4. History
One of the most powerful tools in
molecular biology
Invented by Kary Mullis in
1983, resulting in his Nobel
Prize in Chemistry
First published account appeared
in 1985.
Awarded Nobel Prize for
Chemistry in 1993.
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5. The reaction mixture
1. DNA (purified or a crude extract)
2. Primers specific for the target DNA
3. Free nucleotides (A, G, T, C)
4. DNA polymerase
5. Buffer (containing magnesium)
6. The reaction mixture
1- DNA template that contains the DNA region (target) to be
amplified.
2- One or more primers, which are complementary to the
DNA regions at the 5' (five prime) and 3' (three prime)
ends of the DNA region.
3- A DNA polymerase such as Taq polymerase or another
DNA polymerase with a temperature optimum at around
70 C.
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7. The reaction mixture
, (dNTPs) from which the
DNA polymerase builds the new DNA
, which provides a suitable chemical environment for
the DNA Polymerase
, or ions; generally
Mg2+ is used, but Mn2+ can be utilized for PCR-mediated
DNA mutagenesis, as higher Mn2+ concentration increases
the error rate during DNA synthesis
Monovalent cation ions.
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8. Primers
• On the other hand, the length of a primer is limited by:
the maximum temperature allowed to be applied in order to melt it,
as increases with the of the primer
that are too high, i.e., above , can cause problems:
since the is at such temperatures
• The of a primer is generally from 15 to 40 ,
with a between
calculating:
Tm =4(G+C)+2(A+T)
Software
9. Primers
• The DNA fragment to be amplified is determined by
selecting primers
• 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
• They anneal by adhering to the DNA template at these
starting & ending points,
where the DNA polymerase binds and begins the
synthesis of the new DNA strand
10. Primer 3' terminus
• Primer 3' terminus design is critical to PCR success
since the primer extends from the 3' end
• The 3' end should not be complementary over greater
than 3-4 bases to any region of the other primer
(or even the same primer) used in the reaction
and must provide correct base matching to the
template
• There are computer programs to help design primers
Genrunner
13. The basic protocol
1. Denaturation of DNA to single
strands
2. Annealing of primers to DNA
3. Extension by polymerase
4. Repeat 30-35 times
14. Procedure
The PCR process usually consists of 20 -35
each cycle consists of :
1. The has to be heated to (or 98 C if extremely thermostable
polymerases are used)
in order to separate the strands
This step is called denaturing:
it breaks apart the that connect the two DNA strands
Prior to the first cycle:
the DNA is often denatured for an to ensure that the
and the ,
have completely separated and are now
usually , but up to minutes
Also certain polymerases at this step (
15. Procedure
2. After separating the DNA strands, the temperature is
so :
the primers can themselves to the
the temperature of this stage on the and is
usually their Tm (45-60 C)
A wrong temperature during the annealing step can result in :
primers not binding to the template DNA at all
or binding at random
Time: 1-2 minutes
16. Procedure
3. Finally, the DNA polymerase has to copy the DNA strands
It starts at the annealed primer and works its way along the DNA strand
this step is called elongation
the elongation temperature depends on the DNA polymerase:
Taq polymerase elongates optimally at a temperature of 72º C
• The time for this step depends:
1. both on the DNA polymerase itself
2. and on the length of the DNA fragment to be amplified
as a rule-of-thumb, this step takes 1 minute per 1000 bp
• A final elongation step is frequently used after the last cycle
to ensure that any remaining single stranded DNA is completely copied,
this differs from all other elongation steps, only in that it is
longer, typically 10-15 minutes
17. Primers
forward
5’ 3’
Target DNA
3’ 5’
reverse
26. One One billion in about 2 hours!
• At the end of each cycle, the amount of DNA
has doubled
• By the end of 30 cycles, you will have about 1
billion molecules from the original one you
started with!!
