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Gene Probe Designing And Production
Though gene probe designing and production is one of the most difficult steps, gene probe is essential in many molecular biological techniques. Bioinformatics resources and genetic databases are the starting point of the gene probe designing. However, sometimes it is possible to use related genes of the same family of single species to gain information about DNA sequence that can be useful as a probe.
Similarly, DNA sequence from different species can provide a starting point to produce heterologous gene probe. Sometimes probes already produced and possibly armed with a DNA sequence (from a DNA database) can be used to chemically synthesize a single-stranded oligonucleotide probe. However, this process is accomplished using computer controlled gene sequencers, which link dNTPS together based on the desired sequence. But it is essential to carry out certain checks before probe production to confirm that, the probe is unique with no self-annealing complementary.
In some cases, where little DNA information is available for gene probe designing, knowledge about that particular gene can be gathered by the analysis of the corresponding protein. Thus, isolation and purification of protein and sequence analysis can lead us to determine the gene sequence. As we know genetic code consists of three base pairs which encode a particular amino acid.
Therefore, we can predict the DNA sequence that can code that particular protein and then we can synthesize the appropriate oligonucleotide code. A single amino acid can be encoded by more than one codon, therefore, there will be a possibility that more than one possible oligonucleotide sequence that can encode for the given polypeptide chain.
The larger the polypeptide is, the greater the number of possible oligonucleotides is, that must be synthesized. However, protein should be chosen in such a say that, it contains as many as tryptophan and methionine residues as possible. Since these amino acids have unique codon and, therefore, there will be fewer possible base sequences coding for that part of the protein. Thus obtained synthetic oligonucleotide can, therefore, be used as a probe in many molecular biological procedures.
Labeling of DNA Gene Probe
Gene probe can be visualized or labeled by some means, to detect the presence of gene probe from other gene pools. This procedure uses a complementary sequence, that only binds with particular gene probe and thus identified. There are two ways of labeling gene probes. One traditional way, in which radioactive labeling is done. The other way of labeling is non-traditional, in which some type of enzyme is linked.
A radioactive method of labeling uses 32 phosphorous (32P) most commonly. However, 35S and 3H can also be used for this purpose where they are detected by autoradiography. The labeled probe bound to the sample DNA is placed in contact with x-ray sensitive film. Exposing the film with X-ray, the film is developed and precise location of labeled probe is revealed.
In the case of non-radioactive labeling, different other means of labeling are used. There are two types. 1) direct and 2) indirect labeling. In the direct labeling, enzyme reporters such as alkaline phosphatase are coupled directly to the DNA. However, indirect way of labeling relies on the incorporation of a single nucleotide already attached to a label. Presently there are three types of labels used for this purpose. These are biotin, fluorescein, and digoxigenin. These molecules are linked to nucleotide covalently, using a carbon spacer arm of some atoms.
Then specific binding protein is used as a bridge between nucleotide and reporter protein (enzyme). As, for example, biotin attached DNA fragment is recognized with very high affinity by streptavidin protein. Thus, this DNA fragment can be coupled with the reporter enzyme such as alkaline phosphatase. Which is then detected based on a catalytic reaction of alkaline phosphatase. In these procedures, there is no need to use autoradiography, instead of which either colored products or light-producing reactions are analyzed. This phenomenon is called as chemiluminescence.
End Labeling of DNA Molecules
There are two ways of labeling of DNA; 1) 5′ end labeling and 2) 3′ end labeling. 5′ end labeling includes phosphate group transfers or exchange reaction, where 5′ phosphate of DNA is removed and in its place 32P radiolabeled phosphate group is added. This type of labeling is carried out using two enzymes, alkaline phosphatase (which removes the existing phosphate group from DNA) and polynucleotide kinase (which adds the 32 phosphorous labeled phosphate groups to the 5′ end of the DNA). Thus obtained labeled probe is purified by chromatography and can be used directly.
However, 3′ end labeling is quite less complex as compared to that of the 5′ end labeling. In this case, a new dNTP already labeled with 32P or biotin labeled dNTP is added to the 3′ end of the DNA using terminal transferase enzyme. Although, this method is simple but has a potential problem. Because, new nucleotide added to the existing DNA sequence alters the DNA sequence, which may affect its hybridization with the target sequence. All these labeling makes easy for gene probe designing that is an essential step of detection of the DNA fragments and their analysis.