A number of techniques have been developed by scientists for identifying the specific gene of interest in cDNA as well as genomic library. In this article some of the techniques are discussed.
Screening Specific Clone in cDNA Library:
1. One of the techniques to detect a cDNA clone from its library is by detecting a protein product produced from that specific clone (gene).
2. An expression vector is used for protein to produce. In expression vector, cDNA is inserted between a promoter and a transcription terminator. This helps in the expression of the cDNA gene.
3. Specific proteins which are produced by host colony are detected by using labeled antibodies. Bacterial colonies are transferred to a membrane filter, then cells are lysed so that the proteins present in these cells bind to the membrane filter. This is then incubated with the specific antibodies.
4. If antibodies are labeled with radioactive material, then the antibodies bound to the colonies are detected using a technique known as autoradiogram. In this technique dry filter along with proteins and antibodies are exposed to X ray film in the dark for some time. Antibody bound bacterial colonies will be visible as dark spots on the X-ray film.
5. Once identified, the cDNA can be used to following purposes,
a. This cDNA can be used to analyze the genome of the same or different organism for homologous sequences.
b. cDNA can be used to isolate nuclear gene from a genomic library and this genomic DNA can be used for mRNA production.
Screening Specific Clone in Genomic Library:
1. Technique used to detect a specific clone in a plasmid genomic library or cosmids genomic library is similar to screening a cDNA library.
a. E.coli bacterial cells are transformed with genomic library. These transformed bacterial cells are plated on a selective medium.
b. Bacterial colonies formed on this selective media are then replica plated on a membrane filter on a plate of selective medium, and these transformed cells grow on the membrane filter.
c. Bacterial cells are then lysed; DNA is denatured and made to bind tightly onto the filter.
d. This filter is then incubated with labeled single stranded cDNA probes. These probes form hybrid with complementary DNA molecules which are present in the filter.
e. Filter is washed and the labeled probe is detected using a technique known as autoradiography for a radioactive probe, other nonradioactive probes are detected using chemiluminescent or colorimetric assay.
f. Clones which are detected using probes are then studied further and characterized.
Screening X genomic Library:
Screening X genomic library uses a slightly different screening method than the above one.
1. Transformed bacterial cells are plated on a medium to grow and form a bacterial lawn. Phage is plated on this bacterial lawn, produces plaques that each colony contains a different cloned DNA sequence.
2. Free DNA present in the plaques binds to a membrane filter, which is placed on the surface of the plate.
3. DNA present in the filter are then denatured and exposed to labeled probes and detected using appropriate technique.
4. One detected, DNA of interest is further subcloned into other vectors such as plasmids for further study.
Identifying Gene or cDNA in Libraries Using Oligonucleotide Probes:
1. Synthetic oligonucleotides are very useful in probing genomic library or cDNA library. Some part of the sequence of gene of interest should be known to use this technique. Sequence data which are available for a part of the gene of interest is used to produce complementary probes to the gene of interest. This helps in identifying gene of interest very easily. Sequence for most of the genes can be obtained from GenBank or other sites.
2. The amino acid sequence can also be used to produce a DNA oligonucleotide probes. Amino acid sequence can be obtained by using universal genetic code. Degeneracy in the genetic code indicates that a mixture of oligonucleotides are produces, in such a way that each of the oligonucleotide encodes for the target protein or protein of interest.
3. This oligonucleotide mixture is used as probes to detect the gene of interest from both type of libraries such as cDNA or genomic library. This is a powerful but not perfect method for isolating specific clones from the libraries.
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