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GENE TECHNOLOGY
Objectives:
•To describe how
sections of DNA
containing a desired
gene can be extracted
from a donor organism
using enzymes.
•To explain how isolated
DNA fragments can be
placed in plasmids, with
reference to the role of
ligase.
•To state other vectors
into which fragments of
DNA may be
incorporated.
What is genetic engineering?
• The processes used to obtain a
specific gene and place it in another
organism
• The recipient organism expresses
the gene and is transgenic (or GMO)
• The new DNA is said to be
recombinant DNA because it
contains DNA from another organism
or source
Why do we want to genetically
engineer organisms?
• Improving the recipient organism e.g.
herbicide resistant crops, growth promotion
(the myostatin gene for muscle growth) in
animals
• Creating organisms to make useful
products e.g. insulin or GH production,
production of pharmaceuticals through ewe
milk, beta-carotene production in rice
An overview of the process
1.
2.
3.
4.
5.
Obtain copy of gene (from a donor)
Place gene in a vector
Introduce vector to host cell
Select cells containing donor DNA
Clone the gene
Obtaining a copy of the Gene
Three main methods:
1. make a copy of the gene from its
mRNA using reverse transcriptase
2. Artificially synthesise the gene
3. Cut up the DNA with restrictions
endonucleases and search the DNA
containing the gene – shot gun
approach or use a DNA probe to
identify the DNA before cutting it
Reverse transcriptase
• Reverse transcriptase is a polymerase
enzyme
• It performs the reverse of transcription
• It synthesises DNA from an mRNA
template
• Complementary DNA (cDNA) is DNA made
from mRNA
Making
cDNA from
mRNA
The principle
• mRNA is complimentary to the DNA in a
specific gene
• Reverse transcriptase is able to make a
strand of DNA that is complimentary to
the mRNA
• If the mRNA for a specific gene is
isolated then the gene can be synthesised
using reverse transcriptase
• The DNA formed is called complimentary
or cDNA
• Animation of the Formation of cDNA
b) Synthesising a gene
• The base sequence of the DNA can be
worked out from the aa sequence of the
required protein.
• The gene can then be constructed using
free nucleotides and joining them
together in the right order by a
polynucleotide sequencer
• This is only possible for short genes at
present.
C) Restriction Endonucleases
• Enzymes that cut DNA at specific sites
• Cut the bonds in the middle of the
polynucleotide chain
• Most enzymes make a staggered cut in the two
strands, forming sticky ends which are ssDNA
• Some restriction enzymes cut straight across
both chains, forming blunt ends
• Artificial sticky ends need to be added to blunt
ends
Restriction Enzymes Animation
Steps in Cloning a Gene Animation
Inserting a gene into a vector
Placing the Gene in the vector
• The most common vectors are plasmids (other
e.g.s are certain virus genomes or yeast cell
chromosomes).
• Plasmids are extracted from bacterial cells by
centrifugation
• Restriction endonuclease enzymes are used to
cut the plasmid DNA and the gene from the
donor DNA
• Once cut the plasmids and cDNA are mixed with
DNA ligase
• This allows the sticky ends to form
complimentary base pairs by forming covalent
phosphodiester bonds
• Recombinant DNA is produced
DNA ligase
catalyses the
formation of
phosphodiester
bridges, splicing
the DNA into the
plasmid DNA.
Introducing the Vector DNA into
the Host Cell
• The plasmid is now introduced into a
bacterial host cell to multiply up
• A mutant harmless form of E. coli is commonly
used because it has a doubling time of 30
minutes
• E. coli, the plasmids and ca2+ ions are given a
brief heat shock which temporarily makes the
CSM permeable to DNA
• This process of adding new DNA to a host is
called transformation and produces
transformed or transgenic bacteria
• (there are a range of alternative methods on
p174 – make a list)
Genetic
Engineering
of Human
Insulin as
an example
There are 2 major problems with
this technique.
1. Not all bacteria will take up the
plasmids.
2. Not all the plasmids will have
taken up the foreign DNA i.e. Be
recombinant plasmids.
Solution to Problem 1
• Use plasmids with antibiotic
resistance genes E.g. ampicillin
• Grow the bacteria on agar
containing that antibiotic then
only those that have taken up
the antibiotic resistant plasmids
will survive.
BUT:
• You know the bacteria have
taken up the plasmid but you
still don’t know if they took up
RECOMBINANT plasmids.
• How could we tell if the
bacterium had taken up a
recombinant plasmid?
Solution to Problem 2
• Use a restriction enzyme
will cut through a
different antibiotic
resistance gene e.g.
tetracycline.
• The recombinant plasmids
will have the human insulin
gene inserted in the middle
of the tetracycline
resistance gene, meaning
that it no longer has
resistance.
• Use replica plating to
identify the recombinant
colonies
Replica Plating
The colonies that can grow on the tetracycline are NOT
transformed, so the transformed colonies can be identified
from the original ampicillin plate
Homework
Make notes on the advantage of
bacterial conjugation for taking
up recombinant DNA and the
experiments on pneumonia in mice
and their findings (p176-177)
(note conjugation animation in scheme)