human genetic lab 12

1. Pathological Analysis Dept.
Human Genetics
Lab. 12
3rd
Year
1
P.C.R Technology
What is PCR
It is a molecular technology aim to amplify a single or few copies of the
DNA to thousands or millions of copies. Developed in 1983 by Kary
Mullis, PCR is now a common and often indispensable technique used in
medical and biological research labs for a variety of applications. These
include diagnosis of infectious diseases, DNA sequencing and DNA-
based phylogeny. In 1993, Mullis was awarded the Nobel prize in
Chemistry along with Michael Smith for his work on PCR
PCR application
The name, Polymerase chain reaction, comes from the DNA polymerase
used to amplify (replicate many times) a piece of DNA by in vitro
enzymatic replication.
1. The original molecule (molecules of DNA) are replicated by the
DNA polymerase enzyme, thus doubling the number of DNA molecules.
2. Then each of these molecules is replicated in a second "cycle" of
replication, resulting in four times the number of the original molecules.
3. Again, each of these molecules is replicated in a third cycle of
replication. This process is known as a "chain reaction" in which the

2. Pathological Analysis Dept.
Human Genetics
Lab. 12
3rd
Year
2
original DNA template is exponentially amplified with PCR it is possible
to amplify a single piece of DNA, or a very small number of pieces of
DNA, over many cycles, generating millions of copies of original DNA
molecule
Types of PCR
• Conventional (Qualitative)PCR.
• Multiplex PCR.
• Nested PCR.
• RT-PCR and qRT-PCR.
• Quantitative PCR.
• Hot-start PCR.
• Touchdown PCR.
• Assembly PCR.
• Colony PCR.
• Methylation-specific PCR.
• LAMP assay.
PCR requirement
PCR requires several components and reagents. These components
include:
1. DNA template that contains the DNA region (target) to the 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. DNA polymerase such as Taq polymerase or another DNA
polymerase with a temperature optimum at around 70 C°.

3. Pathological Analysis Dept.
Human Genetics
Lab. 12
3rd
Year
3
4. Deoxynucleoside triphosphates (dNTPs; also very commonly and
erroneously called deoxynucleotide triphosphates), the building blocks
from which the DNA polymerases synthesizes a new DNA strand.
5. Buffer solution, providing a suitable chemical environment for
optimum activity and stability of the DNA polymerase.
6. Divalent cations, magnesium Mg+2
ion is used.
7. Monovalent cation potassium ions.
Procedure
The PCR usually consists of a series of 20 to 35 repeated temperature
changes called cycles; each cycle typically consists of 2-3 discrete
temperature steps. Most commonly PCR is carried out with cycles that
have three temperature steps. The temperatures used and the length of
time they are applied in each cycle depend on a variety of parameters.
These include the enzyme used for DNA synthesis, the concentration of

4. Pathological Analysis Dept.
Human Genetics
Lab. 12
3rd
Year
4
divalent ions and dNTPs in the reaction, and the melting temperature
(Tm) of the primers.
• Denaturation step: this step is the first regular cycling event and consists
of heating the reaction to 94-98 C° for 20-30 seconds. It causes melting
of DNA template and primers by disrupting the hydrogen bonds
between complementary bases of the DNA strands, yielding single
strands of DNA.
• Annealing step: the reaction temperature is lowered to 50 -65 C° for
2040 seconds allowing annealing of the primers to the single - stranded
DNA template. Typically the annealing temperature is about 3-5 degrees
Celsius below the Tm of the primers used. Stable DNA-DNA hydrogen
bonds are only formed when the primer sequence very closely matches
the template sequence. The polymerase binds to the primer- template
hybrid and begins DNA synthesis.
• Extension / elongation step: the temperature at this step depends on
the DNA polymerase used; Taq polymerase has its optimum activity
temperature at 75-80 C°, and commonly a temperature of 72 C° is used
with this enzyme. At this step the DNA polymerase synthesizes a new
DNA strand complementary to the DNA template strands by adding
dNTPs that are complementary to the template. The extension time
depends both on the DNA polymerase used and on the length of the DNA
fragment to be amplified. At its optimum temperature, the DNA
polymerase will polymerize a thousand bases in one minute.

5. Pathological Analysis Dept.
Human Genetics
Lab. 12
3rd
Year
5
• Final elongation: this single step is occasionally performed at a
temperature of 70-74 C° for 5-15 minutes after the last PCR cycle to
ensure that any remaining single - stranded DNA is fully extended.
s
Dr.Hayder Nasser