Title: Polymerase chain reaction PCR
1Polymerase chain reaction (PCR)
2Polymerase chain reaction (PCR)
Polymerase chain reaction (PCR) - One of the
most widely used techniques in molecular biology
and for good reason - Rapid, inexpensive and
simple means of producing relatively large
numbers of copies of DNA molecules - the
technique which allows a small amount of the DNA
molecule to be amplified exponentially
Application
-Hereditary disease -Identification of genetic
finger prints -The diagnosis of infectious
disease -The cloning of genes -Paternity testing
3Amplication of nitrite reductase genes (nirK and
nirS) to detect denitrifying bacteria in
environmental samples.
For DNA sequencing, amplified products were
purified by eluting the bands from an agarose gel
by using the gel extraction kit.
4Basic components of PCR
DNA template
The DNA which will be amplified by the PCR
provide both the energy and nucleosides for the
synthesis of DNA. It is important to add equal
amounts of each nucleotide (dATP, dTTP, dCTP,
dGTP) to the mixture to prevent mismatches of
bases.
dNTP(deoxynucleotide triphosphate)
The role of Mg in PCR forms complexes with
dNTPs that are the actual substrates for Taq
Polymerase. When Mg is too low, primers fail to
anneal to the target DNA. When Mg is too high,
the base pairing becomes too strong and the
amplicon fails to denature completely when you
heat to 94C.
MgCl2
promotes specific annealing of primers to the PCR
template. K binds to phosphate groups on
double-stranded DNA, stabilizing primer annealing.
KCl
5Basic components of PCR
Short pieces of DNA (20-30 bases) that bind to
the DNA template allowing Taq DNA polymerase
enzyme to initiate incorporation of the
deoxynucleotides. Both specific and universal
primers can be used.
Primers
A heat stable enzyme that adds the
deoxynucleotides to the DNA template.
Taq DNA polymerase
keeps the mixture at the proper pH so the PCR
reaction will take place.
Buffer
6dNTP(deoxynucleotide triphosphate)
7dNTP(deoxynucleotide triphosphate)
Deoxy adenosine triphosphate (dATP)
Deoxycytidine triphosphate (dCTP)
Deoxyguanosine triphosphate (dGTP)
Deoxycytidine triphosphate (dCTP)
8PCR mixture
AccuPower? PCR PreMix kit
9PCR cycling program
Initial denaturation 95oC for 3 minutes - make
sure template is fully denatured 1
cycle Amplification 30 cycle 95 oC for 1
minute -denaturing 55 oC for 1 minute -
annealing 72 oC for 2 minutes - extension
Final Extensions 72 oC for 10 minute -
finishing off amplified fragments 1
cycle Soak 4oC until required - storage
10PCR cycling program
Denaturation At temperatures above 90C,
double-stranded DNA denatures or "melts". That
means the weak hydrogen bonds that usually hold
the two complementary strands together at normal
temperatures are disrupted resulting in two
single stranded DNA strands
11PCR cycling program
Annealing
After separating the DNA strands, the temperature
is lowered so the primers can attach themselves
to the single DNA strands. The temperature of
this stage depends on the primers and is usually
5C below their melting temperature (45-60C).
12Primers
Primers are short, artificial DNA strands often
not more than 50 and usually only 18 to 25 base
pairs long that are complementary to the
beginning or the end of the DNA fragment to be
amplified
Examples of bacteria universal primer sequences
areForward 5' GAT CCT GGC TCA GGA TGA AC 3' (20
mer)Reverse 5' GGA CTA CCA GGG TAT CTA ATC 3'
(21 mer)
Estimation of the melting and annealing
temperatures of primer If the primer is shorter
than 25 nucleotides, the approx. melting
temperature (Tm) Tm4(GC) 2 ( AT)
GAGGTAACCACACCAGA ? 4Gs, 5Cs, 7As, 1T Â Â Â Â Â Â Â Tm
4(45)2(71) Â Â Â Â Â Â Â Â Â Â Â
(49)(28) Â Â Â Â Â Â Â Â Â Â Â 3616 Â Â Â Â Â Â Â Â Â Â Â
52?       ?Tm 52?    Â
Annealing temperature should be approx. 5? lower
than the Tm
13The phosphate group of the previous nucleotide is
linked to carbon number 5, and the phosphate
group of the next nucleotide is linked to carbon
number 3. The naming system, 5' to 3' is used to
describe the order of the nucleotides in the DNA
strand.
14(No Transcript)
15DNA is a double-stranded molecule twisted into a
helix (think of a spiral staircase). Each
spiraling strand, comprised of a sugar-phosphate
backbone and attached bases, is connected to a
complementary strand by non-covalent hydrogen
bonding between paired bases.
16PCR cycling program
Extension
Finally, the DNA polymerase has to copy the DNA
strands. It starts at the annealed primer and
works its way along the DNA strand. The extension
temperature depends on the DNA polymerase. Taq
polymerase extends optimally at a temperature of
72C. The time for this step depends both on the
DNA polymerase itself and on the length of the
DNA fragment to be amplified
17PCR cycling program
Exponential template duplication
The process is then repeated by cycling through
the temperatures over and over again (35 to 55
times). Each cycle results in a new DNA duplex,
each strand acting as a potential template for
one or other primer.