Probe marking method.

probe marking methods are:
1,
the
of DNA translation method. Notch translation is a fast, easy, relatively in-cost method for producing high-ratio activity-equally labeled DNA. This technique allows the preparation of sequence-specific probes. When recombining prosury probes are used, although any form of double-stranded DNA can be marked for gap translation. However, it is common to use restrictive nucleic acid in-cut enzymes to insert fragments for enzymatic cutting and gel electrophoresis purification before performing gap translation markers. In addition, hybridization with this probe produces a lower background signal.
2, DNA random citation method. This method is to
DNA fragments using
nucleotides, nucleotides, citants and Klenow fragments of E. coli DNA polymerase I. This method can replace the gap translation to produce an equal marker probe, but also in many ways better than the gap translation method, such as mark nucleotides in the DNA probe doped rate of more than 50%. Because the DNA fragments added during the reaction are not degraded, less than 200 ng of DNA can be added to the random lead reaction, and even 10 ng can be effectively labeled. The size of the DNA fragment does not affect the result of the marker.
markers are equally mixed along the full-length DNA added. Tagged probes can be used directly without removing unindiged nucleotides; Random quotes can be used for smaller DNA fragments (100 to 500 bp), while notch translation works best for large DNA fragments (>1000 bp). The main disadvantage of random quotation method is that the amount of marker probe produced is less than the gap translation, in addition to ring DNA can not be effectively marked, must first use restrictive kernel nuclease lineforming or alkali method or DNA enzyme I. to create gaps.
-body transcription of RNA 3 and RNA. In-body transcription marker RNA probes can be used to commercialize transcriptional granules for preparation. These protons should include RNA start-up sites of SP6, T3, or T7 RNA polymerases, which are adjacent to multiple cloning sites (MCS).
oligonucleotide method of RNA 4 and RNA. Cloned protons pSP64, pGEM-3, pGEM-4 and other SP6RNA initiaters containing adjacent MCS can be used as the basis for the generation of RNA probes from DNA oligonucleotide templates. These two labeling methods produce a large amount of probes, and the resulting probe is not affected by the vector sequence, the need for linearized granulated DNA of about 1 ug, but requires a high purity template.
5, oligonucleotide "follow-up method." End dna nucleotides
transferase
add triphosphate deoxynucleosides to the 3'-OH end of DNA molecule free. This deoxynucleotide connection will form an extended "tail" at the end of probe 3'. In this way, many
genes
can be added to produce high-ratio active probes, suitable for the identification of cloned sequences in the gene pool, point mutation detection of genomic DNA samples and in-place hybridization.
T4 polynucleotides and kinases of oligonucleotides at the end of
6 and 5'
method. T4 polynucleotide kinase transfers γ-32P-ATP γ-phosphorus to the free 5'-OH end. When oligonucleotides are synthesized, they are usually not phosphate and should therefore contain a hydroxyl at the 5' end. Since the probe molecule is only mixed with a
isotope
molecule, the probe activity is related to its length. Short oligonucleotides can be marked with high-ratio activity, while longer probe activity decreases with its length. This kinase marking method is most commonly used for DNA sequence determination.
7, polymerase chain reaction marker. Polymerase chain reaction (polymerase chain reaction,
PCR
) is used to mark more active DNA fragments than this technique, this technology is highly specific, can be a large number of synthetic probe DNA fragments within 1 to 2 h, marker mixing rate can be as high as 70% to 80%. Therefore, PCR marking technology is particularly suitable for large-scale detection and non-radioactive marking. The disadvantage of this method is that a pair of specific PCR quotations need to be synthesized. Good marking results can also be obtained by using small fragments of quotations prepared from the probe DNA.
generally speaking, the detection method of nucleic acid probe should be determined according to the marker of nucleic acid probe. Radioisotopes can be detected by radiation self-developing nucleic acid probes that mark them. Below is the detection of non-radioactively labeled probes:
(1) alkaline
phosphatase
(AKP) display system. ASO-AKP s BCIP (pH9.5) → BCl-OH → Purple. The nucleic acid probe in this system is marked with akP. ASO: Oligonucleotides specific to allegens, BCIP: 5-Bromine-4 chlorine-3-pyridine phosphate, NBT: tetraazole blue, Pi:phosphoric acid.
(2) spicy root peroxidase (HRP) display system. HRP s H2O2 → (HRP: H2O2) and ODA-NH2 s/HRP: H2O2 s →/ODA-N s/Brown s/HRP s/H2O. The nucleic acid probe of this system is marked with HLP. ODA: Neighbor-coriander amine.
(3) ABC display system. DNA-B-SA-enzymes → DNA-B-SA or DNA-B-biotinases → DNA-B-SA-biotinases. The nucleic acid probe of this system is marked with biotin (biotin, B) and then tests the nucleic acid probe using a compound (complex, C) formed by affinity (avidin, A), biotin, and enzymes. SA:streptavidin-chainase affine, ABC is avidin-biotin-enzyme complex-pro-biotin-enzyme complex. Enzymes can be selected with AKP or HRP and then displayed in their respective enzyme coloration systems.
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