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nucleic acid probes have been widely used to screen recombinant clones, species testing of genetic diversity, and evaluation of systemic developmental relationships within and between fungal populations. The earliest radioisotope-labeled nucleic acid probe has the characteristics of high sensitivity, good specificity and strong resolution, but radioisotope markers also have a series of troubling problems, such as high cost, short half-life of the probe, radioactive materials endanger human health and so on. In addition, radioisotope marking experiments require specialized laboratories and corresponding experimental protection facilities, as well as trained professionals, thus limiting the conduct ofexperiments > in general laboratories.
In these detection systems, the most sensitive are the biotin-pro-hydrant detection system and the semi antigen-semi-anti-antigen high-sin detection system. Because biological samples often contain endogenetic biotin and bio-binding proteins, biotin-labeled nucleic acid probes will have some non-specific binding, which will affect the experimental effect. The geo-high-sin detection system, which is also highly sensitive to biotin-affine systems but reduces nonse specific binding, has been accepted and widely used.
manual method can be used to make the line type of geo-high-sin The spacer arm is connected to the dutp to form DIG-11-dUTP, which is incorporated into the DNA probe by random citation or >The RNA probe is labeled by an RNA polymerase encoded with phage information, which is used to in-body transcription to mix DIG-11-dUTP into the RNA probe. The markers of oligonucleotide probes are catalyzed by end transfer enzymes, with DIG-11-dUTP/dATP or DIG-11-ddUTP tails at the end of 3'.
for the intended DNA or RNA, the hybrid part can be detected by the ELISA experimental procedure, i.e. by adding a geocosin-> special that binds to alkalineHeterosexualantibody, which forms an enzyme-linked antibody-semiantigen (DIG) complex with the geococin semiantigen molecule, and then adds the corresponding color substrate, so that the hybrid part can be displayed. 1 the main marking method of the geo-high-sin-labeled nucleic acid probe
1. 1 DNA probe marking method
1. 1. 1 PCR Dop Style is |
uses random citation method to mark DIG-11-dUTP on the DNA strand. For best marking results, pre-label template DNA needs to be treated linearly, and at least one withdrawal with phenol-chloroform and then ethanol precipitation.Template chains of 100 to 10000 bases can be effectively labeled, but template chains larger than 10000 bases need to be restricted by enzyme cutting and digestion before marking. The treated template adds random quotations, which, according to the principle of base complementarity, are evenly incorporated into the newly synthesized DNA chain by extending the lead from the 3' end of the Klenow fragment after the random quotation and the template DNA are re-ignited.
Ind cut translation DNA probe technology is fast and easy, and very mature. Start with the right amount of DNase I to open several single-stranded gaps in the double-stranded DNA, and then E. Coli DNA polymerase I's 5'-3' excision activity and polymerization activity, the single-stranded DNA sequence is removed at the 5' end of the notch and replaced by a newly synthesized dna dna nucleotide chain labeled with geocosin.
this method applies to ring DNA or marks larger than 1kb DNA fragments. Nucleic acid probes for in-place hybridization are most effectively labeled by indicing translation. Because this method can get the right gap by controlling the enzyme activity of DNase I, a probe of the right length is obtained, and the size of the probe is an important parameter for in-place hybridization, and a probe small enough to penetrate tissue or cells.
1. 2. 1 3' end marking method
3' end marking method is the addition of only one DIG-ddU TP at the 3' end of each synthetic oligonucleotide (length 14 to 100bp). When marking oligonucleotides with this method, there is no need for any special oligonucleotide synthesisend transfer enzyme, which is convenient and fast. The synthetic probe is suitable for tests that require the probe to be highly specific and sensitive in general, such as South imprints, Northern imprints, speckled imprints, and bacterial P-phage spot hybridization experiments.
5' end marking method is chemically labeled at the 5' end of oligonucleotide. First connect an amino connection arm residue at the end of 5', purify the synthetic oligonucleotide, and then co-price the DIG-NHS with the amino residue at the end of 5' to form a marker probe. One of the main advantages of such probes is that their 3' end acts as a pronuncing in a DNA synthesis reaction, allowing the reaction product to be labeled.
3' end tailing method is by the end transfer enzyme at the end of probe 3' plus several geo-high-stain base tail. The probe sensitivity of this method tag is about 10 times higher than that of the 3' end marking method, but due to the presence of a long tail, a more serious nonse specific background tends to appear.
2 factors affecting the selection of probe marking methods
< When using the geo-high-sin system, the >can select the probe marking method based on the different uses of the geo-high-sin system, the type of template obtained to prepare the probe, and the sensitivity that the probe can achieve. Overall, the purity of the DNA template, the more efficient the marking. Dna templates should be drawn from phenols and chloroform before marking. In addition, for the method of marking DNA with random citations, it is essential to linearize the template and heating denaturation before marking the reaction, and for oligonucleotides, the tail of DIG-11-dUTP should be purified before adding a DIG-11-dUTP at its 3' end.
3 hybrid
uses a nucleic acid probe labeled with geosin for molecular hybridization, with hybrid dynamics, hybrid conditions similar to radio-labeled probes. The nature of the probe is different, and the hybrid temperature and time are different: DNAP does not contain methamide: the hybrid temperature is 68 degrees C, the time ≥6h ;D NAP contains 50 % methamide: the hybrid temperature is 42 degrees C, the time ≥6h; RNA - RNA hybridization (RNA probe): the hybrid temperature is 68 degrees C and the time ≥ 6h; RNA - DNA hybridization (DNA probe): The hybridization temperature is 50 degrees C and the time ≥ 6h.
p style" text-align:left;" is that it is highly stable. After molecular hybridization, the hybrid fluid still contains a large number of non-refringing geo-high-sin marker probes, as long as the remaining solution is returned to the plastic test tube, stored at -20 degrees C (DNA probe) and -70 degrees C (RNA probe) respectively, and its stability can be maintained for at least one year. When using these probes, simply defrost and heat them up to 95 degrees C, which can be denatured by 10min. If the hybrid fluid contains methamide, it is 68 degrees C denaturation 10min.
In addition, in the hybridization process, in order to avoid interference background due to high probe concentrations, it is necessary to conduct a simulated hybridization before formal hybridization to screen the most appropriate concentration.
