Oligonucleotide probes refer to single-stranded DNA synthesized in vitro by chemical synthesis technology, and the length is generally 30-50bp. Oligonucleotide probes are currently synthesized by DNA synthesizers.
When determining the sequence of an oligonucleotide probe, the probe must be able to specifically bind to the target nucleic acid sequence, and the schematic sequence of the tRNA probe prepared by the in vitro transcription method will not produce a hybridization reaction. If the sequence of the target DNA or mRNA is known, the sequence of the synthetic oligonucleotide can be easily determined according to the principle of base complementarity. If only the amino acid sequence is known, the design of the probe is more complicated, because most amino acids can have several codons, that is, the codons are degenerate. In this case, the amino acid sequence with the least code degeneracy should be selected. The following principles should be observed when designing screening oligonucleotide probes:
1. The length is generally 30-50 bp. If the length is too long, the hybridization time will be longer and the synthesis amount will be low. If the length is too short, the specificity will be poor.
2. The content of (G+C) in the base should be between 40% and 60%. If it exceeds this range, non-specific hybridization will increase.
3. There should be no complementary region in the probe molecule, otherwise there will be a “hairpin”-like structure that inhibits the hybridization of the probe.
Synthetic oligonucleotide probes have the following advantages over cDNA probes:
(1) Simple preparation: The oligonucleotide probe uses nucleotides as raw materials, and can be synthesized in a short time by a DNA synthesizer. The method is simple and does not require complex molecular biology experimental conditions.
(2) Arbitrary sequence: according to the known nucleotide sequence of a specific purpose or target gene, any specific nucleotide fragment can be synthesized and used as a probe.
(3) Easy to penetrate the tissue: Since the oligonucleotide chain is not long and is single-stranded, the penetration force to the tissue is greater than that of the long double-stranded cDNA molecule.
(4) Short hybridization time: due to the short oligonucleotide chain. Its sequence is relatively simple and its molecular weight is small. Therefore, the time for complete hybridization to the same amount of target sites is shorter than that of cloned probes.
(5) Easy to use: The synthetic oligonucleotide probe is deoxyribonucleic acid in nature, which is not sensitive to RNase, so it is more stable than RNA probe; and this kind of probe is a single-stranded molecule, which can be used in tissue section cells. The internal mRNA molecule does not require preheating to melt during hybridization.
Oligonucleotide probes also have certain disadvantages. For example, the hybridization with mRNA is not as stable as RNA-RNA hybridization. Moreover, because of its short molecule, it can only be labeled by end labeling, and there are fewer bound labels, so it is sensitive. low sex. In addition, the cost of synthesizing oligonucleotide probes in large quantities is relatively high.