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1

Define 'Pharmacogenetics' and its objective

- Study of the effect of inherited variations on drug response

- Objective = to link differences in gene structure with differences in
: drug absorption, transport, metabolism
: pharmacological (therapeutic/toxicity) effect

2

Define 'Genotype' and 'Phenotype'

Genotype
: Differences observed at the genetic level

Phenotype
: Differences observed at the enzyme/transporter activity level

3

Which gene products influence drug's PK-PD effect?

- Receptors (drug targets)

- Metabolizing enzymes

- Transporters

4

How does genetic variation effect drug responses?

1. Drug receptors
: altered availability of receptors
: altered affinity of receptor to drug

2. Drug transporters and enzymes
: rate of metabolism
: rate of transport

5

What is the aim of pharmacogenetic studies?

- To understand genetic basis for variation in the therapeutic and adverse drug response

6

What occurs in Phase I and Phase II of liver metabolism?

Phase I
- oxidative reactions
- hydrolysis reactions

Phase II
- conjugation reactions

7

What is the advantage of DNA industry?

- More efficient, cheaper, safer production of therapeutic proteins e.g insulin

- To make rare proteins with therapeutic potential in sufficient quantitiy

- Production of vaccines

- GM food

8

What is the aim of 'Recombinant DNA technology'?

- Analyse function of genes and their products

- Expression/regulation studies

- Production of industrial and pharmaceutical products

9

In which direction do polymerases synthesise in?

5' -> 3' direction

10

What is monocistronic & polycistronic?

monocistronic
: One mRNA, one gene

polycistronic
: One mRNA, 2 or more genes (genes organised in operon)

11

What do the following terms mean regarding transfer of DNA?

1. Transformation

2. Conjugation

3. Transduction

1. Transformation
- uptake of free DNA

2. Conjugation
- transfer of DNA through cell-cell contact

3. Transduction
- transfer of DNA mediated by a virus

12

Characteristics of plasmids

- Most are circular, double stranded DNA molecules

- Replicate independently from chromosomal DNA

- Found in prokaryotes and lower eukaryotes

13

What are plasmids involved in?

- Resistance to antibiotics or toxic metals

- Metabolic functions

- Production of virulence factors

14

What is molecular cloning?

- Obtaining a defined sequence of DNA and produce multiple copies in vivo

15

What are the 3 basic steps in molecular cloning?

- Isolation of source DNA

- Inserting source DNA into a cloning vector

- Introduction of cloned DNA into a host organism

16

What is Polymerase Chain Reaction (PCR)?

- most common technique for obtaining DNA fragments for cloning

- method to amplify section of template DNA

17

What does Polymerase Chain Reaction require and how is it done?

- Water, DNA template, Nucleotide, primers, polymerase, buffer

Three steps (repeated 25-35 times)
1. Denaturation of DNA strands (~30s at 94degrees)

2. Annealing with primers (~30s at 55-65degrees)

3. Elongation with thermostable DNA polymerase (~1min per kb at 72degrees)

18

Recombinant DNA is generated by combining DNA from a source with a vector.

Which specific enzymes are used for 'cutting' and 'pasting'?

Cutting
- Restriction enzymes

Pasting
- Ligase

19

What do restriction enzymes do in cloning?

- recognise palindromic sequence: restriction sites

- Cut both DNA strands, creating sticky and blunt ends

20

What are the 3 important regions of plasmids regarding cloning?

- Replication origin

- Selection marker

- Region where DNA can be inserted

21

What is the procedure of 'Cloning'?

1. Cut source DNA and plasmid with restriction enzymes

2. Mix source DNA and plasmid, and add ligase

3. Use ligation mixture to transform E.coli

4. Grow on agar plates

5. Identify colonies containing recombinant DNA

22

What is 'blue-white' screening?

- plasmid contains lacZ gene, which encodes the enzyme β-galactosidase

- MCS is part of lacZ gene

- if no DNA in MCS, β-gal is active and converts artificial substrate into blue dye

- if foreign DNA in MCS, β-gal is inactive and no blue colour (white)

23

What are the requirements of hosts for cloning and expression?

- Grows rapidly in inexpensive medium

- Non-pathogenic

- Easily takes up DNA

- Is genetically stable

- Allows replication of vector

- Has many tools for genetic manipulation

- Allows high level of expression of genes

24

What is insulin?

- Hormone produced in pancreas; secreted into bloodstream

- Controls blood sugar levels

- Faulty production or ineifficient utilisation leads to diabetes

- Small protein

25

Recombinant human insulin production method 1

- Clone insulin A and B chains seperately in E.coli as fusions with gene encoding β-galactosidase

- Purity fusion proteins and cleave off β-gal

- Combine A and B chains and refold in oxidising conditions in vitro

26

Recombinant human insulin production method 2

- Clone gene for proinsulin, fused to gene encoding β-galactosidase in E.coli

- Extract protein, purify, and cleave off β-gal

- Refold proinsulin

- Cleave proinsulin enzymatically

27

What is Factor VIII?

- Essential Blood Clotting factor

- Used for treatment of haemophilia

- Very large protein

- Largest recombinant protein used commercially

28

How is Cloning of Factor VIII done?

- Very large gene with several introns (requires copies to be made from mRNA)

- initial cloning was done in E.coli

- Plasmid containing F8 gene used to transfect mammalian cell lines

- Plasmid integrates in genone; cell line with highest number of F8 gene copies used for production

29

What is antithrombin?

- Glycoprotein, made in liver

- Inactivates thrombin, Factor Xa and Factor IXa
: regulates normal blood coagulation

- Used in patients with antithrombin deficiency

30

Cloning Key points (JUST READ)

- Recombinant DNA can be made using restriction enzymes (cutting) and ligase (pasting)

- Choice of vector/host system depends on the nature of the protein that has to be produced

- For small proteins that are not post-translationally modified, bacteria or yeast are preferred

- Proteins of mammalian origin that are e.g large and glycosylated need to be produced in insect or mammalian cells, or in whole animals