Experiment A: molecular biology

Question 1

why is glycerol added to the loading dye?

Answer 1

so that the samples are denser than the running buffer and sink to the bottom of the well

Question 2

what are the two conformations that DNA can take?

Answer 2

supercoiled and relaxed

Question 3

explain the migration time of the supercoiled DNA conformation

Answer 3

• due to its supercoiled nature, the DNA fragments become smaller in size
• hence experience less frictional resistance from the gel
• faster migration than other conformations.

Question 4

what effect on the supercoiled and relaxed conformations does adding restriction enzymes have?

Answer 4

both forms are cleaved by restriction enzymes to give the same linear molecule

Question 5

how are restriction endonucleases made?

Answer 5

made naturally by bacteria as a defence against viral infection

Question 6

how does a bacterium prevent the destruction of its own DNA by restriction enzymes?

Answer 6

its marks its DNA by methylation

Question 7

what is the effect of using a restriction enzyme with a short recognition sequence?

Answer 7

the restriction enzyme will cleave any random piece of DNA more often, resulting in smaller fragments of DNA

Question 8

explain the movement of DNA on the gel

Answer 8

DNA molecules migrate towards the anode (positive electrode) due to the negative charge on the phosphate groups

Question 9

what is the difference in migration time between smaller and larger fragments?

Answer 9

migration is inversely proportional to size

ie. smaller fragments move more quickly than larger fragments

Question 10

what is the mechanism of ethidium bromide?

Answer 10

ethidium bromide intercalates between bases and takes on a pink/orange fluorescence when illuminated with UV light at 300 nm

Question 11

why must ethidium bromide be handled using gloves?

Answer 11

• it is a possible carcinogen, shown to be mutagenic to bacteria via the Ames test
• however, this was only after treatment with liver homogenate, which stimulates the metabolic breakdown of the molecule
• lack of detected mutagenicity without liver homogenate
• indicates that ethidium bromide is not directly mutagenic, but that its metabolites are

Question 12

why is bromophenol blue added to the loading dye?

Answer 12

it runs ahead of the smallest fragments, monitoring the process by indicating when the gel electrophoresis is complete

Question 13

at what voltage is gel electrophoresis carried out and for how long?

Answer 13

at 70 V for 40 minutes

Question 14

what is plotted for a standard curve?

Answer 14

distance migrated against log (number of base pairs)

Question 15

what should a standard curve look like?

Answer 15

there should be a linear relationship between the two variables

Question 16

what process occurs at the cathode and at the anode?

Answer 16

cathode - reduction to give a negative charge

anode - oxidation to give a positive charge

Question 17

describe the main properties of gels

Answer 17

• anticonvective - suppress the thermal convection caused by application of the electric field
• act as a sieving medium to retard the progress of molecules
• maintain the finished separation so that a post-electrophoresis stain can be applied

Question 18

what is agarose and why is it used?

Answer 18

• extracted from seaweed
• usually 0.7-2% used
• linear polymer made up of the repeating unit of agarobiose, a disaccharide
• agarose polymer chains form helical fibres that aggregate to form a three-dimensional mesh of channels of diameter 50 nm - > 200 nm (depending on concentration)
• high concentration = low pore diameter
• 3D structure is held together with hydrogen bonds
• broad range of physical/chemical/thermal stability
• lower degree of chemical complexity so it is less likely to interact with biomolecules

Question 19

what is polyacrylamide and why is it used?

Answer 19

• acrylamide subunits + cross linker
• used for nucleic acids and small proteins
• separation of proteins 5-2000 bp

Question 20

how are molecules run in PAGE (polyacrylamide gel electrophoresis)?

Answer 20

either in their native state or levels of biomolecular structure disrupted by a chemical denaturant to leave only the primary structure to be analysed

Question 21

what chemical denaturants are used in PAGE?

Answer 21

• nucleic acids denatured by urea

- proteins denatured by sodium dodecyl sulphate

Question 22

what is the effect of running a denatured molecule in PAGE?

Answer 22

• charge is evenly distributed by unit mass

- eliminates all differences in shape as a factor for separation

Question 23

why is SDS important?

Answer 23

to linearise proteins and impart a negative charge

Question 24

what is the problem with high percentage gels?

Answer 24

high percentage gels are brittle and may not set evenly

Question 25

what is the problem with low percentage gels?

Answer 25

low percentage gels are fragile and are not easy to handle