Molecular Techniques And Diagnosis

Question 0

Describe restriction analysis

Answer 0

Restriction enzymes are bacterial enzymes that recognise specific DNA sequence restriction sites and cut the DNA

Question 1

Describe DNA sequencing

Answer 1

1) fluoresce toy stained ddNTP’s are added to a DNA template with DNA polymerase to create a complimentary DNA strand
2) variety of ddNTP’s means lots of fragments of varied lengths
3) many fragments can be separated by gel electrophoresis and read

Question 2

Describe gene cloning

Answer 2

Bacterial plasmid cut using restricition enzyme
Gene of interest added ->recombiant DNA
Introduced into bacterium (transformation)
Bacteria with recombinant DNA placed in environment to multiply

Question 3

Describe the process of gel electrophoresis

Answer 3

Solution of different fragments placed in well at anode
Introduction of current causes -ve DNA to move towards +ve electrode
Shortest fragments move furthest

Question 4

What can we learn from gel electrophoresis?

Answer 4

Size - deletion/insertion?
Mutations e.g. Sickle cell
Variation - fingerprinting

Question 5

Explain the process of PCR

Answer 5

Heat to 95c - dna denatures to single stranded
Cool to 42 - annealing of primers
Heat to 75*c - addition of bases and elongation.

Uses thermostable taq polymerase

Question 6

Why do we use PCR?

Answer 6

Amplify a specefic DNA fragment
Investigate single base mutations
Investigate small deletions/insertions
Investigate variation, genetic relationships

Question 7

Describe SDS page protein electrophoresis

Answer 7

Seperate by nuclear weight
Detegent SDS denatures proteins -> give -ve charge proportionate to molecular weight
Most negative (heaviest) move furthest

Question 8

Describe isoelectric focussing

Answer 8

Proteins applied to gel with pH gradient move until it reaches its pI (no overall charge)

Question 9

Describe 2d page protein electrophoresis

Answer 9

Can seperate proteins of different weights/same pI
Gel turned 90* snd run for a different property to seperate out bands
I portant for proteonomics

Question 10

Understand the basis for the use of enzyme assays

Answer 10

Used to measure enzyme activity by appearance of product/loss of substrate
Performed at optimal pH,temp and ionic strength
Need appropriate ions/cofactors
Tissue damage increases levels of enzyme in tissue

Question 11

Give examples of some clinically important serum enzymes

Answer 11

Aspartate transaminase (AST) Alanine transferase (ALT) indicate liver damage/disease

Creatine kinase, lactate dehydrogenase indicate MI

Amylase/lipase indicate pancreatitis

G-glutamyl transferase - liver damage/alcohol

Alkaline phosphatase - bone disorders

Acid phosphatase - prostate cancer

Plasma cholinesterase - decrease in liver disease

Question 12

How are antibodies used in western blotting?

Answer 12

Following SDS page, the seperated proteins can be transferred to a nitrocellulose membrane and specefic proteins can be visualised by binding with antibodies conjugated to a label (flourescent)

Question 13

How are antibodies used in immuno assays?

Answer 13

Can detect concentration of a protein by analysing binding of a complimentary antibody

1) primary ab(specefic to protein) is immobilised on a solid support
2) solution to be assayed applied to ab coated surface e.g.microtitre well
3) secondary ab conjugated with enzyme binds to ab/ag complex
4) binding of secondary is measured by assaying for the enzymes activity

Question 14

Describe nuclear targetting

Answer 14

Protein with nuclear localaisation sequence binds to importin
Nuclear pore
Translocates into the nucleas and importin released and exported
Protein alone within

Question 15

Describe mitochondrial targetting

Answer 15

Have n-terminal sequence 
Which is recognised
TOM proteins form channel
moved into imm by chaperones
N-terminal sequence is removed by MPP

Question 16

Describe lysosomal targetting

Answer 16

Pass through golgi
Phosphate group added to OH of C6
Binds to M6P receptor 
Transported to lysosome
Phosphate removed

Question 17

Describe antibiotics that inhibit cell wall synthesis

Answer 17

E.g. Amoxicillin - inhibits penicillin binding proteins, preventing cross linking of peptidoglycan -> cell wall not made

Question 18

Describe antibiotics that inhibit bacterial transcription

Answer 18

E.g. Rifomycin - inhibits prokaryotic DNA transcription into mRNA by inhibiting DNA-dependent RNA polymerase by binding to its beta subunit

Question 19

Describe antibiotics that inhibit bacterial protein synthesis

Answer 19

E.g. Tetracycline - blocks A site on ribosome so tRNA cant bind - aa chain not synthesised

Question 20

Describe the action of antifolates

Answer 20

E.g. Methiotrexate - inhibits synthesis of DNA,RNA and proteins by inhibiting DHFR (which catalyses reaction for synthesis of thymidine)

Question 21

Provide overview of mechanisms of drug resistance

Answer 21

High rate of division
Decreased influx
Increased efflux
Increased transcription of target
Altered target