Probe labelling
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This document discusses nucleotide probes, which are single-stranded DNA or RNA fragments that are labeled and complementary to a target DNA sequence. Probes can range in size from 15 base pairs to several hundred kilobases. They are used to identify a specific DNA fragment through base pairing. Probes must be labeled to be detected, typically through radioactive labeling or fluorescent tags. Labeling can occur on the end of the probe or through polymerase-based incorporation of multiple labeled nucleotides during DNA synthesis. Probes have various uses, including searching DNA libraries and diagnosing genetic disorders through techniques like Southern and Northern blotting.
Probe labelling
- 1. NUCLEOTIDE PROBES Tapeshwar Yadav (Lecturer) BMLT, DNHE, M.Sc. Medical Biochemistry
- 2. NUCLEOTIDE PROBES A probe is defined as a single stranded piece of DNA, labelled (either with radioisotope or with non-radioactive label), the nucleotide sequence of which is complementary to the target DNA. It is a single stranded piece of DNA (sometimes RNA), which can range in size from as little as 15 bp to several hundread kilobases. It can identify, through base pairing, a specific DNA fragment of the library, which contains complementary sequence.
- 3. Probe Type Size Origin DNA 0.1 – 100kb Cell-based DNA Cloning, PCR RNA (or Riboprobe) 1 – 2 kb RNA transcription from plasmid (or phage vectors) Oligonucleotide 15 – 50 nucleotides Chemical Synthesis
- 4. A probe DNA will form complementary base pairing with another DNA strand. mRNA can be used as a probe: it will bind to the DNA fragment that contains exon sequences of it’s gene. RNA probe, termed riboprobe, can be produced by in vitro transcription of cloned DNA inserted into a plasmid vector. Synthetic oligonucleotide probes; constructed by chemical methods, are most commonly used.
- 5. PROBES MUST HAVE A LABEL TO BE IDENTIFIED Probes are labelled with the radioisotopes, such as 32 p or tritium. These probes can be detected by autoradiography, which involves placing the sample in direct contact with the photographic material, usually X-ray film. Alternatively, end-labelling probes with fluorescent tags can be used. The latter are visible under the UV-Lamp.
- 6. TECHNIQUES FOR LABELLING PROBES There are two general ways in which a labelled nucleotide can be incorporated into the structure of the probe: 1) End-Labelling: Addition of a labelled group to one terminal of the probe is done. For eg:- By exchanging a labelled γ-Phosphate from ATP with a phosphate from the 5’- terminal on (single or double- stranded) DNA.
- 7. 2) Polymerase – based Labelling:- Using a DNA Polymerase, multiple- labelled – nucleotides are incorporated into the probe during DNA synthesis. Such a reaction requires dNTPs & it is customary to have one of them to be labelled. Eg: dGTP Because on an average 25% of the nucleotides incorporated are labelled. This type has a higher specific activity than the end-labelling where only terminal nucleotide is labelled.
- 8. USES OF NUCLEOTIDE PROBES: i. To search specific DNA sequences of DNA library, as discussed. ii. In southern & Northern blot techniques, probes are used to identify DNA or RNA fragments respectively. iii. In diagnosis of genetic disorders, such as sickle cell anaemia, thalassaemia, cystic fibrosis etc. NOTE:- Probe gets involved in the formation of heteroduplex with template DNA, and this is the key to usefulness of molecular hybridization.
- 10. WISH U ALL HAPPY NEW YEAR 2072(B.S)