The following points highlight the three types of nucleic acid probes. The probes are: 1. Oligonucleotide Probes 2. DNA Probes 3. RNA Probes.
Type # 1. Oligonucleotide Probes:
These are synthesized chemically as oligonucleotides based on the information available on the amino acid sequence of the protein of interest. These oligo nucleotides can be used as a probe the identification of gene which encode for that particular protein.
However, due to degeneracy of the genetic code, construction of oligonucleotide is carried out with those that are rich in methionine or tryptophan residue or with only two codons. Generally oligonucleotide probe is used to screen cDNA libraries.
Type # 2. DNA Probes:
These are longer than the oligonucleotides. Thus, clones of longer DNA sequence is used as a DNA probes. The sequence obtained from cDNA library (cDNA clones) can be used to probe genomic library to identify genomic clones. The same probe can also be used to reprobe the same cDNA library to identify more cDNA clones. The genomic DNA clones are used to screen cDNA library or a genomic library.
Preparation of DNA Probe:
The DNA probe is prepared by random primer method as follows:
(i) In double stranded DNA containing the sequence that is to act as the probe is denatured and an oligonucleotide sample containing all possible sequences of six nucleotides is added (it is statistical certainty that some of the molecules of the oligonucleotide mixture will hybridize to the unlabelled, denatured probe DNA).
(ii) In the presence of klenow fragment and four deoxyribunucleotides, one of the four deoxyribonucleotides is labelled.
(iii) The bound oligonucleotides act as primers for DNA synthesis.
(iv) The synthesised DNA is labelled and can be used as a probe to detect the presence of a complementary DNA sequence in a source DNA sample (Fig. 13.8).
There are at least two possible sources of probes: one is from cloned DNA, second is the nucleotide sequence of a synthetic probe, based on the probable nucleotide sequence that is deduced from the known aminoacid sequence of the protein encoded by the target gene.
Type # 3. RNA Probes:
RNA probes used only under certain circumstances. Purification of particular RNA is generally used as a specific probe for the corresponding DNA specifies. By employing positive- negative screening or differential hybridized approach, often possible to identify clones for RNA in one population but not so in other population.
The colony is first probed with labelled RNA from one population. After the location of hybridized colonies, labelled RNA is then washed off from the membrane and the membrane is then probed with labelled RNA from the second population and hybridized colonies are identified.
The first RNA probes were mRNAs of a gene that are abundantly expressed in a cell, labelled with 32po4. One of the efficient ways of preparing RNA probes involves transcription from a target gene cloned in a plasmid. Transcription of this gene is initiated from a promoter that is specifically recognised by an RNA polymerase.
Due to unspecificity of RNA pol recognition, specific promoter can be selected. The promoter of a bacteriophage is very specific for the phage polymerase. Some researchers have utilized phage (SP6) T7 promoter and the corresponding enzyme to transcribe a DNA (target gene) cloned in a plasmid vector.
The gene for the phage polymerase and the phage promoter were both cloned in a vector, and a target gene sequence for transcription inserted downstream of the phage promoter. Cells transformed with recombinant DNA are supplied with RNA precursor nucleotides, of which one nucleotide is radio labelled. Transcription of the inserted gene is therefore labelled and may be used as RNA probes (Fig. 13.9).
