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• Organisation of the Human Genome

  • Nucleus
  • DNA
  • RNA
  • Nuclear noncoding RNA (Gomafu, Xist, NEAT1, NEAT2)
  • Messenger RNA
  • Noncoding RNA
  • Cytoplasm
  • Protein
  • Cell
• Protein-coding genes may belong to families - these arise by gene duplication
• Sometimes non-functional gene-related sequence is present = pseudogenes
• The non-protein-coding portion of the genome is of crucial functional importance: both for normal development and physiology and for disease
• Non-coding RNA is functionally important in the regulation of protein coding genes
• How to analyse DNA

  1. Selectively amplify DNA by PCR and cloning

  2. Obtain sufficient quantity of DNA sequence to be analysed
• PCR (Polymerase Chain Reaction)

  1. Template DNA

  2. Denaturation at 94°C-98°C

  3. Annealing at 55°C-70°C

  4. Extension at 68°C-72°C

  5. Cyclical process of heating and cooling
• PCR makes millions of copies of a specific DNA sequence in approximately 2 hours
• Applications of PCR


  • Substitute for cloning
  • Amplify very specific sequences from small amounts
  • Detect DNA sequences not normally present
  • Analyse highly degraded DNA samples
• DNA separation by Gel Electrophoresis

  1. Polyacrylamide gels for single stranded DNA

  2. Agarose gels for 300-20,000 nucleotide molecules

  3. Pulsed-field gel electrophoresis for long DNA molecules
• Analysis of RNA


  1. Reverse transcriptase-polymerase chain reaction (RT-PCR)

  2. Real-time RT-PCR to assess gene expression
• Reverse transcriptase-polymerase chain reaction (RT-PCR)

  Analyses RNA-dependent DNA polymerase activity, RNase H activity, and DNA-dependent DNA polymerase activity to produce ds-cDNA
• Real-time RT-PCR


  Used to assess the 'gene expression' or abundance of individual RNAs in a sample, by monitoring accumulation of amplified DNA via changes in fluorescence
• TaqMan probe


  Binds to gene target in a sequence specific manner, with fluorophore cleaved by Taq polymerase during PCR to allow fluorescence
• Threshold cycle (Ct)


  The PCR cycle where target amplification is first detected, with lower Ct indicating higher initial target quantity
• Hybridisation


  Formation of double-stranded DNA between two single strands with complementary sequences, allowing identification of specific DNA sequences
• Microarray analysis of gene expression

  • Simultaneous analysis of expression profiles of thousands of genes, unlike RT-PCR which tests one gene at a time
• Single Nucleotide Polymorphisms (SNPs) occur about 1 in every 300 nucleotides, so there are around 10 million SNPs in the human genome
• The HapMap project looked at 'common genetic variation', and was a stepping stone for the 1000 Genomes project
• SNPs serve as landmarks in the search for genes associated with disease risk, drug responses, and complex phenotypes
• Genome-wide association studies (GWAS)


  Search the genome for genetic variants (SNPs), looking at thousands of nucleotide variants at the same time
• Array Comparative Genome Hybridisation (aCGH)

  Patient and control DNA are labeled with fluorescent dyes and applied to a microarray, where they compete to hybridize, then the microarray is scanned and analyzed
• aCGH can be used to scan a complete genome for imbalances, and can simultaneously detect aneuploidies, deletions and duplications
• Protein analysis methods


  1. Western blot (immunoblotting)

  2. Immunohistochemistry (IHC)

  3. Immunoassay (Radioimmunoassay (RIA), Enzyme linked immunosorbant assay (ELISA))
• Western blot


  Involves detection of polypeptides after size-fractionation of a tissue lysate on a polyacrylamide gel
• Immunohistochemistry (IHC)


  Studies the overall protein expression across a slice of tissue
• Immunoassay


  Antibodies can be used to quantitate the amount of a protein or antigen in a lysate, using radioisotope or enzyme-linked detection
• Immunohistochemistry studies the overall expression pattern at the protein level within a section of tissue to reveal the amount and cellular location but not protein size
• Western blotting assesses the amount and size of the protein but not its cellular location