Common enzymatic marking techniques for nucleic acid probes

1. Gap Translation The technology was created by Kelly in 1970. The principle is to first use
DNA
ase to create some gaps (nicks) in a chain of double-stranded DNA probe molecules, where a 3' hydroxyl end is formed, and then
nucleotides are catalyzed by E. coli DNA polymerase I.
residue is added to 3'-hydroxyl, and according to E. coli enzyme DNA polymerase I 5'→3'
nucleic acid
excision enzyme activity, this enzyme will gap 5' side nucleotides in turn removed. The result is nick tr-anslation in the gap. According to this principle, if pre-existing nucleotides are replaced with high-intensity radioactive nucleotides (usually α-32PdATP), 32P-labeled DNA with a ratio of up to 108cpm (counted per minute) / sg can be prepared. DNA probes labeled with notch translation meet most hybridization requirements.2. DNA fast-end marker E. coli DNA polymerase i by the dead herb protease cutting can get two
peptide
chains, where the molecular weight of 76kd large fragments called Klenow fragments. The enzyme has a complete polymerase I 5'→3' polymerase activity and 3'→5' nucleic acid exocetic enzyme activity, but lacks 5'→3' nucleic acid exocetic enzyme activity. The Klenow fragment can be used to fill the 3' depression end produced by the restriction enzyme dissolving DNA. Therefore, in this way, the depression 3' end of the double-stranded DNA can be marked. Marking the ends with the α Klenow fragment generally uses only one type of dNTP, adding the reaction's α dNTP depending on the 5' end sequence of the end extension of the DNA, for example, cutting the end of the DNA with Ecor I is marked with the end of the DNA with the dNTP of α-32P. The markup reaction can be carried out immediately after the digestion of DNA by a restriction enzyme, without removing the limit enzyme or inactive, without replacing the buffer, and the end of the DNA with a 3' extension cannot be effectively marked by the Klenow fragment, in order to mark such molecules can be used with T4DNA polymerase.this marking method is used to produce DNA fragments that can be used as a size reference for gel electrophoresis. Because the marked DNA fragment is proportional to its molar concentration and not to the size of the fragment, small and large fragments are marked to the same extent in the restricted enzyme digestion. Therefore, radio-self-development techniques can be used to determine the size of DNA bands not observed by the dyeing of ethyl bromide ingots, especially for the marking of molecular weight markers during South suction hybridization. By selecting the correspondingly labeled dNTP, the method can also mark only one end of the DNA molecule. For example, if the two ends of the DNA fragment are Bam H I and Hind III mucous membrane ends, only the dNTP of "α-32P" or "α-32P" dGTP are added to the reaction, making it available to mark one of the two ends selectively.3. Mark DNa 5' end oligonucleotide probes or short RNA and DNA probes with T4 polynucleotides
kinases
. T4 polynucleotide kinase (PNK) is extracted from E. coli infected with T4 phage, which catalyses the transfer of γ-phosphoric acid from ATP to the 5'-OH end of DNA or RNA. In the presence of excessive ADP, the phosphoric acid exchange reaction can also be promoted, so that PNK transfers the end 5' phosphoric acid of DNA to the ADP to generate α ATP, and then catalytically transfers the marker phosphoric acid on the dNTP to the 5' end of the DNA, thereby rephosphating the DNA and obtaining the marker. Obviously, the end of the PNK-labeled DNA requires γ-32P dNTP, which is different from the enzymatic labeling method described above. Typically, for 5' phosphatized DNA probes, the phosphate group is removed with alkaline
phosphatase
and then used for PNK catalytic 5' end markers, which are more efficient.4. Random quote extension This is the method chosen for synthesizes high-ratio active 32P marker probes with single-stranded DNA or RNA templates. The principle is to make 6 to 8nt long oligonucleotide fragments with denatured DNA or RNA template atrocification, in the DNA polymerase I or reverse transcriptase, with each degeneration to the template oligonucleotide fragments as a leader to trigger the synthesis of dna strands, in response to the α-32P dNTP into the synthesis chain, that is, to be marked. After denaturation, the new synthesis chain (probe fragment) is dissointed from the template, resulting in numerous probe DNA of various sizes. Because the oligonucleotide fragments used are so short that they can react randomly with template DNA at low temperatures, they are called random primers. This random quote can be prepared using calf thymus DNA or fish fine DNA.or cDNA probes marked by random quotation were significantly more active than the notch translation method, and the results were stable. This method is particularly suitable for fuse DNA probes, since random citations come from the nucleo-DNA and have a higher rate of de-ignition from the futose sequence than the primary nuclear sequence. Therefore, for cloned DAN probes, the insertion probe DNA is often cut off and recycled before marking, while the notch translation method can be used directly for the marking of whole particles.5. Polymerase Chain Reaction (polymerase chain reaction,
PCR
) is a new
molecular biology
technology, by Kary.B. Mullis was founded in 1985. The technique uses two oligonucleotide quotations on both sides of the target DNA to be synthesized enzymatically into specific DNA fragments, including template denaturation, re-ignition of the quotation, and repeated cycles of the introduction extension of three steps, resulting in tens of millions of times the amplification of the target DNA sandwiched between the two citants. As a result, PCR technology has become an extremely valuable technology and has been rapidly promoted and applied.PCR technology has many important uses, one of which is to mark high-ratio active DNA probes. PCR technology has a high specificity, can be within 1 to 2h in the amount of synthetic probe DNA fragments, if the substrate added to the substrate of the α dNTP or other markers, then the probe DNA synthesis process can be well marked, 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 to synthesize a pair of specific PCR quotations. The use of small fragments prepared from the probe DNA as a guide can also achieve better marking results.