Download The genetic engineers toolkit

The genetic engineers toolkit
A brief overview of some of the
techniques commonly used.
These tools can be used in
different ways depending on the
job you are doing
Size:
Type:
Restriction enzymes (endonucleases)
Restriction endonucleases cut the DNA at specific
points called recognition sites (where there is a
specific sequence of bases.)
These enzymes were first found in bacteria as a
defense against invading viruses
They leave blunt or sticky ends.
There are over 400 restriction enzymes so genetic
engineers can cut DNA almost any where they
want. they can cut out specific genes using
restriction enzymes.
Blunt end restriction endonucleases
Cut at specific sequence of bases
leaving a blunt end. They are less
specific
Sticky ends leave an overhang and are
therefore more specific
Ligation or joining DNA using DNA
ligase
• If DNA in a vector and DNA fragment are cut with the
same restriction enzyme then the base pairs will
match up and anneal ( base pair matching) and they
can be joined together using ligase
• In the picture a recombinant DNA plasmid is being
created.
PCR (polymerase chain reaction)
• This can take a small piece of DNA and copy it so
you have lots (amplification)and it only takes a
few hours
• It is divides into 3 stages
*Denaturing the DNA Heating it up to
separate the strands
* Annealing- attaching a primer to the
strand.
* Extension- copying the template strand. Taq
polymerase (which does not denature when it
is hot) then completes the double strand.
336 × 475
11KB GIF
A PCR machine
535 × 518
64KB JPG
Gel electrophoresis
This is a way of separating different
DNA fragments out in a mixture
which suspect committed the crime?
How does it work?
• DNA sample is cut up into different lengths
during restriction digestion
• DNA is put into wells (holes) in a agarose gel
matrix
• An electrical current is put through the gel
• Since DNA is negatively charged it moves
towards the positive electrode.
• The DNA may be coloured or made radioactive
so it shows up and is easy to see
• It is a bit like fighting your way through the
jungle. Big bits of DNA find it hard to get
through and so travel slowly. Little fragments
move faster.
Microsatellites
• DNA profiling uses electrophoresis of short
tandem repeats (STRs or microsatellites) as
the whole genome is too big!
eg. CA CA CA CA CA CA CA CA CA CA CA CA
• A lot of DNA consists of long stretches of
repeated nucleotides .
• These vary between individuals and can be
separated using gel electrophoresis.
• Dna profiling usually uses about 10 STR’s
Reverse transcriptase in Gene cloning
• Making lots of copies of a gene or ultimately
its protein product.
• This process may be done before PCR because
it allows for the removal of introns so the DNA
is shorter.
• DNA is transcribed into primary RNA. The
introns are then removed and the exons
joined together to make mRNA.
• The central dogma of DNA
DNA
RNA
Protein
Reverse Transcriptase turns this on its head !!!!
It makes DNA from RNA.
The DNA it makes will be the gene sequence of
base pairs without the introns and any extra
bits.