biotechnology recombinant dna technologymembers.optusnet.com.au/~jbalasuriya/notes/lecture...
TRANSCRIPT
1
2004 Biology Olympiad Preparation Program 2
BIOTECHNOLOGY
2004 Biology Olympiad Preparation Program 3
RECOMBINANT DNA TECHNOLOGY
Recombinant DNA technology involves sticking together bits of DNA from different sources.
Made possible because DNA & the genetic code are universal.
2004 Biology Olympiad Preparation Program 4
Restriction endonucleases Bacteria use restriction enzymes to cut foreign DNA.
Restriction enzymes are specific for a particular restriction site.
Restriction fragments are produced by digesting DNA with a particular restriction enzyme.
EcoRI – Escherichia coli, strain R, enzyme 1. AluI
2004 Biology Olympiad Preparation Program 5
Sticky & blunt ends
- C T T A A - G
G - A A T T C -
G - G C A T C -
- C T T A A - G
- A G - T C
C T – G A -
T T – A A -
- A G - T C
Sticky ends must be compatible for complementary base pairing.
Blunt ends do not have this requirement.
2004 Biology Olympiad Preparation Program 6
Gene cloning – an overview Isolate plasmid DNA
from bacteria
Cut with RE
Isolate human DNA with gene of interest
Cut with same RE
Grow bacteria
Isolate bacterial clone carrying gene of interest
Mix & ligate fragments with DNA ligase
Transform bacteria
2004 Biology Olympiad Preparation Program 7
DNA isolation & restriction
ampR
lacZ
Restriction site
Cut with RE
Gene of interest Cut with
same RE
2
2004 Biology Olympiad Preparation Program 8
Ligation of DNA
Mix
DNA ligase
2004 Biology Olympiad Preparation Program 9
Transformation
Transformation
2004 Biology Olympiad Preparation Program 10
Gene cloning
2004 Biology Olympiad Preparation Program 11
Cell cloning & identification
Plate out bacteria on solid nutrient media
Ampicillin +
X-gal
Only transformed bacteria can grow
Β-galactosidase hydrolyses X-gal into a
blue substance
Colonies with disrupted lacZ genes
appear white
∴ White colonies have human DNA inserts – pick these
2004 Biology Olympiad Preparation Program 12
Clone identification Transfer some cells to filter
Lyse cells & Denature DNA
Wash filter with probe
Autoradiography
DONE!
2004 Biology Olympiad Preparation Program 13
Complementary DNA Are we able to take a gene
straight from a human and expect bacteria to express it (make proteins from it) properly?
Eukaryotic genes have introns which prokaryotes cannot splice.
The protein product would most likely be incorrect.
Hence the importance of cDNA if we want to express eukaryotic
genes in bacteria.
3
2004 Biology Olympiad Preparation Program 14
DNA ANALYSIS & PRACTICAL APPLICATIONS
2004 Biology Olympiad Preparation Program 15
DNA libraries Since we can cut, copy and paste DNA, we can make
libraries of different DNA fragments.
Cut with RE Cut with RE
Plasmid library Phage library
Fragments of kangaroo DNA in ‘books’ of library
2004 Biology Olympiad Preparation Program 16
Polymerase chain reaction
Denature DNA
2 strands after 1st cycle
Primers anneal
DNA polymerase adds nucleotides to make new DNA strands
2004 Biology Olympiad Preparation Program 17
Polymerase chain reaction
2004 Biology Olympiad Preparation Program 18
Polymerase chain reaction Takes just 30 cycles to make over 1 billion copies of the DNA.
PCR is fast, efficient and specific.
Specificity determined by the primers used.
PCR is a technique widely used to amplify DNA from a wide variety of sources.
Only requires a small amount, sometimes just a single cell, of starting material.
2004 Biology Olympiad Preparation Program 19
Gel electrophoresis Electrophoresis separates macromolecules (DNA, proteins)
according to their charge and size.
DNA has phosphate groups, making it negatively charged.
DNA is run on an agarose gel which provides a solid, porous medium for the molecules to move through.
Wells
4
2004 Biology Olympiad Preparation Program 20
Southern blotting Southern blots allow the detection of particular DNA sequences.
2004 Biology Olympiad Preparation Program 21
Northern & Western blotting
Northern blots detect and analyse RNA.
Western blots detect and analyse protein.
Both use the same principle as Southern blotting – i.e. electrophoresis, transferral to filter, probe binding.
2004 Biology Olympiad Preparation Program 22
Restriction fragment length polymorphisms
RFLPs allow DNA comparison of different individuals.
Person A: 3 fragments electrophoresed:
Person B: 2 fragments electrophoresed:
Detectable difference between length of restriction fragments, which are dissimilar (polymorphic) between individuals.
2004 Biology Olympiad Preparation Program 23
DNA fingerprinting A DNA ‘fingerprint’ is a specific pattern of bands that is
essentially unique to the individual.
These bands can be obtained by analysing RFLPs.
2004 Biology Olympiad Preparation Program 24
DNA fingerprinting
2004 Biology Olympiad Preparation Program 25
Forensics
DNA fingerprints are now accepted as compelling evidence by legal
experts and scientists.
Probability of 2 people having identical DNA fingerprints is
between 1/100,000 and 1/1 billion.
Who does the bloodstain belong to?
5
2004 Biology Olympiad Preparation Program 26
Sanger sequencing DNA polymerase cannot move past a ddNTP.
+ dNTPs + ddATP
A T A T T A C G
Etc…
2004 Biology Olympiad Preparation Program 27
Sanger sequencing
2004 Biology Olympiad Preparation Program 28
Modern DNA sequencing Fragments run on a single lane
Colours read by laser Original
sequence deduced
2004 Biology Olympiad Preparation Program 29
Chromosome maps Computers can piece together DNA sequences into a whole
chromosome map bioinformatics.
Genomics – the study of genomes based on their DNA
sequences.
Genome organisation. Control of gene expression.
Growth & development. Evolution.
2004 Biology Olympiad Preparation Program 30
Plant gene technology - Agrobacterium
Agrobacterium causes crown gall disease in plants it
infects by introducing its Ti plasmid into the plant cell. Agrobacterium
Ti plasmid
Gene of interest
Allow recombinant Agrobacterium to infect plant cell
Transformed plant cell
Tissue culture
Mature plant
2004 Biology Olympiad Preparation Program 31
Plant gene technology – gene gun DNA coated onto tiny
gold or tungsten particles
Pellets are placed inside a gene gun
Pellets are shot into plant tissue
Some pellets will enter the nucleus, and some foreign DNA will successfully incorporate into the plant cell genome.