L11: Isolation of DNA

Question 1

How is DNA isolatied

Answer 1

Through cell lysis and DNA purification (add high salt buffer, wash with ethanol buffer and elute with low salt)

Question 2

What is the purpose of cell lysis?

Answer 2

To release DNA from the cell by breaking down the cell membrane

Question 3

What are the 3 types of cell lysis?

Answer 3

Biological, physical and mehchanical lysis

Question 4

Explain biological lysis

Answer 4

Uses enzymes to disrupt cell membrane. Different enzymes used for different cells

• for plants: cellulose
• for bactria: lysozyme (mainly gram+ as targets are peptidoglycans)
• for euk. cell: sappanin

Question 5

Explain the different mechanisms of physical lysis

Answer 5

• osmotic pressure: excess water moves into cell when cells placed in hypotonnic solution and cell bursts
• free thaw: repeated cycles of freezing and thawing ruptutres cell membranes through ice crystal formation

Question 6

Explain mechanisms of mechanical lysis

Answer 6

• Grinding: pestle+motar usde to disrupt plant cells and hard tissue (e.g. mice or human organ tissue)
• Bead mill: used to beat and grind tough samples
• Vortex: used with beads or alone for small cell number
• Shearing:
• -Homogeniser: tissue suspensions are forced through narrow space
• -Rotor- stator: uses a combo of turbulence and mechanical shearing
• -Syringe-needle: purification of longer DNA fragments

Question 7

Name 2 methods used for DNA purification

Answer 7

Phenol-Chloroform extraction and commercial kits

Question 8

How is DNA purified by Phenol-Chloroform extractin

Answer 8

1. lysed cells are mixed with equal volumes of a phenol:chloroform mixture
2. centrifugation occurs, giving 2 distinct phases as the the phenol:chloroform mixture doesn’t mix with water
3. 0.3M sodium acetate & 2.5 volumes ethanol can be usd to precipitate DNA from salt and sugar to concentrate it

Question 9

Name advantages of using phenol chloroform extraction

Answer 9

• very cost effective
• fast
• big sample in one go
• DNA still has salts& impurities so purifies with alcohol

Question 10

Name cons of using phenol chloroform extraction

Answer 10

• dangerous to us and environement
• chloroform is an easy anaesthetic
• phenol can cause v serious chemical burns

Question 11

How can DNA be purified using commercial kits?

Answer 11

1. Column contains a silica membrane that binds DNA in the presence of a high concentration of salt
2. Impurities such as salts are washed away
3. Low salt buffer, such as water or 10mM Tris, used to release DNA from membrane and collect it

Question 12

Why may commercial kits be prefered to purify DNA?

Answer 12

• They aren’t as hazardos
• Less time consuming
• Results in purer DNA

Question 13

What are the functions of restriction endonucleases (REs)?

Answer 13

• Used to make recombinant DNA molecules (cloning)

- Used to cut DNA into defined fragments (DNA fingerprinting & mutation analysis)

Question 14

What enzyme is dubbed as ‘molecular scissors’?

Answer 14

Restriction endonucleases

Question 15

How do REs cut DNA in precise locations?

Answer 15

By making one cut in each of the sugar-phosphate backbones of the double helix (breaks bond between 3’ O and P) at their recognition site in the presence of Mg2+

• hydrolyses phosphate group
• cuts ends have a 5’ phosphate

Question 16

State the naming convention

Answer 16

Bacterial genus → species → strain → type

Question 17

What are the 2 main types of REs and how do they work?

Answer 17

Blunt end: cuts in the middle of sequene

Sticky ends: cuts from one end to the opposite

Question 18

The recongition sites for REs are often what?

Answer 18

Palindromes (read the same backwards and forwards)

Question 19

What is the function of MCSs (multiple cloning sites)?

Answer 19

They indicate all restriction sites and have to be in the correct reading frame

Question 20

What is ‘star activity’?

Answer 20

The relaxation/ alteration of the specificity (chemical or drug intervention from outside the host)

Question 21

When does star activity occur?

Answer 21

When reaction conditions differ significantly from the optimum for the enzyme e.g. low ionic strength, high pH

Question 22

Explain the use of gel electrophoresis

Answer 22

Gel agorase acts as a molecular sieve with DNA travelling downwards from large to small.
-DNA is -vely charged so migrates to positive electrode

Question 23

How is DNA fragment size determined?

Answer 23

• The size of the product of interest is compared with MW DNA ladder.
• Can also graph log DNA size of the markers by distance migrated creating a straight line