probe labelling

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  1. 1. NUCLEOTIDE PROBES Tapeshwar Yadav (Lecturer) BMLT, DNHE, M.Sc. Medical Biochemistry
  2. 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. 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. 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 its 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. 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. 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 -Phosphate from ATP with a phosphate from the 5- terminal on (single or double- stranded) DNA.
  7. 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. 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.