Trends in Biotechnology

110516 Constructing and Screening a DNA Library

Constructing and Screening a DNA Library.

DNA libraries help to map and sequence genomes, and are screened for the target DNA.

Genomic Library.Contains DNA fragments that

represent an entire genome.

Created by the following steps: a) Total nuclear DNA is isolated and cut with a restriction enzyme. b) A cloning vector is also cut with the same enzyme. c) The two DNAs are mixed in a test tube and placed into host cells. d) The host cells are selected for the recombinant DNA by antibiotics.

e) Colonies (if not using a bacteriophage) or plaques (if using a bacteriophage) on bacterial plates represent successful transformation.f) A collection of colonies or plaques represents a library. g) Can calculate how many clones are needed to represent a genome.

Fig. 3.15 The steps

involved in the

construction of a DNA library

(Genomic).

cDNA Library.Made from mRNA, and shows only

genes expressed by a cell at a given time.

Reduces the amount of DNA to be cloned because all the genome is not being used.

Made this way: a) Get mRNA from cells, use the enzyme reverse transcriptase to make one strand of DNA from the mRNA.b) Degrade mRNA with a ribonuclease (an enzyme that breaks down RNA) or an alkaline ( 알칼리의 ) solution. c) Makes the second DNA strand with DNA polymerase.d) Add double-stranded DNA pieces, called “DNA linkers,” to the new DNA, and put the recombinant DNA into a vector.

The RNA that is used has already been processed and does not contain regulatory elements such as promoters. Fewer clones represent a library, making screening less labor-intensive.

Fig. 3.16 The

synthesis of a cDNA.

Fig. 3.16 The synthesis of a

cDNA.

Screening Libraries.DNA inserts are found by a

screening process called “nucleic acid hybridization.”

A known DNA sequence is used as a probe to find the clones or plaques with the DNA sequence we want.

Screening libraries are made this way (Figure 3.17): a) Bacterial colonies are transferred from a bacterial plate to a nylon or nitrocellulose membrane. b) Membranes are treated to lyse the cells.

c) DNA on the membrane is denatured, and single-strand DNA probes that are labeled attach to the desired DNA. d) Unattached probe is washed off, and the label gives off light to expose photographic film.

Fig. 3.17 Colony hybridization is used to identify bacterial cells that

have a specific recombinant plasmid.

Fig. 3.18 Probe hybridization (cDNA or DNA) to signal-stranded DNA in cloned

DNA.

Expression Libraries.1. Made with a cloning vector that contains the required regulatory elements for gene expression, such as the promoter region (Table 3.2). 2. Can insert into host cells to produce a protein or create a library.

3. Useful for finding a clone with the gene or cDNA of interest. 4. DNA probes or antibodies (a process called “antibody binding”) can be used to find a DNA sequence. Antibodies can also be used to find proteins by Western blotting.