DNA Extraction Flashcards
LO
*** What are the purposes of DNA extraction?
* What are the types of extraction?
* What are the steps involved?
* Advantages and disadvantages?
* Differential extraction **
What are types of sample and evidence collections?
Buccal swabs
Blood samples (IV and finger)
What are the different types of forensic samples?
- 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
Why do we have to isolate DNA for DNA extraction
Swabs and blood samples can have other things that could act as inhibitors on/in them
What are the three main goals of extraction?
What are some inhibitors to DNA extraction?
Name a method used for extraction and what it is and how it works
**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 **
What is the chelex-100 extraction procedure?
- 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
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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
What are the advantages and disadvantages to chelex-100 extraction?
Tell me about the use of solid phase extraction (SPE)
- 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
Tell me the QIAmp DNA investigator procedure
What are the advantages and disadvantages to SPE?
What is differential extraction and when is it used?
- 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
What are the two lysing procedures in differential extraction?
o Female epithelial cells- SDS, proteinase K
o Male sperm cells- SDS, Proteinase K and DTT
What does differential extraction depend on?
- 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
Differential extraction procedure
