Labelling of probes

Content
Genetic Engineering and Biotechnology 1.  Recombinant DNA and PCR (Cloning and Amplification of DNA)
Restriction enzymes in cloning
Techniques used in recombinant DNA 
Cloning vectors for recombinant DNA
Plasmids as vectors
Bacteriophages as vectors
Plant and animal viruses as vectors
Transposons as vectors
Artificial chromosome vectors for cloning large DNA segments
Construction of chimeric DNA
Palindromes and staggered cleavage
Adding poly dA at the 3' ends of the vector and poly dT at the 3' ends of DNA clone
Blunt end ligation by T4 DNA ligase
Cloning in bacteria and eukaryotes
Cloning in bacteria
Cloning in eukaryotes
Molecular probes 
Labelling of probes
Applications of molecular probes
Construction and screening of genomic and cDNA libraries
Gene amplification : PCR and its applications
cDNA library from mRNA
Colony (or plaque) hybridization for screening of libraries
Gene Amplification : PCR and Its Applications
The basic polymerase chain reaction (PCR)
Different schemes of PCR
Labelling of probes
The detection of homologous sequences after hybridization with the probe is like finding a needle in the haystack. Therefore, for the success of DNA probe assay, it is necessary to develop simple, safe and sensitive techniques for their use. As probes transmit no signal of their own, they have to be (i) labelled either with radioactive isotopes (e.g. 32P dCTP) or with non-radioactive signal molecules (e.g. biotin or digoxigenin) without impairing the hybridization ability of these probes. These signal molecules may include fluorescent antibodies and enzymes that produce colour changes in dyes and chemiluminescent catalyst, (fig 39.16).

Use of labelled DNA probe for identification of homologous DNA sequence through DNA hybridization.
Fig 39.16 Use of labelled DNA probe for identification of homologous DNA sequence through DNA hybridization.


After hybridization with radioactively labelled probe, hybrids are detected by autoradiography (Fig. 39. 3). 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.


Southern blotting method of analysing DNA segments that share homology with a nucleic acid probe.
Fig. 39. 3. Southern blotting method of analysing DNA segments that share homology with a nucleic acid probe.

Recent advances in nucleic acid technology now offer alternatives to radioactively labelled probes. One procedure that is becoming increasingly popular is biotin labelling of nucleic acids. After hybridization and washing, detection of hybrids is done by a series of cytochemical reactions which finally gives a blue colour whose intensity is proportional to the amount of biotin in the hybrid. There are several advantages of using biotinylated probes. For example, these assays employ non-toxic materials, whose half-life is longer. These probes can be prepared in advance in bulk and stored at - 20°C for repeated uses.

Digoxigenin is another chemical derived from plants and used for non- radioactive labelling of probes. An antibody associated with an enzyme (anti- digoxigenin-alkaline phosphatase conjugate) is used for the detection of the presence of digoxigenin. The probe may be labelled with digoxigenin-11-dUTP supplied with a digoxigenin kit (these kits are available from any commercial firm, e.g. Boehringer Mannheim). The labelled and denatured probe may be used for hybridization with denatured DNA on Southern blots or for in-situ hybridization. After hybridization the membrane or the slide may be washed and the membrane or the slide is transferred into detection buffer containing 20 μg/ml of anti-digoxigenin fluorescein and 5% (w/v) BSA (bovine serum albumin). The system is incubated for 1h at 37°C, and then the membrane/slide is washed in detection buffer three times (8 min each at 37°C). Alkaline phosphatase activity can be detected using 017 mg/ml BCIP (5-bromo 4-chloro 3-indolyl phosphate) and 0-33 mg/ml NBL (nitroblue tetrazolium) as dye substrate.