Applications of nucleotides in molecular diagnostics:

Polymerase Chain Reaction (PCR): PCR is a fundamental technique that amplifies specific DNA regions. It utilizes nucleotides as substrates for DNA polymerase enzymes, which synthesize complementary DNA strands to the target sequence. By repeatedly cycling through heating, annealing, and extension steps, PCR exponentially increases the amount of target DNA, enabling its detection and analysis.

Nucleic Acid Hybridization: This technique relies on the specific base pairing between complementary nucleotide sequences. Single-stranded DNA probes, containing nucleotides complementary to the target sequence, are used to identify and bind to specific DNA or RNA molecules in a sample. The probes can be labelled with fluorescent dyes or other detectable markers, allowing visualization and quantification of the target molecules.

Sanger Sequencing: This traditional DNA sequencing method utilizes dideoxynucleotides (ddNTPs), modified nucleotides that lack a 3′-hydroxyl group essential for further phosphodiester bond formation. By incorporating ddNTPs during DNA synthesis, the chain extension terminates at specific points, revealing the sequence of the DNA strand.

Next-Generation Sequencing (NGS): NGS technologies employ sequencing-by-synthesis approaches. Nucleotides labelled with fluorescent dyes are sequentially incorporated into growing DNA strands. The detection of each incorporated nucleotide allows for the identification of the corresponding base in the target sequence. NGS offers high-throughput sequencing of entire genomes or targeted regions, aiding in genetic analysis, variant detection, and personalized medicine.

In Situ Hybridization (ISH): This technique involves hybridizing probes directly to cells or tissues fixed on slides. By employing nucleotide probes specific for a particular gene or mRNA, ISH enables the visualization of gene expression patterns within cells or tissues.