It is one of the commonly used techniques for producing a radioactive probe. A purified phage or plasmid vector containing a cloned genomic or cDNA sequence is treated with a small amount of pancreatic DNase which hydrolyzes the phosphodiester bonds between nucleotides. At very low concentration the DNase produces only scattered “nicks” in one or other strand of the duplex DNA. DNA polymerase and radioactively labeled deoxynucleotides are also added to the DNA sample. Using the unharmed strand as template, the DNA polymerase synthesizes a new second strand using exposed 3’ end at a nick site as primer, which then displaces the existing DNA from the 5’ end of the nick. Radioactive nucleotides are incorporated into the new strand, so, a single standard probe is created when the duplex DNA is denatured (Fig. 3(a) and 3(b)).

Gene probes, cloned or PCR-amplified, and oligonucleotide probes can be random-primed labeled with radioactive isotopes and nonradioactive labels (e.g., DIG). Random-primed labeling of DNA fragments (double- or single-stranded DNA) was developed by Feinberg and Volgestein as an alternative to nick translation to produce uniformly labeled probes. Double-stranded DNA is denatured and annealed with random oligonucleotide primers. The oligonucleotides serve as primers for the 5’ to 3’ polymerase (the Klenow fragment of E. coli DNA polymerase I), which synthesizes labeled probes in the presence of a labeled nucleotide precursor (Fig. 4).

Single-stranded oligonucleotides are usually endlabeled using polynucleotide kinase (kinase endlabeling). The label is provided in the form of a³²P at the γ-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 labeling 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 (Fig. 5).

Nucleic acid probes can be labeled using radioactive isotopes (e.g., 32P, 35S, 125I, 3H). Detection is by autoradiography or Geiger–Muller counters. Radiolabeled probes used to be the most common type but are less popular today because of safety considerations as well as cost and disposal of radioactive waste products. However, radiolabeled probes are the most sensitive, as they provide the highest degree of resolution currently available in hybridization assays. High sensitivity means that low concentrations of a probe–target hybrid can be detected; for example, 32P-labeled probes can detect single-copy genes in only 0.5 µg of DNA.

Compared to radioactive labels, the use of nonradioactive labels have several advantages like safety, Higher stability of probe, Efficiency of the labeling reaction, Detection in situ, Less time taken to detect the signal. Concern over laboratory safety and the economic and environmental aspects of radioactive waste disposal have been key factors in their development and use. Some examples are as follows:

The restriction fragment length polymorphisms (RFLPs) can be studied in a set of related species using a random or a specific DNA probe. The similarities and differences can be used to infer phylogenetic relationships. This has actually been done in a number of cases both in plants and in animals (Gill et al. 1991; Fukuchi et al. 1993; Moore et al. 1991; Mason-Gamer et al. 1998; Deshpande et al. 1998; Drinkwater et al. 1991; Mburu and Hanotte, 2005; Guimaraes, 2007).

DNA polymorphisms (many forms) are differences in DNA sequence that result from point mutations, random deletions or insertions or the presence of varying number of repeated copies of a DNA fragments (tandem repeats). A polymorphism in the coding region of a gene may be detected as an alteration in the amino acid sequence of the encoded protein. It is now possible to detect polymorphisms in unexpressed regions of DNA by this analysis. Genetic disorders like sickle cell anemia, Thalassemia’s, Huntington’s disease and cystic fibrosis were identified through RFLP mapping analysis which demonstrates power of RFLP/Linkage analysis.

Specific molecular probes can be used for isolation of specific genes. These probes may be available either from same species or from another species can be used for isolation of genes. If probes obtained from one species used for isolation of gene from the same species they are called as homologous probes. If probes obtained from another species used for isolation of genes in other species they are defined as heterologous probes.

In situ hybridization (ISH) is a technique which permits detection of DNA or RNA sequences in cell smears, tissue sections and metaphase chromosome spreads. The method is based on the formation of double stranded hybrid molecules which form between a DNA or RNA target sequence and the complementary single standard labeled probe. In a number of cases rye chromosomes in wheat background have been identified using this technique.

DNA fingerprinting is a way of identifying a specific individual, rather than simply identifying a species or some particular trait. It is also known as genetic fingerprinting or DNA profiling. Inventor of this technology is Sir Alec Jeffreys in 1985. DNA fingerprinting is currently used both for identifying paternity or maternity and for identifying criminals or victims (Table 1 & 2).

A change to the DNA sequence is a mutation, e.g. deletion, insertion, substitution. Changes in certain gene sequences can cause inherited diseases and their detection by probes can be diagnostic:

Cystic fibrosis (due to a 3 bp deletion).