Principles of nucleic acid extraction

What are the principles of nucleic acid extraction?

1. tissue disruption/homogenisation

2. cell lysis

3. separation of DNA from other cellular components

4. DNA precipitation

5. DNA washing

6. DNA resuspension/elution for downstream processing

What occurs during tissue disruption/homogenisation?

tissue is ground to separate cells from each other and break down cell walls

e.g., motar and pestle

use dry ice or liquid nitrogen

What occurs during cell lysis?

cell/nuclear membranes must also be disrupted to release DNA

extraction buffer containing a detergent and/or salts

these break down lipids in the membranes 

What occurs during the separation of DNA from other cellular components? 

to get a clean sample of DNA, it’s necessary to remove as much of the cellular debris as possible 

a protease is added to degrade DNA-associated proteins and other cellular proteins 

What occurs in DNA precipitation?

the DNA is separated by precipitation out of solution

using alcohol 

DNA is soluble in water but insoluble in the presence of salt and alcohol 

What occurs in DNA washing?

the precipitated DNA is pelleted (via centrifugation) or bound to a membrane 

wash with alcohol (70% ethanol) to remove contaminants such as salts that may impact downstream applications

What occurs in DNA resuspension/elution?

the DNA is then resuspended in solution or eluted from the column using 

1. water

2. TE buffer

3. Tris-Cl buffer 

What are column based/commerical kits?

• selective adsorption of DNA/RNA to silica gel membrane 

• same basic principles of extraction

• no precipitation required 

• DNA/RNA is eluted using low salt buffer 

advantages of kits: 

• increased purity 

• high throughput

• simple and reliable

• quick 

How is DNA measured using agarose gel electrophoresis? 

• DNA fragments are drawn through pores in agarose gel using an electrical field 

• negatively charged DNA migrates through the pores of an agarose gel towards the positively charged end of the gel when an electrical current is applied 

• this separates the fragments based on size 

• a dye is added that binds to the DNA - and can be visualised under UV 

• this method is QUALITATIVE

How is DNA/RNA measured using a spectrophotometer? 

nucleic acids have a peak absorbance at 260nm

measure the absorbance at 260nm to determine concentration ng/ul

purity of sample - proteins

ratio of OD 260/230 

DNA ~1.8; RNA `2.0 = pure sample