Methods in molecular genetics 1

Question 1

Summary

5 Methods and Techniques used in Molecular Genetics:

Answer 1

DNA/RNA extraction and purification

DNA/RNA quantification

DNA/RNA separation

DNA/RNA identification

DNA/RNA sequencing

Question 2

Nucleic acids quantification

Answer 2

Spectrophotometry

Question 3

Nucleic acids separation

Answer 3

Chromatography

Elecrophoresis

Question 4

Nucleic acids detection and identification

Answer 4

Blotting

Arrays

Question 5

Nucleic acids sequencing

Answer 5

Termination (Sanger) sequencing

Next Generation sequencing

Question 6

Nucleic acid isolation and purification

Tissue disruption

Answer 6

Grinding tissues in a mortar
Needs to be under liquid nitrogen N2
OR
Using lysozyme to destroy bacterial cell wall

Question 7

Nucleic acid isolation and purification

Tissue homogenization

Answer 7

Buffer systems: used to solubilise and protect

nucleic acids.

Question 8

Nucleic acid isolation and purification

2 points

Answer 8

1. Differential (phase) extraction
   Uses a combination of aqueous and organic solvents to separate nucleic acids from other substances.
2. Ion exchange cartridges
   Use charged resins that retain nucleic acids
   which are eluted after all the contaminants
   are washed. [DNA, RNA negatively charged. Silica positive charge = retain DNA and RNA]

-> analysis

Question 9

Nucleic acid quantification

What is Spectrophotometry
4 points

Answer 9

1. light absorbance
2. Nucleic acids absorb light at 260nm
3. Extinction coefficient (EC) at 260 nm: Light absorbance (optic density) in 1 ml solution in 1cm path.
   e. g. Double stranded DNA: 50 (1OD: 50 ug/ul)
4. UV light through cuvette -> Optic Density

Question 10

Nucleic acid quantification

Spectrophotometry allows

3 points

Answer 10

1. To calculate the amount/concentration of nucleic acids (DNA, RNA)

OD x EC = x ug/ul

2. To study the secondary structure of DNA. Pairing of single stranded DNA.
3. To check purity of DNA and RNA.

Determined by the A260/280 ratio: OD(260nm)/OD(280nm)

OD (260): nucleic acids
OD (280): proteins

Question 11

Nucleic acid separation

What is Electrophoresis

3 points

Answer 11

1. the motion of charged particles into fluids
   under the influence of an electric field.
2. DNA and RNA are separated based on their size
3. DNA and RNA are uniformly negatively charged.
   They move towards the anode (+).

Question 12

Nucleic acid separation

3 types of electrophoresis

Answer 12

1. Horizontal agarose gel-electrophoresis

Separation of DNA and RNA using Agarose gels.

2. Vertical polyacrylamide gel-electrophoresis (PAGE)

Uses slide polyacrylamide gels for resolving
DNA and RNA fragments.

Higher resolution: one base pair difference

3. Capillary electrophoresis

The separating gel is contained within a thin capillary.

DNA or RNA molecules are driven through
the gel under a very high voltage: 30 kV.

The capillary (gel) can be used hundreds of times.

Question 13

Nucleic acid separation

Ion-exchange chromatography

4 points

Answer 13

1. Separates molecules based on their charge.
2. The charged molecules are first retained on a resin having opposite charge to them.
3. All non retained molecules are washed through.
4. The retained molecules are then eluted using a salt solution.

Question 14

Nucleic acid separation

Size-exclusion chromatography

Answer 14

1. Separates molecules based on their size.
2. The small molecules are retained in the resin particles.
3. The large molecules pass through between the resin particles.

Question 15

DNA and RNA detection

4 points

Answer 15

1. Fluorescent intercalating agents:

Ethidium bromide: orange fluorescence under UV light.

SYBRGreen: green fluorescence under UV light.

2. Dyes: Hoechst, silver
3. Radioactive labelling, 32P(ATP)
4. Fluorescent labelling: Cy3, Cy5, 6-FAM, Rox, TAMRA, etc

Question 16

DNA/RNA sequence detection and identification

What are blotting techniques
5 points

Answer 16

1. techniques by which a specific DNA or RNA sequence is detected/identified in a complex sample.
2. DNA or RNA are first separated by gel electrophoresis.
3. DNA or RNA are transferred from the gel onto a synthetic membrane (nitrocellulose or nylon) using capillarity- or electro-transfer.
4. Target DNA is then detected using a labelled probe which is incubated with the membrane and let to hybridize to the target sequence.
5. The membrane is then washed to remove non hybridized probe then exposed to an X-ray film to detect the hybridized probe.

Question 17

DNA/RNA sequence detection and identification

blotting techniques summary

4 points

Answer 17

1. DNA separation by gel-electrophoresis
2. DNA transfer onto a membrane
3. The membrane is incubated with a labelled probe
4. The membrane is exposed to an X-ray film

Question 18

DNA/RNA sequence detection and identification

southern vs northern vs western blotting

3 points

Answer 18

1. southern: detection of DNA sequences

probe usually DNA sequence

2. Northern: detection of RNA sequences

probe usually DNA sequence

3. Western: detection of proteins using antibodies

Question 19

DNA/RNA sequence detection and identification

Gene chips (gene arrays)

4 points

Answer 19

1. A collection of DNA spots arrayed on a solid matrix. Each spot contains
   a probe for a gene.
2. The chips are hybridized with DNA or RNA samples for detection and quantification of particular sequences.
3. Macro-chips:
   Contain hundreds of macroscopic spots (probes) on glass or membrane supports.
4. Micro-chips, microarrays:
   Contain thousands of microscopic spots (probes) on glass supports.

Question 20

DNA sequencing

Sanger & Coulson method

3 points

Answer 20

1. Sequencing reaction:

Single stranded DNA to be sequenced

A primer is required

dNTPs (dATP, dCTP, dGTP, dTTP) required.

Labelled ddNTPs (ddATP, ddCTP, ddGTP, ddTTP) (big dye terminator) required.

DNA polymerase I

2. Electrophoresis:
   PAGE, capillary electrophoresis
3. Fragment detection –> sequence

Question 21

High throughput sequencing: Next Generation sequencing

5 points

Answer 21

1. Parallel sequencing of millions of short DNA fragments in a liquid or solid matrix.
2. Sequencing by synthesis using DNA polymerase.
3. Direct or indirect sequence detection via fluorescence
   or change in pH.
4. High throughput allowing sequencing of large genomes
   and transcriptomes.
5. Cost efficiency.

Question 22

High throughput sequencing: Next Generation sequencing

Steps

Answer 22

1. Library preparation for NGS
2. Amplification by PCR
   (The library is amplified by Polymerase Chain Reaction (PCR) to increase the amount of the template.)
3. Sequencing
4. Sequence assembly

Question 23

High throughput sequencing: Next Generation sequencing

454 pyrosequencing

3 points

Answer 23

1. 4 Enzymes: DNA polymerase, ATP sulfurylase, Luciferase, Apyrase.
2. DNA is bound to beads, amplified by Polymerase Chain Reaction and
   pyrosequenced in picotitre plates by sequential addition of nucleotides.
3. High efficiency and low cost: up to 600 million bp sequenced in 10 hours.

Question 24

High throughput sequencing: Next Generation sequencing

Illumina NGS

4 points

Answer 24

1. Parallel sequencing of millions of short DNA fragments on
   a solid matrix.
2. Sequencing by synthesis using DNA polymerase and
   fluorescent nucleotides.
3. High throughput allowing sequencing of large genomes
   And transcriptomes.
4. cost efficent