L9 - Isolation of DNA & genes

Question 1

Why would we want to isolate DNA?

Answer 1

For genetic manipulations

For DNA analysis 
– Scientific 
– Medical 
– Forensic 
– Ecological 
– Archaeological

Question 2

Steps of DNA isolation?

Answer 2

1. Cell lysis
2. DNA purification from the cell extract
3. Concentrate DNA
4. Measurement of DNA purity & concentration

Question 3

What is cell lysis?

Answer 3

Release the DNA from the cell by breaking down its cell membrane

Question 4

3 methods of cell lysis

Answer 4

Biological methods
Physical methods
Mechanical methods

Question 5

Biological methods of cell lysis

Answer 5

Use enzymes to disrupt cell membranes – different enzymes for different cells

Plants = cellulase
Bacteria = lysozyme
Eukaryotic cells = sappanin

Question 6

Physical methods of cell lysis

Answer 6

Osmotic pressure – excess water moves into the cell when cells are placed in hypotonic solution
Messy – isn’t really a technique we want to use

Freeze-Thaw – repeated cycles of freezing & thawing ruptures the cell membranes through ice crystal formation

Question 7

Mechanical methods of cell lysis

Answer 7

Grinding

Shearing

Question 8

Grinding as a mechanical methods of cell lysis

Answer 8

Pestle & mortar – used to disrupt plant cells & hard tissue

Bead mill – used to beat & grind tough samples

Vortex – used with beads or by itself for small cell numbers

Question 9

Shearing as a mechanical methods of cell lysis

Answer 9

Homogeniser – cell or tissue suspensions are forced through a narrow space causing cells to lyse

Rotor-Stator – uses a combination of turbulence & mechanical shearing

Syringe-needed – purification of longer DNA fragments

Question 10

DNA purification by Phenol-Chloroform extraction

Answer 10

Lysed cells or tissue are mixed with equal volumes of a Phenol-Chloroform mixture

Centrifugation = 2 distinct phases as the Phenol:Chloroform mixture doesn’t mix with water
– Aqueous layer = buffer & DNA

DNA concentration – 0.3M Sodium Acetate & 2.5 volumes Ethanol can be used to precipitate DNA from salt & sugar to concentrate it

Question 11

DNA purification by Phenol-Chloroform extraction

CONS

Answer 11

Don’t want to inhale chloroform

Phenol can cause serious chemical burns

Question 12

DNA purification using commercial kits

Answer 12

Column contains a silica membrane that binds DNA in the presence of a high concentration of salt

1) When DNA added to high salt conc buffer, DNA will bind to the silica membrane
2) Everything else filtered out by centrifugation
3) Wash it with ethanol to cleanse all impurities from the DNA
4) Need to elute DNA from the silica membrane
5) low salt buffer such as water or 10mM Tris-Cl, pH 8.5 is used to release the DNA from the membrane & collect it

Question 13

DNA purification using commercial kits VS DNA purification by Phenol-Chloroform extraction

Answer 13

Using these kits is not as hazardous, is less time consuming & results in purer DNA than phenol-chloroform extraction

More expensive & can only use small volumes

Question 14

What are restriction endonucleases?

Answer 14

They are molecular scissors that cut DNA at precise locations

Restriction = act on specific DNA sequences ‘restriction sites’
Endonucleases = cleave the phosphodiester bind within a polynucleotide 

There are over 3000 different ones

Question 15

What are restriction endonucleases used for in the lab?

Answer 15

To make recombinant DNA molecules – cloning

To cut DNA into defined fragments – DNA fingerprinting & mutation analysis

Question 16

How do restriction enzymes cut DNA?

Answer 16

Make 1 cut in each of the sugar phosphate backbones of the double helix (breaks bond between 3’ O & P) at their recognition site in the presence of Mg2+
– Hydrolyses the phosphate group
– Cut ends have a 5’ phosphate

Question 17

Naming convention for restriction enzymes

Answer 17

Named for bacterial genus, species, strain & type

Example: EcoRI
• Genus: Escherichia
• Species: coli
• Strain: R
• Type: I

Question 18

Different types of restriction endonucleases

Answer 18

Recognition sites for restriction enzymes are often palindromes

Palindromes are the same forwards & backwards

Blunt ends – cuts in half through both DNA strands – no overhanging sections

Sticky ends – creates overhangs at each end that can stick to complementary sequences

Question 19

What enzymes are needed to stick strands back together?

Answer 19

Ligase

Question 20

What is star activity?

Answer 20

When it starts to cut the DNA up everywhere – relaxation or alteration of the specificity – non-canonical site cleavage

When reaction conditions differ significantly from the optimum for the enzyme

Question 21

What is electrophoresis?

Answer 21

Migration and separation of charged particles (ions) under the influence of an electric field

Separates DNA fragments

Question 22

Agarose in electrophoresis gels

Answer 22

Agarose is from seaweed

Forms a molecular sieve in the gel

The higher the concentration the smaller the pores – harder for the DNA to get through

Question 23

Electrophoresis method

Answer 23

DNA is negatively charged so migrates to the anode (positive electrode)

Smaller DNA fragments migrate quicker

Visualise results with intercalating dyes

Always run with a ladder so you know the molecular weights of the fragments

Graph of log DNA size of the markers by distance migrated creates a straight line

Can then plot the distance the product of interest has travelled on the line & estimate the DNA size