DNA extraction

Question 1

DNA isolation, DNA purification DNA extraction

Answer 1

aims to maximise yield of DNA from a sample and extract pure DNA

Question 2

3 Main steps

Answer 2

1) Disruption of cell membranes, cell lysis
2) protein denaturation
3) Separation of DNA from denatured proteins and other cell debris

(denaturation, extraction, annealing)

Question 3

Different methods of DNA extraction- which one used will depend on sample type

Answer 3

• Sodium hydroxide/ tris-hcl
• Chelex
• Column extraction
• Magnetic beads
• Phenol chloroform
• Differential extraction

Question 4

Sodium hydroxide

Answer 4

• Quick and dirty
• 3 steps:
• Add alkaline NaOH, solubilises membrane proteins
• Incubation 75 degrees bursts open cells
• Add tris hcl- stops reaction

Question 5

Chelex 100

Answer 5

• Chelating resin supplied as beads
• A 5% solution is usally used
• Incubation to rupture cells (with or without proteinase K)
• Chelex has a high affinity for metal ions such as as magnesium
• Magnesium ions are co factors for DNAase enzymes and therefor chelex inactivates the enzymes, protecting DNA
• Beware carry over into PCR

Question 6

Proteinase K

Answer 6

• Used in many DNA extraction protocols
• Broad specificity proteinase
• Removes contaminating proteins and nucleases from cells

Question 7

Column extraction

Answer 7

• Process begins with lysis
• DNA binds to silica membrane in column
• A series of wash steps is carried out
• Change of pH at end of protocol releases DNA from membrane into solution (elution)

More steps so more can go wrong as it’s a longer process

• Expensive
• But gives a purer sample at the end

Question 8

Magnetic Beads

Answer 8

• Similar principle to column extraction, different format
• Easily automated

1) addition of magnetic beads
2) specific binding
3) supernatant removal
4) wash & elution

Question 9

differential extraction

Answer 9

• 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

Question 10

contamination

Answer 10

forensic samples may have low levels of DNA present

• anticontamination procedures vital
• any contamination introduced at extraction stage will be carried though whole down- stream process