CMB2000/L12 Isolation of DNA & Genes

Question 1

Give 2 reasons why DNA is isolated.

Answer 1

For genetic manipulations
For DNA analysis

Question 2

Give the 4 steps of DNA isolation.

Answer 2

Cell lysis
DNA purification from cell extract
Concentrate DNA
Measurement of DNA purity and concentration

Question 3

Describe how cell lysis/extraction of DNA can be achieved. (3)

Answer 3

Biological methods
Physical methods
Mechanical methods

Question 4

Give 3 detergents used for biological extraction of DNA.

Answer 4

Cellulase (plants)
Lysozyme (bacteria)
Sappanin (eukaryotic cells)

Question 5

What does lysostaphin target?

Answer 5

Cross-links of peptidoglycan layer of cell wall in Staphylococcus

Question 6

Describe a physical method of cell lysis.

Answer 6

Osmotic pressure
Excess water moving into cell when cells placed in hypotonic solution
Freeze-thaw
Repeated cycles of freezing and thawing ruptures cell membranes through ice crystal formation

Question 7

Give 2 mechanical methods of cell lysis and their apparatus.

Answer 7

Grinding - pestle & mortar, bead mill, vortex
Shearing - homogeniser, rotor-stator, syringe-needle

Question 8

Describe DNA purification by phenol-chloroform extraction. (3)

Answer 8

Lysed cells or tissue mixed with equal vols phenol:chloroform mixture
Centrifugion forms 2 phases
0.3M Sodium acetate and 2.5 vols ethanol precipitates DNA from salt and sugar

Question 9

Describe DNA purification using commercial kits. (4)

Answer 9

Lyse cells
Add high salt buffer
Wash with ethanol buffer
Elute with very low salt

Question 10

Explain how commercial kits purify DNA.

Answer 10

Silica membrane of column binds DNA in high [salt]
Impurities washed away and low salt buffer (water or 10mM Tris.Cl pH 8.5) used to release DNA from membrane and collect

Question 11

Compare phenol-chloroform extraction and commercial DNA purification kits.

Answer 11

Kits are not as hazardous
Less time consuming
Results in purer DNA

Question 12

Give 2 ways to measure the quantity and quality of DNA.

Answer 12

UV absorbance
Fluorescence dyes
Agarose gel electrophoresis
Capillary electrophoresis
Diphenylamine method

Question 13

What is the absorbance of pure DNA?

Answer 13

> 1.8 at 260/280

Question 14

What are restriction endonucleases and what do they do?

Answer 14

Molecular scissors that cut DNA in precise locations

Question 15

Why are restriction endonucleases used in the laboratory?

Answer 15

To make recombinant DNA molecules (cloning)
To cut DNA into defined fragments (DNA fingerprinting and mutation analysis)

Question 16

How do restriction enzymes cut DNA?

Answer 16

Make one cut in each of the sugar phosphate backbones of the double helix (between 3’O and P) at recognition site
In presence of Mg2+
Hydrolyses phosphate group
Cut ends have 5’ phosphate

Question 17

How are restriction enzymes named?

Answer 17

Bacterial genus, species, strain and type

Question 18

Define star activity.

Answer 18

Relaxation or alteration of the specificity
Chemical/drug intervention from outside the host

Question 19

Why can star activity occur?

Answer 19

Low ionic strength
High pH
High (>5% v/v) glycerol concentrations
Presence of Mg2+

Question 20

How is Southern blotting different to Western blotting?

Answer 20

Southern blotting uses DNA not proteins
Uses hybridisation probes not Abs

Question 21

Describe Armstrong’s experiments in chickens.

Answer 21

Looked for AvBD gene expression levels in GIT using qPCR
Analysed environmental effects
Extracted genomic DNA from 10 tissues in chickens and pooled blood samples
Looked for SNPs and AvBDs
Cloned AvBDs into plasmid
Hyper-expressed in E. coli and tested antimicrobial potency

Question 22

What is the aim of electrophoresis?

Answer 22

Separate DNA fragments

Question 23

How does polymerised agarose gel act as a molecular sieve?

Answer 23

It is porous and allows for movement of DNA

Question 24

Which dye is used to visualise DNA in agarose gel electrophoresis and why?

Answer 24

Nancy Red
It is safe

Question 25

What properties does DNA migrate according to in agarose gel electrophoresis?

Answer 25

Charge (-ve)
Size
Shape

Question 26

Give 2 ways to determine the size of agarose gel electrophoresis products.

Answer 26

Compare size of product of interest in DNA ladder
Look by eye and ‘guess’
Plot a log10 graph and band migration distance