DNA Extraction

Question 1

How to obtain DNA from an animal cell

Answer 1

1. open the cell membrane to gain access to the nucleus
2. need to break down nuclear membrane
3. DNA has chaperone proteins, which help protect it.
4. need to break down chaperone proteins and purify DNA in an environment that protects it

Question 2

what type of DNA is from extraction?

Answer 2

Nuclear/Mitochondrial DNA.

After extraction, you have DNA from everywhere including contaminated DNA

Question 3

Principles of Extraction

Answer 3

Vary based on evidence type.
Method of choice=yields sufficient quantity, good quality, and high purity of DNA
Always isolating total cellular DNA

Question 4

Steps of DNA processing

Answer 4

Extraction, Quantitation, Amplification, Electrophoresis, Analysis

Question 5

What is the goal of cell/tissue disruption?

Answer 5

gain access to the cells containing DNA

Question 6

What are the techniques to obtain DNA?

Answer 6

1. cell/tissue disruption
2. lysis of cellular & organelle materials
3. removal of proteins
4. DNA storage
   (step 1 & 2 often occur at the same time; except with bone, teeth, and hair)

Question 7

What do you do during the removal of proteins?

Answer 7

Remove any and all impurities that will interfere with analysis.
AKA “washing steps”

Question 8

Why, Where, and How do you store samples of DNA?

Answer 8

why: prevent degradation
where: cold temperatures (-20 to 80 c)
How: kept in buffer that inactives nucleases

Question 9

Types of Contamination

Answer 9

Individual to sample
sample to sample
amplified to non amplified

Question 10

What are some systematic checks used?

Answer 10

• 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

Question 11

What happens during the lysis of cellular/organic membrane step?

Answer 11

• 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.

Question 12

What are the three methods of extraction?

Answer 12

• Phenol Chloroform (organic): solvent base
• Chelex: boiling base
• Silica based methods

Question 13

Organic Extraction: Cell Lysis/Tissue Disruption

Answer 13

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)

Question 14

Organic Extraction: Removal of Protein/ Impurities

Answer 14

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.

Question 15

Pros & Cons of Organic Extraction

Answer 15

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

Question 16

Chelex Procedure

Answer 16

1. washing
2. boiling
3. centrifugation

Question 17

Washing Step

Answer 17

(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

Question 18

Boiling

Answer 18

• 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

Question 19

Chelex: Protein Removal and Centrifugation

Answer 19

samples are centrifuged (spun down really fast) so chelex and cell debris move to the bottom.
DNA is in the supernatant

Question 20

Pros and Cons of Chelex

Answer 20

PROS

• Simple
• rapid
• one tube
• cheap

CONS

• single stranded DNA
• very fickle

Question 21

Silica Based Method

Answer 21

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

Question 22

Silica Based Method: Cell lysis and protein digestion

Answer 22

Break open cell/organelle membranes through Pro K. DNA is released.

Question 23

Pros and Cons: Silica

Answer 23

PROS

• high quality DNA
• double stranded DNA
• amenable to automation

CON

• expensive
• multiple tube transfers

Question 24

Differential Extraction

Answer 24

(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.