Molecular Probes Overview

Molecular Probes

• Molecular probes are small DNA/RNA segments used to detect complementary sequences in nucleic acid samples.
• Antibodies (Abs) are probes used to recognize specific protein sequences.
• Both DNA and RNA can be used as probes. Single-stranded DNA (ssDNA) probes are more convenient and preferable. Denatured double-stranded DNA (dsDNA) is also used. RNA probes are typically single-stranded (ss).

Preparation of Molecular Probes

• Genomic DNA probes:
  • Extract DNA.
  • Digest with restriction enzymes.
  • Run electrophoresis (AGE/PAGE).
  • Isolate DNA.
  • Clone into a vector.
  • Multiply in bacteria.
• cDNA probes: Synthesized from isolated mRNA using reverse transcriptase. Then cloned and used as probes.
• Synthetic oligonucleotides: Probes with known sequences synthesized chemically using automated DNA synthesizers.
• RNA probes/riboprobes: DNA template cloned into an expression vector. The vector has a different and specific prokaryotic promoter beyond the two ends of the DNA insert. Recombinant vector is linearized and transcribed using appropriate RNA polymerase to obtain RNA molecules that are complementary to one or the other strand of the DNA insert.

Labelling of Probes

• Radioactive labelling: Commonly used radioisotopes include 32P, 3H, 35S, and 125I. Methods include nick translation, oligonucleotide labelling, and riboprobe preparation.
• Non-radioactive labelling: Fluorophores and haptens are commonly used. Examples of haptens include biotin and digoxigenin.
  • Fluorophores: Directly detected by fluorescence spectroscopy. Classical dyes like rhodamine, fluorescein, and cyanine derivatives are widely used.
  • Biotin-labelled probes: Prepared through nick translation. Detection of hybrids is done through cytochemical reactions that produce a blue color, with intensity proportional to the amount of biotin in the hybrid.
  • Digoxigenin-labelled probes: Digoxigenin (DIG) is widely used for immune detection.

Applications of Probes

• Identification of recombinant clones with the desired DNA insert.
• Confirmation of DNA insert integration into the host genome.
• Development of Restriction Fragment Length Polymorphism (RFLP) maps.
• DNA fingerprinting for identifying plant varieties, criminals, and determining parental relationships.
• In situ hybridization for determining locations of specific sequences in chromosomes.
• Accurate diagnosis of diseases caused by parasites, pathogens, or defective viruses.
• Preparation of genome maps of eukaryotes.