DNA Extraction

Question 1

LO

Answer 1

*** What are the purposes of DNA extraction?
* What are the types of extraction?
* What are the steps involved?
* Advantages and disadvantages?
* Differential extraction **

Question 2

What are types of sample and evidence collections?

Answer 2

Buccal swabs
Blood samples (IV and finger)

Question 3

What are the different types of forensic samples?

Answer 3

• Body fluids
• Swabs
• Clothing
• ‘Touch’
• Hairs
• Nails
• Bone
• Teeth
• Each sample needs a different extraction method
  e.g., bone contains lots of Ca2+ which can interfere with downstream processes so would need to go through decalcification processes first

Question 4

Why do we have to isolate DNA for DNA extraction

Answer 4

Swabs and blood samples can have other things that could act as inhibitors on/in them

Question 5

What are the three main goals of extraction?

Answer 5

Question 6

What are some inhibitors to DNA extraction?

Answer 6

Question 7

Name a method used for extraction and what it is and how it works

Answer 7

**Chelex-100 extraction **

• **Chelating **material from Bio-Rad
  o Made of styrene-divinylbenzene co-polymers containing paired iminodiacetic ions
• Under alkaline conditions will bind metal ions (especially Mg2+)
• Prevents DNAse from degrading DNA at high temperature lysis
• Polar resin beads bind cellular components whilst DNA and RNA remains within an aqueous phase
• Centrifugation draws beads to the bottom of the tube, allowing removal of **DNA within the supernatant **

Question 8

What is the chelex-100 extraction procedure?

Answer 8

• Samples are vortexed to release cells in water
• Osmotic shock causes cell lysis
• Samples are centrifuged to separate cellular material from contaminants, DNA at bottom of tube
• 5% Chelex bead suspension is added, and sample is re-suspended
• Heat (56 and 100˚c) and alkalinity degrade proteins (lyse)
  o Lack of Mg2+ prevents DNA damage
• Centrifugation draws beads, material away from aqueous phase DNA (Separation)
  o DNA isolated is single stranded (due to heating to 100˚c and has been denatured)
  o DNA from mitochondria will also be released

Question 9

What are the advantages and disadvantages to chelex-100 extraction?

Answer 9

Question 10

Tell me about the use of solid phase extraction (SPE)

Answer 10

• Selection of DNA extraction methods based on the ‘bind-wash-elute’ principle- the most popular produced by **Qiagen **
• DNA is selectively bound to silica membranes whilst proteins and other cellular components are actively removed
• Cells lysed with high concentrations chaotropic salts (chaotropic agent is a molecule in water solution that can disrupt the hydrogen bonding network between water molecules)- helps destabilise proteins and allows DNA to interact with silica
• A series of wash steps enhance the binding of DNA to silica whilst removing lysed cellular material and contaminants
• Changes in pH allow the elution of DNA from the membrane

Question 11

Tell me the QIAmp DNA investigator procedure

Answer 11

Question 12

What are the advantages and disadvantages to SPE?

Answer 12

Question 13

What is differential extraction and when is it used?

Answer 13

• The isolation of DNA from two types of cells simultaneously without mixing their contents (only works if one of the cells is a sperm cell)
• Used in sexual assault cases

Question 14

What are the two lysing procedures in differential extraction?

Answer 14

o Female epithelial cells- SDS, proteinase K
o Male sperm cells- SDS, Proteinase K and DTT

Question 15

What does differential extraction depend on?

Answer 15

• Relies on the fact that sperm cell membranes are full of di-sulphide bonds impervious to degradation without DTT
• Will not work for azoospermia males or highly degraded samples

Question 16

Differential extraction procedure

Answer 16

Question 17

What are the controls to differential extraction?

Answer 17

*** Regent extraction negative: **check for contamination in any of the extraction components
o buffers, SDW, swabs
* Item extraction negative: e.g., check that any DNA extracted is from the stained area as other areas of item do not produce usable DNA
* Generally, **no positive control **used- however, method should be validated for extraction from that matrix

Question 18

Summary

Answer 18

• 3 main goals of extraction
  o Lyse the cells to release DNA
  o Separate the DNA molecules from other cellular materials
  o Isolate the DNA so that it Is compatible for downstream analysis
• Chelex-100 is a quick, cheap method
  o Used for reference samples
• Spin column based SPE (e.g., QIAmp DNA investigator)- better DNA recovery and inhibitor removal
  o Used for forensic samples
• Can isolate both epithelial and sperm DNA using differential extraction