Identifying Mutations (Diagnostics L3)
- Created by: Former Member
- Created on: 04-11-19 12:05
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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
PCR
- 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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