recombinant DNA and cloning vectors

Question 1

the 4 recombinant vectors in the molecular tool kit

Answer 1

plasmids
bacteriophages
viruses
artificial chromosomes

Question 2

describe plasmids

Answer 2

found in many bacteria
has restricted host range
transferable by transformation and conjugation

Question 3

describe phages

Answer 3

bacteria viruses = lambda

transfer of antimicrobial resistance through a mechanism called transduction

Question 4

describe viruses

Answer 4

non primate lentiviruses = vectors used to integrate DNA in mammalian cells

baculoviruses = vectors used in combination with recombinant expression in insect cells

Question 5

describe artificial chromsomes

Answer 5

yeast artificial chromosomes = introducing large segments of DNA

Question 6

why are plasmids essential to molecular tool kit

Answer 6

discrete circular DNA molecules found in many
genetic info is maintained in bacteria
are genetic elements that exist and replicate independently of bacterial chromosomes and are extra chromosomal
can be exchanged between bacteria within restricted host range

Question 7

features of plasmid vectors

Answer 7

1. can be linearised at one or more sites in non-essential stretches of DNA
2. can have DNA inserted into them
3. can be re-cicularised without loss of ability to replicate
4. modified to replicate at high multiplicity
5. contain selectable markers
6. most are relatively small 4-5kb in size

Question 8

why use plasmids as recombinant tools

Answer 8

• add functionality over simple DNA and facilitate experimental or functional genomics
• expression of recombinant gene in living organism
• add or modify control elements
• alter properties of gene product fuse to peptide tag, make useful as therapeutic

Question 9

recombinant proteins in clinical use - facilitate production of recombinant drugs

Answer 9

human insulin - diabetes
interferons - viral hepatitis or MS
erythropoietin - kidney disease, anaemia
factor XIII = haemophilia
tissue plasminogen activator = embolism, stroke

Question 10

requirements for plasmid in prokaryotic system

Answer 10

• ability to replicate in bacteria
  -maintained at high copy no.
  selectable contains an antibiotic marker
  easy to manipulate

Question 11

what control elements are required for expression in bacteria

Answer 11

gene coding insufficient
shine-dalgarno sequence RBS recognition of AUG
bacterial promoter not enough
transcriptional terminator

Question 12

constitutive promoter

Answer 12

always on

allows culture of cells to express foreign protein to high level

Question 13

inducible promoter

Answer 13

molecular switch
allows large cultures to be grown without expressing foreign protein
induced in response to defined signal

use lac operon

Question 14

requirements for DNA insert

Answer 14

DNA easy to manipulate
copy of coding sequence
must contain start codon and stop codon

Question 15

difference in pro and euk expression vectors

Answer 15

pro sequence = shine-dalgarno
euk sequence = kozak

euk = introns can be tolerated but not necessry

terminators diff

Question 16

describe the green fluorescent protein

Answer 16

was identified and cloned from jelly fish

green colour derives from intrinsically green fluorescent protein, non toxic and biochemically inert