Nucleic acid extraction

Question 1

What are the different types of nucleic acids?

Answer 1

• Genomic/chromosomal (DNA)
• Plasmids
• Different RNAs -> mRNA, rRNA, tRNA and miRNA (this is much smaller and binds mRNA to prevent protein production)

Question 2

What are the rough stages of Nucleic Acid Extraction (NAE)?

Answer 2

1. Cell disruption
2. Removal of membrane lipids, proteins and other nucleic acids
3. Nucleic acid purification/binding from the bulk
4. Nucleic acid concentration

Question 3

How is cell disruption generally carried out?

Answer 3

Chemically or mechanically, varying with sample type. These can also be used in combination.

Main relevant examples may include:
- Alkali treatment for plasmid DNA
- Detergent treatment for general use
[the above two make the membrane soluble using chemical techniques]
- Homogenisation (blade or pestle) for animal tissues
[this shreds the cells mechanically]

Question 4

What is a potentially unexpected benefit of Nucleic Acid Extraction?

Answer 4

Recombinant proteins can be recovered, as well as important cellular constituents used in the bio-pharmaceutical industry other than just nucleic acids.

Question 5

How can NAE methods usually be divided?

Answer 5

1. Chemically driven methods
2. Solid-phase methods

Both contain similar steps.

Question 6

What are two examples of chemically-driven NAE methods? Give the advantages and disadvantages of each.

Answer 6

1. GuSCN-phenol-chloroform -> high purity and yield of DNA/RNA, but requires hazardous chemicals
2. Alkaline extraction -> fast, reliable and relatively easy, but produces medium purity and potential fragmentation of genomic DNA

Question 7

What do chemically driven NAE methods rely on?

Answer 7

The biochemical properties of the cellular components.
These may extract DNA or RNA exclusively.

Question 8

What are solid-phase extractions based on?

Answer 8

The specific properties (e.g., polar, ionic etc.) of the liquid and stationary phases which are used to selectively separate the target analyte from solution.

Question 9

What are two examples of solid-phase NA extractions? What are the advantages and disadvantages of each?

Answer 9

1. Silica matrices -> high purity DNA and easy to perform/reproduce, but unable to recover small DNA fragments and one time use
2. Cellulose matrix -> easy to use and store, but the extraction protocols are complex and prone to error

Question 10

What is an example procedure for the extraction of DNA from the sea anemone A. viridis?

Answer 10

Cell disruption:
1. Remove two whole tentacles from the anemone, dry and transfer to a 2 ml reaction tube
2. Add 250 micro-l of DNA extraction buffer (this begins the lysis)
3. Homogenise using a pestle, keeping the pestle cone in the liquid to ensure no air is drawn in (prevents foam formation)

Removal of membrane lipids, proteins and other nucleic acids:
4. Add 750 micro-L of chloroform under the fume head and shake for two minutes vigorously (this causes the DNA to enter the aqueous phase (liquid))
5. Centrifuge at maximum speed (up to 14k rpm (revolutions per minute)) for five minutes (this enables the aqueous separation above to properly occur)
6. Transfer 500 micro-L of aqueous phase to a new 2 ml reaction tube (remember the aqueous phase is the part which contains DNA)
7. Add 500 micro-L isopropanol and mix gently (this causes the DNA to precipitate)
8. Centrifuge again at max speed for 20 mins (precipitated DNA forms a pellet at the base)
9. Remove and discard the supernatant (the liquid above the solid residue)

Nucleic acid purification/binding from bulk [requires an alcohol]:
10. Add 700 micro-L of EtOH 70% (ethanol) to wash the pellet (allows for purification)
11. Centrifuge at max speed for 5 mins
12. Remove the supernatant and invert the tube on absorbent paper to dry the pellet (this contains the DNA)

Nucleic acid concentration:
13. Dissolve the pellet in 200 micro-L 1xTAE (electrophoresis) - this concentrates the DNA