Labelling of dna
- 1. V.SNEGA I M.sc BIOTECHNOLOGY BON SECOURS COLLEGE FOR WOMEN THANJAVUR
- 2. A probe is normally a short sequence of nucleotide bases that will bind to specific regions of a target sequence of nucleotides. The degree of homology between target and probe results in stable hybridization.
- 3. Probes can range in size from as short as 10 nuicleotide bases (molecular weight of 3,300) to as long as 10,000 bases or more (molecular weight of 3,300,000). The most common size range for most probes is between 14 and 40 bases. For statistical uniqueness, a minimum of 20 nucleotide bases are usually needed for a probe.
- 4. Which probe is best???? Short or long?????
- 5. Short probes tend to hybridize nucleic acids at very high rates (in minutes). where as longer probes may require reaction times of hours to achieve a stable hybridization. Short probes do have some disadvantages. They are subject to more nonspecific hybridizations , are limited in specificity , and are more difficult to label. Long probes hybridize more stably than short probes at high temperatures and low salt concentrations(low stringency).
- 6. - A nucleic acid probe Is a short , single- stranded molecule of radioactively labeled or fluorescently labeled DNA or RNA.
- 7. The base sequence of the probe and the conditions under which the probe is used determine its specificity.
- 8. What ever label is used, it has to be attached to, or incorporated into, the nucleic acid probe. Here are 5 methods of labelling probes. 1) Nick translation 2) Primer extension 3) RNA polymerase 4) End labelling 5) Direct labelling
- 9. It involves introducing single- strand breaks (nicks by endonuclease) in the DNA, leaving exposed 3’ hydroxyl termini and 5’ phosphate termini. Nick serves as a start point for introducing new nucleotides at the 3’ hydroxyl side using DNA polymerase. As a result, the nick will be moved progressively along the DNA (translated’) in the 5’ 3’ direction. The synthesis reaction allows the incorporation of labelled nucleotides in place of the previously existing unlabeled.
- 11. Based on hybridization of a mixture of all possible hexanucleotides. The starting DNA is denatured and then cooled slowly so that the individual hexanucleotides can bind to suitably complementary sequences within the DNA strands. DNA synthesis occurs in the presence of the four dNTPs, at least one of which has a labelled group.
- 13. DNA of interest is denatured to give single-strands. Random primer sequences are added (or sequences unique to a particular sequences of interest). DNA polymerase is added together with labelled nucleotides. Complementary DNA strands are synthesized starting from the primer sequences and incorporating the labelled nucleotides. There is partial filling in of the gaps between the primers. DNA is then denatured to release the labelled probe molecules.
- 15. Single-stranded oligonucleotides are usually end-labelled using polynucleotide kinase ( kinase end- labelling). Label is provided in the from of a 32P at the gama- phosphate position of ATP and the polynucleotide kinase catalyses an exchange reaction with the 5’-terminal phosphates. The same procedure can also be used for labelling double- stranded DNA. Here fragments carrying label at one end only can then be generated by cleavage at an internal restriction site, generating two differently sized fragments which can be separated by gel electrophoresis and purified.
- 17. o The preparation of labelled RNA probes (riboprobes) is most easily achieved by in vitro transcription of insert DNA cloned in a suitable plasmid vector. o The vector is designed so that adjacent to the multiple cloning site is a phage promoter sequence , which can be recognized by the corresponding phage RNA polymerase. o By using a mix of NTPs , high specific activity radiolabeled transcripts can be generated. o Labelled sense and antisense riboprobes can be generated from any gene cloned in such vectors and are widely used in tissue in situ hybridization. o DNA template + RNA polymerase + labelled ribonucleotides ======= labelled RNA probe.
- 19. Radiolabels Non-radioactive labels Chemiluminescence Fluorescence Antibodies
- 20. Probe nucleic acid can be labelled using radioactive isotopes, e.g 32p, 35S ,125I ,3 H. Detection is by autoradiography or geiger-mullur counters. Radiolabelled probes used to be the most commen type but are less popular today because of safety considerations. However,radiolabelled probes are the most sensitive ,e.g .32p labelled probes can detect single –copy genes in only 0.5 mg of DNA. High sensitivity means that low concentrations of probe – target hybrid can be detected.
- 21. These are harmless than radiolabels and do not require dedicated rooms , glassware and equipment or staff monitoring , etc.but they are not generally as sensitive. Some examples: Biotin this labels can be detected using avidin or streptavidin which have high affinities for biotin . Enzymes the enzymes is attached to the probe and its presence usually detected by reaction with a substrate that changes colour .Used in this way the enzymes is sometimes referred to as a “reporter group”. Examples of enzymes used include alkaline phosphatase and horseradish peroxidase.
- 22. In this method chemiluminescent chemicals attached to the probe are detected by their light emission using a luminometer. Fluorescence chemicals attached to probe fluorescence under UV light. This type of label is especially useful for the direct examination of microbiological or cytological specimens under the microscope- a technique known as fluorescent in situ hybridization (FISH).
- 24. Antibodies an antigenic group is coupled to the probe and its presence detected using specific antibodies . Also ,monoclonal antibodies have been developed that will recognize DNA-RNA hybrids.
- 25. Probe and target hybridize with each other but how this is brought about can vary. There are 4 main formats;
- 26. Target (usually) is bound to a solid support such as a microtitre tray or filter membrane. Probe is added in solution and binds to target (if present ) on solid support. After washing to remove unbound probe, hybridization is detected on the solid support using whatever method is appropriate for the probe label.
- 27. Both the probe and the target are in solution. Because both are free to move , the changes of reaction are maximized and , therefore , this format is generally faster than others.
- 28. In this format probe solution is added to fixed tissues, sections or smears which are then usually examined under the microscope. The probe label, e.g a fluorescent marker, produces a visible change in the specimen if the target sequence is present and hybridization has occurred. However, the sensitivity may be low if the amount of target nucleic acid present in the specimen is low.This can be used for the gene mapping of chromosomes, and for the detection of microorgnisms in specimens.
- 29. After size fractionation of nucleic acids by electrophoresis, they are transferred to a filter membrane which is then probed. The presence of target is confirmed by detection of probe on the filter membrane, e.g radiolabelled probe can be detected by autoradiography and the bands in the original gel determined.
- 30. 1.Southern blots: Detection of gel –fractionated DNA molecules transferred to a membrane. This includes restriction fragment length polymorphism (RFLP) analysis. 2.Northern blots: As above but used for RNA. 3.Dot blots: Detection of unfractionated nucleic acid immobilized on a membrane. 4. Colony and plaque blots: Detection of immobilized nucleic acid on a membrane that has been released from lysed bacteria or phages.
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