2) Isolation Of DNA and Genes

Question 1

Physical Methods of cell rupture

Answer 1

• osmotic pressure: excess water moves into cell when cells are placed in hypotonic solution, and cells burst
• Freeze-Thaw: repeated cycles of freezing and thawing ruptures cell membranes. Ice crystals form and pierce the cell membrane. When you defrost it, the cell membrane is still there. But if you re-freeze it, ice crystals form in different places. If it is thawed again, there will be no cell membrane left to retain the cell structure. Cell material is released.

Question 2

Biological methods

Answer 2

Lytic enzymes (e.g. Lysozymes)

Question 3

Mechanical (grinding) methods

Answer 3

• mortar and pestle: used to disrupt plant cells, and hard tissue
• Bead mill: used to beat and grind tough samples
• Vortex: used with beads or by itself for small cell numbers
  (Friction can cause heat, so have to make sure that samples are kept cool)

Question 4

Mechanical (shearing) methods

Answer 4

• Homgeniser: cell/tissue suspensions are forced through a narrow space
• Rotor-Stator: designed with an out stationary tube (stator) and an inner turning shaft (rotor) which is connected to a motor. At the bottom of the rotor-stator are slots on both the tube and shaft. It uses a combination of turbulence and mechanical shearing
• Syringe-needle: can allow for purification of longer DNA fragments (Other methods can break DNA into smaller fragments)

Question 5

Mechanical (Pressure) method - Sonication

Answer 5

• Pressure is created by a probe the rapidly expands and contracts at high frequencies
• High frequency oscillation is due to oscillating current applied to certain crystals on the probe (sodium potassium tartrate)
• when probe contracts, -ve pressure causes liquid to flow up with the probe, while expansion of the. Crystals pushes liquid, and liquid turns into a zone of microscopic shockwaves
• liquid can’t flow as fast as crystals oscillate, during the contractions, small vacuum cavities form. When crystals expand, cavities rapidly implode creating shockwaves (cavitation)
• disrupts most cells in seconds
• not good for solid tissue

Question 6

French pressure Cells press

Answer 6

• Forces cells through a narrow valve under high pressure
• used to break the resilient plasma membrane and cell walls of bacteria and other microorganisms
• More flexible cell don’t burst in the valve, but after going from high to low pressure, they expand and burst
• any surviving cells are fired against a steel wall
• sample sizes are small and throughput is low
• samples other than single cells or microbial cultures have to go through a pre-homogenisation step first

Question 7

Isolating DNA by Phenol-Chloroform

Answer 7

• Lysed cells/tissue are mixed with equal volumes of Phenol/Chloroform mixture
• Mixture is centrifuged and 2 distinct phases form (lower organic phase, separated by an upper aqueous phase by denatured proteins). This in because phenol/chloroform is hydrophobic
• proteins and hydrophobic lipids partition into lower organic phase, whilst nucleic acids (and other contaminants e.g. Salts and sugars) remain in the upper aqueous phase
• upper aqueous phase is pipetted off
• 0.3M sodium Acetate and 2.5 volumes Ethanol are used to precipitate DNA from salt and sugar and concentrate it
• The DNA pellet is the redissolved in EDTA buffer

Question 8

The role of Sodium Acetate in phenol/chloroform isolation of DNA

Answer 8

• in solution sodium acetate breaks up into NA+ and [CH3COO]-
• Na+ binds neutralises PO3- (-ve backbone), making DNA less hydrophilic, so less soluble in water.
• can also use NaCl, LiCl or Ammonium Acetate

Question 9

Role of ethanol in phenol/chloroform isolation of DNA

Answer 9

• electrostatic attraction between Na+ and PO3- is ditacted by Coulumb’s law which is affected by the dielectric constant of the solution.
• ethanol (100%) has a low dielectric constant so it is easier for Na+ and PO3 to come together
• it is also used to wash excess salt of (70%)

Question 10

In phenol/chloroform isolation of DNA, how does the pH of solution affect the phase that DNA ends up in?

Answer 10

If the mixture is acidic, DNA will precipitate in the organic phase, whilst RNA remains in the aqueous phase, because DNA is more readily neutralised than RNA

Question 11

How is RNA contamination dealt with after phenol/chloroform isolation of DNA?

Answer 11

Use RNase enzymes

Question 12

What are the dangers of phenol/chloroform?

Answer 12

• phenol burns

- chloroform is a carcinogen

Question 13

Isolating DNA using commercial kits

Answer 13

• column contains silica membrane that binds DNA in the presence of high con of salt ( salt alters pH of solution so that DNA can bind
• Impurities (e.g. Salts) are washed away, then a low salt buffer (e.g. Water or 10mM Tris-Cl, pH 8.5) is used to elute DNA from membrane and collect it
• Lysis buffers with these kits often contain chaotropic salt - helps to denature proteins and provides optimal pH and salt conc of the binding solution so the nucleic acid will bind to the silica membrane as solution passes through

Question 14

Chaotropic

Answer 14

Molecule in water that can disrupt the hydrogen bonding network between water molecules

Question 15

Measuring quantity and quality of DNA

Answer 15

• Measure abosorbance of known vol of sample at 260nm and 280nm
• A260/A280 of 1.7 - 1.9 = pure DNA
• ratio >1.9 may have RNA contamination
• ratio <1.7 may have protein contamination

Question 16

Restriction Endonucleases

Answer 16

• Enzymes produced by bacteria to protect them against viral DNA infection
• Restriction = act on specific sites (4-6 bps)
• Endonuclease = cleave within DNA strands
• recognition sites are palindromic
• produce sticky ends or blunt ends
• Cleave at the recognition site through phosphodiester backbone in the presence of Mg2+

Question 17

Uses of restriction endonucleases

Answer 17

• can be used to make recombinant DNA
• restriction digests of large genomes run as smear
• Southern blotting can be used to transfer DNA to a membrane and probe for a specific sequence

Question 18

Electrophoresis

Answer 18

• works on basis that DNA fragments are -very charged and can be separated based on size
• Polymerised agarose is porous, allowing DNA to move through it
• DNA is placed at the -very charged end and pulled towards the +very charged end.
• smaller fragments travel further
• DNA is visualised using intercalated g dyes (orange/red fluorescence in UV light)
• Pulse field gel electrophoresis is used for large DNA fragments that are difficult to separate
• Size of fragment can be determined by comparing with DNA ladder
• A graph can be drawn by plotting log DNA size of markers against distance migrated to create a straight line. Graph can then be used to determine fragment size

Question 19

In electrophoresis, what could cause bands that appear larger than expected?

Answer 19

Concatamers - long continuous DNA molecules that contain multiple copies of the same DNA sequence linked in series
Open ring structure - this structure is less compact than a linear structure

Question 20

In electrophoresis, what could cause a band to appear smaller than expected?

Answer 20

Supercoil structure - this structure is less compact than a linear structure