Identifying Mutations (Diagnostics L3)

  • Created by: Hpgrice
  • Created on: 04-11-19 12:05

Types of Mutations

  • Base substitutions
  • Small Deletions (one or few bases)
  • Large deletions (whole exons or genes)
  • Small insertions
  • Duplications
  • Expansions of tri/ tetra nucleotide repeats
  • Imprinting Errors e.g. DNA methylation

Human Genome Variaiton Society (HGVS) Nomenclature

  • Internationally recognsised standard which allows changes to be named:
    • at the DNA level: genomic (g) or cDNA (c) 
    • at the protein level (p. prefix)
    • at the RNA level (r. prefix)
    • mitochondrial DNA (m. prefix)
  • The nucleotide fo a variant is written as c.76A>G counting from the A of the ATG start codon (using cDNA reference sequence)
  • An amino acid change is written as p.Gly251Arg
  • Deletions: c.76_79delAATC
  • Duplications: c.76_79dupAATC
  • The bases in introns are not numbered the same. Sequence numbers stops at the end of an exon and continues at the start of the next exon.
  • The intron bases are named using +1, +2 or -1, -2 etc. For example: if the last base in the exon is 326 then the intron bases would be named 326+1, 326+2, 326+3 and so on. If closer to the next exon then they would be named 327-1, 327-2, 327-3 and so on
    • The mutation would be described as: c.326+1G>A or c.327-1G>T

Basic Techniques for Molecular Analysis

  • DNA extraction
  • PCR
  • Agarose gel and capillary electrophoresis
  • Southern blotting
  • DNA sequencing

DNA Extraction

  • Usually blood samples but can use other tissues such as blood spots, buccal swabs, separated cells, paraffin blocks, solid tissue, amniocytes, chorionic villus biopsies
  • Mostly automated extraction


  • In vitro replication of a specific segment of DNA
  • newly synthesised molecules are the templates for repeated rounds of replication
  • exponential reaction
  • Detect PCR products using gel electrophoresis and stain with DNA specific stain e.g. Ethidium bromide
  • Can fluorescently label
  • Capillary electrophoresis can be used to separate out DNA by charge to size ratio. Fluroescent laser detection
    • quantitative
    • easier to automate
    • much better sizing
    • multiplex to higher degree and can use for sequencing
  • Advantages:
    • small amount of starting material
    • highly specific and sensitive
    • rapid
  • Limitations
    • only applicable to small stretches of DNA
    • cotamination can be a problem because sensitive
    • some sequence knowledge is required
    • additional downstream steps often required
    • standard PCR is not quantitative

Southern Blotting

  • Step 1: Prepare electrophoretic gel
    • whole genomic DNA -> Digest with restriciton enzyme ->separate by electrophoresis
  • Step 2: Transfer to blotting membrane
  • Step 3: Prepare probe for gene/ DNA fragment of interest
    • radioactively label using 32P-dCTP and DNA polymerase
  • Step 4: Hybridise probe to blotting membrane
    • add radioactive probe to membrane
    • incubate at 68 degrees C for several hours
  • Step 5: Autoradiography
    • wash unbound probe away


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