3-5.4.1 DNA probes:

Conventional DNA probes are isolated by cell-based DNA cloning or by PCR. In cell-based DNA cloning, the starting DNA can range from 0.1 kb to a range of ~10kb in length and is often double-stranded. PCR-derived DNA probes are usually 10 kb long and are usually double-stranded. These DNA probes are labeled using labeled dNTPs during in vitro synthesis.

3-5.4.2 RNA probes:

Detection of specific nucleic acid sequences can be accomplished by hybridization with a labeled RNA probe. RNA probes are sequences of a variable length that are used to detect the presence of complementary nucleotide sequences in a sample. RNA probes are first labeled with radioactive nucleotides or with modified nucleotides that can be detected by fluorescence or chemiluminescence. They can be used for Northern blotting, RNase protection assays, Southern blotting, downstream of polymerase chain reaction (PCR), and in situ hybridization analysis.

3-5.4.2 Synthetic probes:

These probes are chemically synthesized which can generate short single stranded stretches 15–50 bases in length. They are usually highly specific as they are designed particularly for target DNA. Probes with degenerate sequences can also be synthesized using protein sequences. Degeneracy is introduced when parallel synthesis of oligonucleotides is done with similarity at some sites and difference at others. These probes are usually labeled using 32 P isotope or other labeled group at the 5′ end.

3-5.5 Labeling Of Probes:

Probes can be labeled at specific location within the oligonucleotides or internally at multiple sites. Some probes are of defined length and some are heterogeneous populations of labeled molecules.

In vivo labeling: DNA and RNA can be directly labeled inside tissue culture cells by adding labeled deoxynucleotides in culture plate in vivo. This method is restricted only to prepare labeled viral DNA from virus-infected cells and to study RNA processing events.

In vitro labeling: It is a more versatile method involving in vitro labeling of purified RNA, DNA or oligonucleotide using DNA polymerasesfor incorporation of labeled nucleotides.

3-5.5.1 L abeling during synthesis:

In vitro labeling of DNA can be done by various methods as follows:

a) Nick-translation

b) Random primed labeling

c) PCR-mediated labeling- Labeling of RNA is generally accomplished by an in vitro transcription system.These procedures require DNA or RNA polymerases to add labeled nucleotides to synthesize in vitro probes. It requires at least one labeled nucleotides among four nucleotides.