And Denaturation Of

Add labeled dna probe

Probe binds to DNA from occasional colonies

FIGURE 22.24 Screening a DNA Library by Probing

The first step in screening a DNA library is to grow colonies of bacteria containing the library inserts on agar. A large number of different transformed bacteria are grown, so that all genes in the library have a reasonable chance of being present. Next, the bacterial colonies are transferred to a membrane or filter. The filter is applied to the top of the bacterial colonies and carefully lifted off. A portion of each bacterial colony will stick to the filter while the rest of the colony stays on the agar plate. Once on the filter, the bacteria are lysed open and the DNA is denatured. The single-stranded DNA stays bound to the filter, and the majority of the bacterial components are washed away. The library filters are covered with a solution of a radioactively labeled single-stranded DNA probe, allowed to hybridize, and then the excess probe is washed away. Placing a piece of photographic film over the filter identifies the hybrid molecules. When the probe hybridizes to a library insert, a black spot appears on the photographic film. By lining up the original bacterial colonies with the photographic film, the corresponding library insert can be isolated from the bacteria.

Probe binds to DNA from occasional colonies

FIGURE 22.24 Screening a DNA Library by Probing

Non-coding introns are a nuisance in many procedures. They can be avoided by making cDNA copies of the gene using reverse transcriptase.

protein, and isolating all the antibodies from a sample of the rabbit's blood. Producing an antibody is costly and a long process, so instead of directly conjugating this antibody to the detection system, a second antibody is produced in another animal, such as a goat. The secondary antibody recognizes all rabbit antibodies; therefore, it can be used for any primary antibody made in a rabbit. The secondary antibodies are available from companies and are relatively inexpensive. Secondary antibodies also amplify the signal, since usually two secondary antibody molecules bind to each primary antibody. Double the color will be produced using the two antibody system.

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