedure used depends on what is known about the gene.

The first step in screening is to plate out the clones of the library. If a plasmid or cosmid vector was used to construct the library, the cells are diluted and plated so that each bacterium grows into a distinct colony. If a phage vector was used, the phages are allowed to infect a lawn of bacteria on a petri plate. Each plaque or bacterial colony contains a single, cloned DNA fragment that must be screened for the gene of interest.

One common way to screen libraries is with probes. We've seen how probes can be used to find specific fragments of DNA on an electrophoretic gel (see Figure 18.5). In a similar way, probes can be used to find cloned fragments of DNA in bacteria or phages. To use a probe, replicas of the plated colonies or plaques in the library must first be made. Figure 18.15 illustrates this procedure for a cos-mid library.

Buffer

Buffer

A special column contains short oligo(dT) chains linked to cellulose.

^ mRNAs have poly(A) tails.Total cellular RNA is isolated from cells and passed through the column.

^The poly(A) tails of mRNA molecules pair with the oligo(dT) chains and the mRNA is retained in the column,.

.whereas the rest of the RNA passes through.

.leaving only mRNA with poly(A) tails.

A special column contains short oligo(dT) chains linked to cellulose.

^ mRNAs have poly(A) tails.Total cellular RNA is isolated from cells and passed through the column.

^The poly(A) tails of mRNA molecules pair with the oligo(dT) chains and the mRNA is retained in the column,.

.whereas the rest of the RNA passes through.

^ The mRNA can then be washed from the column by adding a buffer that breaks the hydrogen bonds between the poly(A) tails and the oligo(T) chains,.

.leaving only mRNA with poly(A) tails.

mRNA

Poly(A) tail

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