DNA extraction Flashcards
what two other terms can DNA extraction also be referred to
DNA isolation
DNA purification
what are the two main aims
maximise yield of DNA from a sample
extract pure DNA
what are the three main steps
- disruption of cell membranes, cell lysis
- protein denaturation
- separation of DN from denatured proteins and other cell debris
sodium hydroxide method
- add alkaline NaOH = solubilises membrane proteins
- incubation 75 degrees = bursts open cells
- add Tris-HCl = stops reaction
chelex 100 method
chelating resin supplied as beads
5% solution used
incubation to rupture cells
chelex has high affinity for metal ions
magnesium ions are co-factors for DNAase enzymes and there chelex inactivates the enzymes, protecting DNA
beware carry-over into PCR
proteinase K method
used in many DNA extraction protocols
broad specificity proteinase
removes contaminating proteins and nucleases from cells
column extraction method
process begins with lysis
DNA binds to silica membraned in column
a series of wash-steps carried out
change of pH at end of protocol, releases DNA from membrane in solution
disadvantages of column extraction
more steps
longer process
more can go wrong
expensive
advantage of column extraction
purer sample at the end
magnetic beads method
similar to column extraction but different format
easily automated
addition of magnetic beads
specific binding
supernatant removal
wash and elution
differential extraction method
DNA from 2 different types of cells can be extracted without mixing
often used in sexual assault cases to separate sperm cells from epithelial cells
sperm cells more resilient to membrane disruption
contamination
forensic samples may have low levels of DNA present
anti-contamination procedures vital
any contamination introduced at extraction stage will be carried through whole down-stream process
