Download PCR Polymerase Chain Reaction

PCR
Polymerase Chain Reaction
Solubiosysteemit
S-114.2500
Harjoitustyö
Maria Sipilä
30.11.2005
Contents:
1. Introduction
2. Gene cloning in bacteria
3. The other way to multiply genes: PCR
3.1 Tools for PCR
3.2 The principle of PCR
3.3 The Melting Point Temperature
3.4 Annealing State Temperature
3.5 What time does it take?
3.6 Problems with primers
3.7 Allocated mutagenesis
4. Other techniques and applications for PCR
5. Sources
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1. Introduction
Gene multiplying is needed for researching for example diseases or
producing some protein. There are two main ways: Gene cloning in
bacteria and polymerase chain reaction.
First I’m going to discuss the basic of gene cloning in bacteria and then I’ll
go a bit deeper with PCR.
2. Gene cloning in bacteria
The main idea is to transpose a gene in some vector into a bacterium and
then let it divide until there’s
enough bacteria (and genes
also). Then gene is isolated, or
kept in plasmides to have
genebanks.
Just plasmides are
mainly used as segmentation
places. Bacteria have also
solute DNA but plasmides are
easier to handle (they are easy
to get off from cells and the
extra DNA is easy to add into Picture 1. Gene’s multiplying in cells.
Violet is transposed gene.
them). Plasmides can also move
to another cell.
When you clone gene in bacteria, you will need: restriction
enzymes (they cut the stripes from an exact place leaving an unpaired base
strand, normally from fore to eight bases), ligase enzymes (they “glue” the
unpaired strands with reversed bases), DNA-polymerase enzymes (they
douplicate the amount of DNA) and reverse transcriptases (which make
cDNA from mRNA because bacteria don’t know how to remove introns
which would lead to an inactive protein).
There are several ways to get the wanted gene in to a cell:
plasmid vectors, virus vectors, micro injection and using electricity or
chemical agent to brake the cell membrane.
3. The other way to multiply genes: PCR
PCR was invented by Kary Mullis (USA) in 1983. He and another scientist
get the Nobel prize in ten years later. PCR is nowadays used and is almost
invaluable tool for gene researchers.
The superiority in PCR is its quickness and cheapness compared
to other processes. It needs only little model-DNA to produce a huge range
of copies. Mutations in primers can make ligase anzymes attach to it and
so the gene is ready to transpose to a cell.
PCR, however, needs unleast some information of the gene’s
DNA order to make the primers. It’s possible to use same sort of gene and
“guess” the primers: this will sometimes work. Because PCR is so
sensitive, it is also very susceptible to surroundings’ impurity.
3.1 Tools for PCR
To get PCR work, you need: a small amount of model DNA, DNA
polymerace enzymes, nucleotides and primers. There are two different
kind of primers, one for both strands. Primers are often about 20
nucleotides long.
3.2 The principle of PCR
First:
Clean the sample from proteins and
other dirts
Second: Separate the strands by heating up to
95°C (called the melting point =Tm)
Third: Primers attach to strands and build
new ones (called annealing Ta=+55+72°C)
Fourth: The strands douplicate at their
favourite temperature (+72°C)
Then the prosess stars all over again…
Picture 2. The principle of PCR
3.3 The Melting Point Temperature
Denaturation is the progress when the two strands separate each other. The
temperature of this is called the melting point temperature (Tm). It depends
on the number of guanine and cytocine (the more, the higher temperature)
and also on the length of primers (the longer, the higher). An average
length for primer is 2 µm.
Tm can be calculated:
Tm = 81,5 °C + 0,41*(%G + %C) – 550/n
where n is the number of nucleotides.
Usually for primers to work, ∆ Tm < 2°C.
3.4 Annealing State Temperature
Annealing state temperature is the stage where primers fasten the strands
and the building begings (from the 3’end). The temperature is conditional
on the concentration of primers and the composition of nucleotides. This
stage last normally only few seconds, but it is often programmed to 0,5-2
min long.
3.5 What time does it take?
Denaturation: 30 - 60 sec
Annealing: 30 - 60 sec
Doupling: 30 - 60 sec
There can be made only 25-35 cycles, otherwise there will be errors. At
the end of program the temperature will be 72°C for 5 minutes to get every
DNA strand ready.
3.6 Problems with primers
If primers are negligently
Picture 3. The hairpin structure
designed, there may occur
some unwanted structures.
These can be prevented
using some computer
programs which calculate
the possibility of those
structures to form.
One usual this kind of structure is the hairpin structure. This
happens when the primers 3’end and 5’end pairs, or the other end pairs
with the body.
Another structure
is primer dimers
where the primers
bind themselves
and then form the
rest
of
the
structure. Commonality to both of
these structures is
that only if the
3’end is involved to
Picture 4. The primer dimer structure
the structure, only
then will the primer be useless.
There may also be nonspesific products because of false
adhesion of primers. They are possible if only few bases are wrong or Ta is
too low.
3.7 Allocated mutagenesis
Picture 5. Amino acid table
As said before, if there is
one or two “false” basepair
on a strand, it doesn’t
prevent the primer to bind.
This can be useful for
reforming
proteins:
a
certain base can be traced
out or change a base to
another
to
improve
toleranse for cold or hot.
A silent mutation
is called an event, where
the base order is changed,
but the peptide order isn’t.
This is possible because
there is several different
triple codons for example for arginine, when changing one base from
strand won’t change the peptide.
4. Other techniques and applications for PCR
PCR has also went forwards: there has been developed several of methods
to improve the mechanics of PCR. To name a few, non-symmetric PCR
produces one-stranded DNA for sequencing; inverced PCR copies some
unknown piece of DNA-strand between 2 known ones. There has been
also invented primers to recognize genetic diseases and so on…
5. Sources:
Ulmanen, Tenhunen ym. Geeni Biologia, WSOY, Porvoo 2000
http://www.mcb.uct.ac.za/pcrcond.htm
http://www.edu.fi/oph/abc/dna/pcr1.html
http://www.mcb.uct.ac.za/hybridn.htm#INTRODUCTION
Pictures:
Picture 2: Ulmanen, Tenhunen ym. Geeni Biologia, WSOY, Porvoo 2000
Picture 3 & 4: http://www.edu.fi/oph/abc/dna/pcr1.html
Picture 5:
http://www.biology.lsu.edu/heydrjay/1201/Chapter17/SCI_Amino_Acid_
CIRCLE.jpg