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Polymerase Chain Reaction - Coggle Diagram
Polymerase Chain Reaction
Uses
identify:
disease causing
virus/bacteria
deceased person
criminal suspect
used in
pathogen
sequencing programmes
forensics
DNA from tiny drop of blood amplified for genetic fingerprinting
archeology
diagnosis of human infectious
diseases (eg Lyme disease)
What does it do?
amplify number of copies of a specific region of DNA
so enough DNA produced to be tested
example enzyme -
Taq polymerase
from bacterium that lives in hot springs
∴ withstand high temps needed for DNA strand separation)
definition
used to amplify target DNA
sequences
present in DNA source
can produce many copies of
selected section of DNA
in very short time
Advantages
highly
specific
easily
automated
capable of amplifying minute
amounts of samples
1. Denaturation (95°C)
mixture
heated
(DNA, bases, primers + polymerase enzymes)
breaks H bonds between compl. base pairs
separates 2 strands
of double stranded DNA
which contains target sequence
why important sample DNA
remains as 2 separate strands?
each strand acts as
template
against which
free nucleotides
2. Annealing (55°C)
'joining together'
mixture
cools
so 2
primers
can
anneal
the start of each strand of the complementary
DNA region (can
attach
onto exposed
bases on separated strand)
primers
D: short pieces of DNA exactly complimentary
to flanking sequence
prevent the DNA
strands re-joining
act as signals to the polymerase
enzymes to start adding nucleotides
3. Extension (72°C)
once new piece of double stranded DNA formed
polymerase enzyme
(opt temp =72°C)
extends primers
into new complementary strands
now 4 strands of DNA
how DNA polymerase enzyme works
to form new double strand?
nucleotides
position themselves
in place opposite
exposed bases
on template strands
compl. base pairs held by
H bonds
condensation reactions
create
PHOSPHODIESTER
bonds
between newly joined nucleotides
Conditions
what is needed?
3 steps repeated
30-40 cycles
carried out in
automated cycler
heat + cool tubes with reaction
mixture in a very short time
flanking regions must be known
(exact sequences that lie on either sides of desired region of DNA or gene)
enzyme -
DNA polymerase
been extracted from thermophilic
bacteria (thermostable)
Free
deoxyribonucleotides
Primers
short strands of DNA complementary
to sequence at start of each strand of region to be amplified
exact temps involved + length of time
sample is kept @ each temp -
depend on the
type
of DNA being amplified.
4. Repeated heating &
cooling cycles
multiply target DNA exponentially
as new double strand separates to form 2 templates for further synthesis