PCR & NAH Applications

• PCR (Polymerase Chain Reaction)


  Allows small samples of DNA to be amplified infinitely
• PCR


  • DNA sample can be pure or a small part of a mixture of materials
• RT-PCR (Reverse-Transcription-PCR)


  RNA can also be analysed and amplified. RNA sample is transcribed into cDNA, which then undergoes usual PCR
• Real-Time PCR (qPCR)


  Amplified DNA product is measured after each cycle. Fluorescent molecule in each reaction fluoresces, and fluorescence increases as the product increases
• Real-Time PCR (qPCR)


  • Closed-tube reactions and data evaluation reduces contamination risk
• Applications of Real-Time PCR (qPCR)


  • Relative, semi-quantitative determination of the number of particular DNA/RNA copies present within a diverse sample
  • Measurement of gene copy number (gene dosage)
  • Quantitate changes in gene expression by measuring changes in cellular mRNA (RT-PCR)
• How Real-Time PCR (qPCR) works

  1. DNA dye-based method (fluorescence): a fluorescent dye binds to the dsDNA as it amplifies. Fluorescence intensity increases as the product increases.

  2. Probe-based method: labelled probe that binds to the target sequence. Probe contains a reporter dye and a quencher. Upon hybridisation, Taq polymerase extends the DNA, displacing the probe and separating the 2 molecules, resulting in fluorescent light emission
• Real-Time PCR (qPCR) relies on specific enzymatic properties of DNA polymerase and real-time fluorescence detection. Some polymerases (i.e. Taq) harbour 5' to 3' exonuclease activity
• In Real-Time PCR (qPCR), the number of cycles it takes depends the number of copies present at the start. More copies = fewer cycles needed to accumulate enough product
• In Real-Time PCR (qPCR), the results can be compared to determine relative starting concentration of material
• Digital PCR enables precise, sensitive quantification of DNA/RNA, but is more expensive and time-consuming compared to Real-Time PCR (qPCR)
• How Digital PCR works

  1. Sample partitioning: PCR sample is partitioned into thousands of droplets, each containing 1, 0, or a few template molecule (micro-reactors)

  2. Each droplet acts as an individual PCR reaction

  3. Post-amplification, droplets containing the target sequence are detected by fluorescence and scored as positive

  4. Poisson statistical analysis quantifies the starting template quantity
• Applications of Digital PCR


  • Enables detection of rare allele variation, such as SNPs. Unlike qPCR, signal from wild-types doesn't dominate and obscure the signal from rarer sequences
• Nucleic Acid Hybridisation (NAH)


  Uses synthesised labelled oligonucleotide probes to detect the presence of DNA/RNA
• How Nucleic Acid Hybridisation (NAH) works


  1. Denatured sample is fixed to a solid support

  2. Oligonucleotide probes are added and hybridise to complementary sample DNA

  3. A wash is performed to remove excess probes that are obsolete
• Applications of Nucleic Acid Hybridisation (NAH)


  • In situ hybridisation: can be used to detect presence of DNA/RNA in cells/tissues following fixation. Probes are usually fluorescently labelled (FISH). Widely used to detect specific gene transcription and localise genes to specific chromosomes in developing embryos
  • Microarrays: thousands to millions of known oligonucleotide probes, that are complementary to all the known protein-coding genes in the genome, are bound to a solid surface, known as the chip. The chip is bathed with fluorescently labelled DNA/RNA, isolated from a sample. Hybridisation: complementary base pairing between the sample and chip-immobilised fragments produces light through fluorescence that can be detected by a computer
• Microarrays are used in large-scale applications, e.g. GWAS, transcriptome analysis, because it's cheaper than sequencing
• Microarrays are very commonly used for large-scale gene expression studies
• A microarray is a silicon grid, with numerous spots. Each spot has many copies of a complimentary oligonucleotide attached. Enough spots so that all protein coding genes are covered