Selection of DNA Clones

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Once a cDNA or genomic DNA library has been prepared, it can be screened to identify a particular DNA fragment by a technique called in situ hybridization. The cloned bacterial colonies, yeast colonies, or phage plaques containing the recombinant DNA are transferred onto nitrocellulose or nylon filters by replica plating (Figure 23-9). The filter is then treated with NaOH, which both lyses the bacteria and denatures the DNA, allowing single-stranded DNA (ssDNA) to bind to the filter. The filter with bound DNA is then incubated with a radioactive probe specific for the gene of interest. The probe will hybridize with DNA in the colonies or plaques on the filter that contain the sought-after gene, and they can be identified by autoradiography. The position of the positive colonies or plaques on the filter shows where the corresponding clones can be found on the original agar plate.

Various radioactive probes can be used to screen a library. In some cases, radiolabeled mRNA or cDNA serves as the probe. If the protein encoded by the gene of interest has been purified and partly sequenced, it is possible to work backward from the amino-acid sequence to determine the probable nu-cleotide sequence of the corresponding gene. A known sequence of five or six amino-acid residues is all that is needed to synthesize radiolabeled oligonucleotide probes with which to screen a cDNA or genomic library for a particular gene. To cope with the degeneracy of the genetic code, peptide segments containing amino acids encoded by a limited number of codons are usually chosen. Oligonucleotides representing

Treat filter with NaOH to lyse bacteria or phage and to

Treat filter with NaOH to lyse bacteria or phage and to

Lyse Bacteria

Wash and expose filter to photographic film ssDNA bound to filter

Colonies containing gene of interest

Petri dish with colonies of bacteria containing recombinant plasmids or plaques from recombinant X phage

Wash and expose filter to photographic film

Position of desired colonies detected by autoradiography ssDNA bound to filter

Colonies containing gene of interest

Position of desired colonies detected by autoradiography

FIGURE 23-9

Selection of specific clones from a cDNA or genomic DNA library by in situ hybridization. A nitrocellulose or nylon filter is placed against the plate to pick up the bacterial colonies or phage plaques containing the cloned genes. After the filter is placed in a NaOH solution and heated, the denatured ssDNA becomes fixed to the filter. A radioactive probe specific for the gene of interest is incubated with the filter. The position of the colonies or plaques containing the desired gene is revealed by autoradiography

Cleave with restriction enzymes

Gel electrophoresis

Cleave with restriction enzymes

Gel electrophoresis

Hybridize Bacteria

Blotting:

capillary action transfers DNA from gel to filter

The Southern-blot technique for detecting specific sequences in DNA fragments. The DNA fragments produced by restriction-enzyme cleavage are separated by size by agarose gel elec-trophoresis. The agarose gel is overlaid with a nitrocellulose or nylon filter and a thick stack of paper towels. The gel is then placed in an alkaline salt solution, which denatures the DNA. As the paper towels

Alkaline solution

Filter

Blotting:

capillary action transfers DNA from gel to filter

Autoradiography

Filter

Alkaline solution

Autoradiography Steps

Hybridize with labeled DNA or RNA probe

Autoradiography

Hybridize with labeled DNA or RNA probe

FIGURE 23-10

The Southern-blot technique for detecting specific sequences in DNA fragments. The DNA fragments produced by restriction-enzyme cleavage are separated by size by agarose gel elec-trophoresis. The agarose gel is overlaid with a nitrocellulose or nylon filter and a thick stack of paper towels. The gel is then placed in an alkaline salt solution, which denatures the DNA. As the paper towels soak up the moisture, the solution is drawn through the gel into the filter, transferring each ssDNA band to the filter. This process is called blotting. After heating, the filter is incubated with a radiolabeled probe specific for the sequence of interest; DNA fragments that hybridize with the probe are detected by autoradiography. [Adapted from J. Darnell et al, 1990, Molecular Cell Biology, 2nd ed, Scientific American Books]

all possible codons for the peptide are then synthesized and used as probes to screen the DNA library.

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