Pharmceutical Biotechnology
Section 1
PCR
The structure of DNA (Deoxyribonucleic acid):
•DNA is a polymer called ‘polynucleotide”. The monomer units of DNA are nucleotides.
•Each nucleotide consists of
5-carbon sugar (deoxyribose)
Nitrogen containing base attached to the sugar
Phosphate group.
Deoxyribose =
OH H at position 2`
The structure of DNA, Types of nucleotides:
Four different types of nucleotide, differing only in the nitrogenous base
A is for adenine
G is for guanine
C is for cytosine
T is for thymine
The structure of DNA: Base pairing and complementary sequences:
- Composed of two strands that are held together by base pairing.
-The two chains of the double helix have complementary sequences; This is called complementary base
pairing where Adenine pairs with Thymine (A T), Guanine pairs with Cytosine (G C).
- Composed of parallel strand and anti-parallel strand (one oriented 5’– 3’ and the other 3’ – 5’)
** If sequence 5’-ATGTC-3’ is present on one chain, the opposite chain must have the complementary
sequence 3’-TACAG-5’
The structure of DNA, DNA backbone:
The DNA backbone:
The DNA backbone is a polymer of an alternating sugar-phosphate sequence. The deoxyribose sugars
are joined at both the 3'-hydroxyl and 5'-hydroxyl groups to phosphate groups in ester links, which
known as "phosphodiester" bonds.
Synthesis of DNA:
DNA replication (addition of nucleotides to form a new strand) occurs through
DNA polymerase III.
DNA polymerase III can only add nucleotides to the 3’ end of the DNA. This
causes the NEW strand to be built in a 5’ to 3’ direction.
Synthesis
in 5’ to 3’
direction
Polymerase Chain Reaction (PCR)
It is used to selectively amplify a particular sequence of DNA In Vitro.
Sequence may represent a small part of a large and complex mixture of DNAs
It considered as DNA photocopier
Invented by Kary Mullis in 1983 who was awarded Nobel Prize in 1993.
Reaction components of Polymerase Chain Reaction (PCR)
1) Target DNA - contains the sequence to be amplified
2) Forward and reverse Primers; oligonucleotides that define the sequence to be
amplified.
PCR Targets; The targets in PCR are the sequences of DNA Primers are designed on
each end of the region of DNA.
PCR Primers (15-30 nucletotides)
Primer is a short strand of nucleic acid that serves as a starting point for DNA
replication.
3) dNTPs – deoxy nucleotide triphosphates: DNA building blocks.
4) Thermostable DNA Polymerase
5) Mg++ ions - cofactor of the enzyme
6) Buffer solution – maintains pH and ionic strength of the reaction solution
suitable for the activity of the enzyme.
Polymerase of PCR & Why called PCR?
Taq DNA Polymerase
Taq stands for Thermus aquaticus.
Taq DNA polymerase is heat stable polymerase.
It is called “polymerase” because the only enzyme used in this reaction is DNA
polymerase.
It is called “chain” because the products of the first reaction become substrates
of the following one.
Steps of Polymerase Chain Reaction (PCR):
1- Denaturation: in which the DNA strands are separated by heating to 95°C (break of
hydrogen bonds).
2- Annealing: the process of allowing the primer sequences to form hydrogen
bonds by cooling the DNA.
3- Extension:
Taq DNA polymerase binds to the template DNA and
starts adding nucleotides that are complementary to
the first strand at 72 °C.
Denaturation: 94°- 95°C 1 min.
Primer Annealing: 55°- 65°C 45 Sec.
Extension of DNA: 72° 1-2 min.
Number of Cycles: 25-40
Amplification process of Polymerase Chain Reaction (PCR)
Applications of Polymerase Chain Reaction (PCR)
1- In cloning of genes:
Forward primer with the suitable restriction site.
According to that of the vector at which it will be liagted.
Reverse primer with the suitable restriction site.
According to that of the vector at which it will be
liagted.
Applications of Polymerase Chain Reaction (PCR)
2- Can detect infectious pathogens at very early stages” detect a viral genome in a
drop of blood” even in presence of very few copies.
3- PCR is used in forensic medicine.
Questions:
1- In PCR, DNA polymerase starts its activity in ………….step.
2- The DNA polymerase used in PCR is called …………………..
3- The components of PCR reaction are :…., …., …., ……, ……., and ……
4-The binding of primers to the DNA in PCR occurs in……….step.
5 - Denaturation step in PCR is important to ….
Thanks for your attention
Questions
