How to obtain DNA from an animal cell
- open the cell membrane to gain access to the nucleus
- need to break down nuclear membrane
- DNA has chaperone proteins, which help protect it.
- need to break down chaperone proteins and purify DNA in an environment that protects it
what type of DNA is from extraction?
Nuclear/Mitochondrial DNA.
After extraction, you have DNA from everywhere including contaminated DNA
Principles of Extraction
Vary based on evidence type.
Method of choice=yields sufficient quantity, good quality, and high purity of DNA
Always isolating total cellular DNA
Steps of DNA processing
Extraction, Quantitation, Amplification, Electrophoresis, Analysis
What is the goal of cell/tissue disruption?
gain access to the cells containing DNA
What are the techniques to obtain DNA?
- cell/tissue disruption
- lysis of cellular & organelle materials
- removal of proteins
- DNA storage
(step 1 & 2 often occur at the same time; except with bone, teeth, and hair)
What do you do during the removal of proteins?
Remove any and all impurities that will interfere with analysis.
AKA “washing steps”
Why, Where, and How do you store samples of DNA?
why: prevent degradation
where: cold temperatures (-20 to 80 c)
How: kept in buffer that inactives nucleases
Types of Contamination
Individual to sample
sample to sample
amplified to non amplified
What are some systematic checks used?
- Reagant Blank (negative control)
- use of pre/post PCR room
- separate processing of evidence and reference sample
- use of DNA free reagants
- lab employees submit DNA samples
What happens during the lysis of cellular/organic membrane step?
- Once cells are accessible, need to disrupt cell membrane, organelles, and proteins protecting DNA.
- Performed with Salts, Detergents, Enzymes, and Heat.
- need to be performed with a buffer to protect from nucleases.
What are the three methods of extraction?
- Phenol Chloroform (organic): solvent base
- Chelex: boiling base
- Silica based methods
Organic Extraction: Cell Lysis/Tissue Disruption
Breakdown cell membrane and proteins protecting the DNA using proteinase K (pro k) and a 56 c incubation.
(proteinase is an enzyme that breaks down protein)
Organic Extraction: Removal of Protein/ Impurities
to remove the proteins a Phenol-Chloroform mixture is added to separate from the aqueous layer by moving to the bottom of the tube. DNA is found at the top (aqueous layer), lipids at the bottom (organic), and proteins at the interface.
Once DNA is in the aqueous solution, use salts and ethanol to remove impurities. DNA is precipitated out and then can be eluted into a buffer for safe storage.
Pros & Cons of Organic Extraction
PROS
- large, double stranded DNA
- good quality
- good yield
- often considered the “gold standard” in terms of quality and yield.
CONS
- laborious
- hazardous
- multiple tube transfer
Chelex Procedure
- washing
- boiling
- centrifugation
Washing Step
(This step is only necessary if there is a known inhibitor like indigo dye for blood.)
Basically, the sample is “washed”, meaning it is diluted with a buffer that also dilutes the inhibitors
Boiling
- a 5 to 10% solution of Chelex is added to samples
- Initially, samples are incubated at 56 c to soften and separate clumps of cells
- followed by an incubation of 90 to 99 c, which breaks down the cellular/nuclear membrane, and chaperone proteins
- leaves DNA (single stranded because of heat) out in solution
Chelex: Protein Removal and Centrifugation
samples are centrifuged (spun down really fast) so chelex and cell debris move to the bottom.
DNA is in the supernatant
Pros and Cons of Chelex
PROS
- Simple
- rapid
- one tube
- cheap
CONS
- single stranded DNA
- very fickle
Silica Based Method
DNA is reversibly adsorbed on to the silica surfaces in the presence of chaotropic salts…salts promote an environment were DNA wants to stick to the membrane
Silica Based Method: Cell lysis and protein digestion
Break open cell/organelle membranes through Pro K. DNA is released.
Pros and Cons: Silica
PROS
- high quality DNA
- double stranded DNA
- amenable to automation
CON
- expensive
- multiple tube transfers
Differential Extraction
(used in sexual assault cases, there is a mixture of cells that need to be effectively separated.)
- Sperm Heads (acrosome) are extremely strong and can NOT be broken down by Pro K.
- incubation of the sample will only open epithelial cells and the enzyme DTT can open sperm heads.
