Radiolabelled Probes, Conventional Labelling, Proportion Nucleotides, Nucleic Acid Molecule, Double Strand DnA, Nick Translation, Klenow Fragemnt

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Chimeric DNA Molecular Probes and Gene Libraries Chimeric DNA Molecular Probes and Gene Libraries Introduction Restriction Enzymes for Cloning Technique of Restriction Mapping Restriction Clevage and Gel Electrophoresis Construction of a Restriction Map Use of Partial Digests end Labelling and Hybridization in Restriction Mapping Construction of Chimeric DNA Cloning of Plasmid Vectors Directional Cloning of DNA Fragments with Heterologus Protruding Termini Adding Poly dA at 3' Ends of DNA Clone Blunt End Ligation by T4 DNA Ligase Cloning in Phage λ and Cosmid Vectors Cloning in phage λ Vectors Cloning in Cosmid Vectors Hosts for Cloning Vectors Bacteria as Hosts Eukaryotes as Hosts Molecular Probes Preparation of Probes Genomic DNA Probes

Home >> Biotechnology and Genomics >> Chimeric DNA Molecular Probes and Gene Libraries >> Radiolabelled Probes

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Radiolabelled Probes

Traditionally radioactively labelled probes are used for a variety of experiments. ³²p is the most commonly used radioisotope, although other labels such as ³H, ³⁵S and ¹²⁵I have also been used. Conventional labelling replaces a proportion of nucleotides in a nucleic acid molecule with ³²p derivatives (e.g: ³²PdCTP) or adds ³²p to the end of the molecule.

Atleast three methods are available for preparation of a lebelled probe from a double stranded DNA: (i) nick translation involves creation of nicks in the probe DNA followed by extension of the broken ends using a labelled deoxyribonucleotide with, the help of DNA polymerase 1 (or 'Klenow fragment' of this enzyme);

(ii) oligonucleotide labelling involves the use of short random oligonucleotides, which are used as primers for copying the probe DNA in presence of labelled deoxyribonucleotides; (iii) riboprobe preparation involves synthesis of labelled RNA with the help of RNA polymerase, using DNA probe as template, in presence of a labelled ribonucleotide.

Single stranded DNA or RNA probes, which consist of only one of the two complementary strands of a given sequence, are also used and have an advantage over the more traditional double stranded probes. The absence of the complementary strand in a single-stranded probe eliminates the possibility of forming non-productive hybrids composed of reannealed probe.

These probes are particularly useful in experiments involving hybridization between RNA and DNA, as required in nuclease S 1 mapping (S 1 enzyme will degrade unhybridized single-stranded DNA and will allow mapping of exon sequences that are found in mRNA).

Riboprobes described above are single stranded probes, but single stranded DNA probes can also be synthesized. A DNA segment cloned in a phagemid or M13 vector is used as template for the synthesis of radiolabelled single stranded DNA. A. sequence complementary to a sequence of the vector is used as a primer and the four dNTPs, of which one should be labelled, are used for DNA synthesis by Klenow fragment of E. coli DNA polymerase I.

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Non radioactive Labelling of Probes and the use of Three labelled probes in DNA hybridization

In all above cases, labelling involves incorporation of labelled dNTPs at internal positions of DNA. However, in some cases, when the probe (double stranded or single stranded) is small (-20 mer), end labelling of 5' or 3' termini of DNA is needed. Such end lebelled probes are used (i) for nuclease-S 1 mapping of RNA,

(ii) as radiolabelled primers in primer extension reactions and (iii) for in-gel hybridization (e.g. in oligonucleotide fingerprinting. The double stranded DNA is generally labelled at the 3' termini of both the strands using either E. coli Klenow fragment or T4 DNA polymerase, and the oligonucleotides (single stranded) are labelled at their 5' termini using T4 polynucleotide kinase.

After hybridization with labelled probe, hybrids are detected by autoradiography. 32p has the advantage over other radioisotopes, since it has high specific activity. However, in general, radioisotopes have some disadvantages. They are difficult to handle and expensive to dispose off.

Detection by autoradiography, while sensitive, may take a long time if there are few counts in the hybrid. Furthermore, radioisotopes have a short half life (e.g. 32p has a half-life of 14.3 days) and therefore experiments should be completed preferably within one half life.

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c DNA Probes Synthetic Oligonucleotides as Probes RNA Probes or Riboprobes Labelling of Probes Radiolablled Probes Non Radioactive Probes Biotin Labelled Probes Digoxigenin Labelled Probes Alternatives to Biotin and Digoxigenin Labelling Amplification of DNA Probe Signals Techniques Used in Molecular Probing Southern Nothern and Western Blotting Southern Blotting Northern Blotting Western Blotting Dot Blots and Slot Blots Applications of Molecular Probes Construction and Screening of Genomic and cDNA Libraries Genomic Library by Shotgun Experiment cDNA Library from mRNAs Colony or Plaque Hybridization for Screening of Libraries Chromosome Walking and Characterization of Chromosome Segments Chromosome Jumping or Hopping Libraries BAC Libraries and Assembly of BACs into Contigs