Colony and Plaque Hybridisation

Once a cDNA or genomic library has been prepared, the next task requires the identification of the specific fragment of interest. In many cases, this may be more problematic than the library construction itself, since many hundreds of thousands of clones may be in the library. One clone containing the desired fragment needs to be isolated from the library and therefore a number of techniques, mainly based on hybridisation, have been developed.

Colony hybridisation is one method used to identify a particular DNA fragment from a plasmid gene library (Figure 1). A large number of clones are grown up to form colonies on one or more plates, and these are then replica plated onto nylon membranes placed on solid agar medium. Nutrients diffuse through the membranes and allow colonies to grow on them. The colonies are then lysed, and liberated DNA is denatured and bound to the membranes, so that the pattern of colonies is replaced by an identical pattern of bound DNA. The membranes are then incubated with a pre-hybridisation mix containing non-labelled non-specific DNA such as salmon sperm DNA to block non-specific sites. Following this denaturation, the labelled gene probe is added. Under hybridising conditions, the probe will bind only to cloned fragments containing at least part of its corresponding gene. The membranes are then washed to remove any unbound probe and the binding detected by autoradiography of the membranes. If non-radioactive labels have been used then alternative methods of detection must be employed. By comparison of the patterns on the autoradiograph with the original plates of colonies, those that contain the desired gene (or part of it) can be identified and isolated for further analysis. A similar procedure is used to identify desired genes cloned into bacteriophage vectors. In this case, the process is termed plaque hybridisation . It is the DNA contained in the bacteriophage particles found in each plaque that is immobilised onto the nylon membrane. This is then probed with an appropriately labelled complementary gene probe and detection undertaken as for colony hybridisation.

Fig1. Colony hybridisation technique for locating specific bacterial colonies harbouring recombinant plasmid vectors containing desired DNA fragments. This is achieved by hybridisation to a complementary labelled DNA probe and autoradiography.

PCR Screening of Gene Libraries

In many cases it is possible to use the PCR to screen cDNA or genomic libraries con structed in plasmids or bacteriophage vectors. This is usually undertaken with primers that anneal to the vector rather than the foreign DNA insert. The size of an amplified product may be used to characterise the cloned DNA and subsequent restriction mapping is then carried out (Figure 2). The main advantage of the PCR over traditional hybridisation-based screening is the rapidity of the technique, as PCR screening may be undertaken in 3–4 h, whereas it may be several days before detection by hybridisation is achieved. The PCR screening technique gives an indication of the size rather than the sequence of the cloned insert; however, PCR primers that are specific for a foreign DNA insert may also be used. This allows a more rigorous characterisation of clones from cDNA and genomic libraries.

Fig2. PCR screening of recombinant vectors. In this figure, the M13 non-recombinant has no insert and so the PCR undertaken with forward and reverse sequencing primers gives rise to a product 125 bp in length. The M13 recombinant with an insert of 100 bp will give rise to a PCR product of 125 bp + 100 bp = 225 bp and thus may be distinguished from the non-recombinant by analysis on agarose gel electrophoresis.

Screening Expression cDNA Libraries

 In some cases the protein for which the gene sequence is required is partially characterised and in these cases it may be possible to produce antibodies to that protein. This allows immunological screening to be undertaken rather than gene hybridisation.

Such antibodies are useful since they may be used as the probe if little or no gene sequence is available. In these cases it is possible to prepare a cDNA library in a specially adapted vector termed an expression vector, which transcribes and translates any cDNA inserted into it. The protein is usually synthesised as a fusion with another protein such as β-galactosidase. Common examples of expression vectors are those based on bacteriophage such as λgt11 and λZap or plasmids such as pEX. The precise requirements for such vectors are identical to vectors that are dedicated to producing proteins in vitro . In some cases, expression vectors incorporate inducible promoters that may be activated by, for example, increasing the temperature, allowing stringent control of expression of the cloned cDNA molecules ( Figure 3).

Fig3. Screening of cDNA libraries in expression vector λgt11. The cDNA inserted upstream of the gene for λβ-galactosidase will give rise to a fusion protein under induction (e.g. with IPTG). The plaques are then blotted onto a nylon membrane filter and probed with an antibody specific for the protein coded by the cDNA. A secondary labelled antibody directed to the specific antibody can then be used to identify the location (plaque) of the cDNA.

The cDNA library is plated out and nylon membrane filters prepared as for colony/ plaque hybridisation. A solution containing the antibody to the desired protein is then added to the membrane. The membrane is washed to remove any unbound protein and a further labelled antibody that is directed to the first antibody is applied. This allows visualisation of the plaque or colony that contains the cloned cDNA for that protein and this may then be picked from the agar plate and pure preparations grown for further analysis.

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مواضيع ذات صلة

Analysing Genes and Gene Expression

Hybridisation and Gene Probes

Cloning Vectors: Vectors Used in Eukaryotes

Addition of Telomeric Sequences by Telomerase Prevents Shortening of Chromosomes

Centromere Sequences Vary Greatly in Length and Complexity

Interphase Polytene Chromosomes Arise by DNA Amplification

Cloning Vectors : Cosmid-Based Vectors

Cloning Vectors : Phagemid-Based Vectors

Cloning Vectors : Virus-Based Vectors

Cloning Vectors : Plasmids

The TATA Box, Initiators, and CpG Islands Function as Promoters in Eukaryotic DNA

Chromosome Painting and DNA Sequencing Reveal the Evolution of Chromosomes