W1L1 - Nucleic Acid Extraction

Question 1

Molecular Diagnostics

Answer 1

Class of diagnostic test that detects variations in DNA or RNA

Question 2

Uses of Molecular Diagnostics

Answer 2

Determines:

• if individual has disease
• if individual is pre-disposed to have a disease
• if treatment option may or may not be useful for someone with pre-existing disease
• identifies bacteria, viruses, certain cell types etc.

Question 3

Types of Molecular Diagnositcs

Answer 3

Haematology
Immunology
Microbiology
Cytology
Biochemistry
Histology

Question 4

Nucleic Acid Extraction

Answer 4

For most samples, the nucleic acid to be analysed (DNA/RNA) must be purified
First a lysate is produced by disrupting tissue
Lysate may then be centrifuged to separate soluble material from insoluble material
RNase A added if performing DNA analysis and contaminating RNA is an issue
Nucleic acids then purified from the lysate

Question 5

A lysate is produced by disrupting the tissue. This can be

performed using one, or a combination, of what?

Answer 5

Proteases
- proteinase K
Detergents
- SDS
Tissue homogeniser
Chaotropic salts
Phenol (RNA isolation only)

Question 6

What are the two methods for nucleic acid extraction?

Answer 6

Phenol-chloroform extraction

Column-based methods

Question 7

Phenol-Chloroform Extraction

Answer 7

Equal volume of phenol-chloroform mixture added to lysate
Mixed well then centrifuged
Sample separates into an upper aqueous phase and lower organic phase
Nucleic acids in aqueous phase (due to negatively charged phosphate backbone making them polar)
Proteins found in organic phase (hydrophobic core of proteins interacts with phenol)
Proteins at interface as well due to hydrophilic regions

Question 8

Phenol-Chloroform Extraction and pH

Answer 8

pH of the phenol affects the partitioning of nucleic acids into aqueous or organic phase
At neutral/ slightly basic pH
- both DNA and RNA negatively charged (polar) and found in aqueous phase
Acidic pH
- negative charge of NA phosphate backbone neutralised by H ions, DNA moves into organic phase
- RNA ss so free nucleotides present able to form H bonds with water
- RNA moves into aqueous phase

Question 9

Nucleic Acid Precipitation

Answer 9

After either phenol-chloroform or column-based, nucleic acid are precipitated out of aqueous phase
Sodium acetate plus alcohol added
Nucleic acids have negatively charged backbone, therefore positively charged ions from acetate counteract negative charge
DNA is polar so its soluble in water
Alcohol is less polar than water and DNA isn’t soluble in it

Question 10

Column-Based Method of Nucleic Acid Isolation

Answer 10

Column-based NA isolation methods based on the binding of NA to silica in the presence of chaotropic salts and alcohol

Question 11

Column-Based Steps

Answer 11

1. Sample is lysed using chaotropic salt, proteinase K, SDS, homogenisation
2. Alcohol added and lysate loaded into spin column then centrifuged
3. NA binds to silica in membrane, while protein/salts don’t
4. Column washed to remove residual contaminants
   - chaotropic salts, then alcohol
5. NA are eluted from silica using water or TE buffer

Question 12

Comparison of Phenol-Chloroform and Column-Based

Answer 12

Phenol-Chloroform
Advantages
- inexpensive
- separates DNA from RNA
Disadvantages
- low throughput
- handling of hazardous chemicals
Column-based
Advantages
- fast
- safe
- easy 
- low to high throughput
Disadvantages
- more expensive

Question 13

Why is RNA extraction difficult?

Answer 13

Less stable than DNA because:

• single stranded
• susceptible to hydrolysis at basic pH

Question 14

What happens after nucleic acid extraction?

Answer 14

DNA/RNA needs to be checked for quantity, purity and integrity
Quantity and purity checked spectrophotometrically
Amount of NA can also be determined running on gel
Integrity uses electrophoresis on agarose gel