230=1,073,741,824
27. The basic protocol—what’s in the tube
5’ 3’
Target DNA
3’ 5’
A
B Free
primers nucleotides
Mg2+ Mg
2+
Buffer
Taq DNA Mg2+ containing
Mg2+
polymerase Mg2+ magnesium
Mg2+
28. 4/28/2012 Free Template from www.brainybetty.com 28
29. Optimising the PCR Reaction
C G Denaturation -
- Annealing -
Primer extension -
--
Ramp -
dNTP -
DNA Template DNA -
PCR -
-
Tm -
30. PCR optimization
1. For the preparation of reaction mixture, a laminar flow
cabinet with UV lamp is recommended
2. Fresh gloves should be used for each PCR step
3. As well as displacement pipettes with aerosol filters
4. The reagents for PCR should be prepared separately and used
solely for this purpose
5. Aliquots should be stored separately from other DNA
samples
6. A control reaction (inner control), omitting template
DNA, should always be performed, to confirm :
a. the absence of contamination
b. or primer multimer formation
31. Applications of PCR
1. the detection of hereditary diseases
2. the identification of genetic fingerprints
3. the diagnosis of infectious diseases
4. the cloning of genes
5. paternity testing
6. and DNA computing
32.
33. How It Works
• Heating/cooling
• Capillary surface area
Intake
• Single chamber
– holds 32 capillaries
• Photohybrids measure
fluorescence at
530, 640 and 705nm
41. The use of multiple, unique primer sets within a single PCR reaction ,
to produce amplicons of varying sizes specific to different DNA sequences
• By targeting multiple genes at once,
additional information may be elicited from a single test run that otherwise
:
would require several times the reagents and technician time to perform
• Annealing temperatures for each of the primer sets ,
must be optimized to work correctly within a single reaction
and amplicon sizes should be separated by enough difference,
in final base pair length to form distinct bands via gel electrophoresis
42. Multiplex PCR
• PCR reactions can be devised in which several
targets are amplified simultaneously often used
in diagnostic applications.
44. Nested PCR
is intended to reduce the contaminations in
products due to the amplification of
unexpected primer binding sites
• Two sets of primers are used in two successive
PCR runs
the second set intended to amplify a secondary
target within the first run product
• This is very successful, but requires more
detailed knowledge of the sequences involved
45. RT-PCR
RT-PCR (Reverse Transcription PCR) is the method
used to amplify, isolate or identify a known sequence
from a cell or tissues RNA library
• Essentially normal PCR preceded by transcription by
Reverse transcriptase (to convert the RNA to cDNA)
this is widely used in :
1. expression mapping, determining when and where
certain genes are expressed
2. detection of RNA viruses
46. Colony PCR
Bacterial clones (E.coli) can be screened for
the correct ligation products
• Selected colonies are picked with a sterile
toothpick from an agarose plate,
and dabbed into the master mix or sterile
water,
primers (and the master mix) are added
the PCR protocol has to be started with an
extended time at 95ºC
48. Uses of PCR
PCR can be used for a broad variety of experiments and analyses:
1. Genetic fingerprinting
• is a forensic technique used to identify a person by comparing his or
her DNA with a given sample
• An example is blood from a crime scene being genetically compared
to blood from a suspect
• The sample may contain only a tiny amount of DNA ,
(obtained from a source such as blood, semen, saliva, hair, or other
organic material)
49. Uses of PCR
2. Detection of hereditary diseases
• The detection of hereditary diseases in a given genome is a
long and difficult process, :
which can be shortened significantly by using PCR
• Each gene in question can easily be amplified through PCR by
using the appropriate primers :
and then sequenced to detect mutations
50. Uses of PCR
3. Viral diseases
• can be detected using PCR through amplification of
the viral DNA
• This analysis is possible right after infection,
which can be from several days to several months
before actual symptoms occur
• Such early diagnoses give physicians a significant
lead in treatment
• Treatment evaluation, viral load
• Genotyping, viruses, bacteria
51. Uses of PCR
4. Mutagenesis
• Mutations can be introduced into copied DNA sequences,
in two fundamentally different ways in the PCR process
• Site-directed mutagenesis allows the experimenter to introduce a mutation
at a specific location on the DNA strand
• Usually, the desired mutation is incorporated in the primers used for the
PCR program
• Random mutagenesis, is based on the use of error-prone polymerases in the
PCR process
the location and nature of the mutations cannot be controlled
• One application of random mutagenesis is :
to analyze structure-function relationships of a protein
• By randomly altering a DNA sequence:
one can compare the resulting protein ,
with the original and determine the function of each part of the protein
52. Uses of PCR
• 5. Genotyping of specific mutations
• Through the use of allele-specific PCR,
one can easily determine which allele of a mutation or
polymorphism an individual has
• Here, one of the two primers is common,
and would anneal a short distance away from the mutation,
while the other anneals right on the variation
• The 3' end of the allele-specific primer is modified,
to only anneal if it matches one of the alleles
