synonymNucleic acid probe(Nucleic acid probe) generally refers to RNA probe
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RNA probe refers to the labelednucleotide sequenceComplementary combination of a single chaincDNAorcRNAMolecules.The probes used in RNA hybridization can be divided into three types according to their sources: specific cDNACRNA probeAnd laborSynthetic oligonucleotideProbe.
RNA probe is a kind of promising nucleic acid probe. Because RNA is a single stranded molecule, its hybridization reaction efficiency with target sequence is extremely high.The early RNA probes used were cellsmRNAProbe andVirusesRNA probes, these RNAs areGene transcriptionOr the virus is labeled in the process of replication, the labeling efficiency is often not high, and is restricted by many factors.
classification
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Several common RNA probes and their characteristics:
one
CRNA probe: It is obtained by in vitro transcription using cDNA as a template.Because it is a single strand probe, it also avoids the renaturation problem between two strands when using double strand cDNA probes for hybridization reaction.The hybridization between cRNA and RNA is more stable than that between cDNA and RNA.Hybrids formed between cRNAs and RNAs are not affected by RNA enzymes.Therefore, after hybridization reaction, RNA enzyme can be used to remove the unconjugated probe.Because of these advantages, the hybridization saturation level of cRNA probe is 8 times higher than that of double stranded DNA probe, so it is widely used in in situ hybridization.The disadvantage of cRNA probe is that the preparation process of the probe is relatively complicated, and betterMolecular Biology ExperimentEquipment, which is sensitive to RNA enzyme and easy to be damaged, should be careful to avoid RNA enzyme pollution during operation.[1]
two
Single chaincDNA probeBecause there are no introns and other highly repetitive sequences in the cDNA, it overcomes the defect of double stranded cDNA probe renaturation between two strands in the hybridization reaction, thus improving the sensitivity of hybridization reaction.However, due to the difficulty in preparing single stranded cDNA probes, they have been rarely used in RNA in situ hybridization.[1]
three
Oligonucleotide probe: the synthetic oligonucleotide probe is made from nucleotides through the DNA synthesizer, which avoidsEukaryotic cellThe adverse effects of highly repeated sequences in.Because most oligonucleotides are short in sequence and do not need to be purified, tissue penetration is excellent.The probe designed according to the specific sequence of the target gene has strong specificity.The disadvantage of synthetic oligonucleotide probes is that the probe length must be appropriate. If the probe is too long, it may cause internal mismatch hybridization, and if the probe is too short, it may form non-specific binding.The hybridization between it and mRNA is not as stable as that of cRNA RNA. Moreover, the probe is shorter, and it carries fewer markers with lower sensitivity.[1]
application
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These RNA probes are mainly used for research purposes, not for detection.For example, when filteringRetrovirusHuman immunodeficiency virus (HIV)genomeDNAWhen cloning, because there is noDNA probeIt can be used to successfully screen multiple HIV genomes using the full set of HIV labeled mRNA as a probeDNA cloning。Another example is that in the process of nuclear run on transcription assaynucleusSeparated, and then in α - 32P-ATPTranscription in the presence ofisotopeThe mixed mRNA andCellulose nitrateThe DNA of a specific gene on the filter membrane can be hybridized to reflect the transcriptional state of the gene, which is a reverse probe experiment technology.
Other information
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Current development
In recent yearsIn vitro transcriptionWith the continuous improvement of technology, one-way and two-way in vitro transcription systems have been established.The system is mainly based on a new type of carrier pSP and pGEM, which are used inPolyclonal siteSP6 on both sidesPromoterAnd T7 promoterSP6 RNA polymerase orT7RNA PolymeraseCan be carried out under the action ofRNA Transcription, if aExogenous DNAFragment, one of the two strands of this DNA can be used as a template for transcription to generate RNA.This in vitro transcription reaction is very efficient, and nearly 10 μ g of RNA can be synthesized within 1 hourradioactivityorBiotinNTP, the synthesized RNA can be efficiently labeled.This method can effectively control the length of the probe and improve theMarkersOfUtilization。
Other applications
It is worth mentioning that by changingforeign geneInsert direction or select differentRNA polymeraseCan control the transcription direction of RNA, that is, which DNA strand is used as templateTranscriptional RNA。This method can obtain synonymous RNA probes (which are in the same sequence as mRNA) andAntisense RNAProbe (complementary to mRNA), antisense RNA, also known as cRNA, can be used forAntisense nucleic acidIn addition, it can also be used to detect the mRNA expression level.In this case, because the probe and target sequence are single strand, the efficiency of hybridization is several times higher than that of DNA DNA hybridizationOrder of magnitude。In addition to detecting DNA and mRNA, RNA probes have an important application in researchgene expressionIt is often necessary to observe the transcriptional status of the gene.stayProkaryotic expressionIn the systemExogenousGenes not only carry out forward transcription, but also sometimes existReverse transcriptionThis phenomenon is often an important reason for the low expression of foreign genes.In addition, in theEukaryotic systemSome genes also have reverse transcription, produce antisense RNA, and participate in the regulation of self expression.In these cases, accurate measurement of forward and reverse transcription levels cannot be usedDouble chainDNA probeOnly RNA probes or single strand DNA probes can be used.
