BIOTECHNOLOGY RECOMBINANT DNA TECHNOLOGYmembers.optusnet.com.au/~jbalasuriya/Notes/Lecture 11.pdf · RECOMBINANT DNA TECHNOLOGY Recombinant DNA technology involves sticking together

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2004 Biology Olympiad Preparation Program 2

BIOTECHNOLOGY


RECOMBINANT DNA TECHNOLOGY

Recombinant DNA technology involves sticking together bits of DNA from different sources.

Made possible because DNA & the genetic code are universal.


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


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.


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


DNA isolation & restriction

ampR

lacZ

Restriction site

Cut with RE

Gene of interest Cut with

same RE

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Ligation of DNA

Mix

DNA ligase


Transformation

Transformation


Gene cloning


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


Clone identification Transfer some cells to filter

Lyse cells & Denature DNA

Wash filter with probe

Autoradiography

DONE!


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.

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DNA ANALYSIS & PRACTICAL APPLICATIONS


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


Polymerase chain reaction

Denature DNA

2 strands after 1st cycle

Primers anneal

DNA polymerase adds nucleotides to make new DNA strands


Polymerase chain reaction


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.


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

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Southern blotting Southern blots allow the detection of particular DNA sequences.


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.


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.


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.


DNA fingerprinting


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?

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Sanger sequencing DNA polymerase cannot move past a ddNTP.

+ dNTPs + ddATP

A T A T T A C G

Etc…


Sanger sequencing


Modern DNA sequencing Fragments run on a single lane

Colours read by laser Original

sequence deduced


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.


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


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.

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BIOTECHNOLOGY RECOMBINANT DNA TECHNOLOGYmembers.optusnet.com.au/~jbalasuriya/Notes/Lecture 11.pdf · RECOMBINANT DNA TECHNOLOGY Recombinant DNA technology involves sticking together