The document describes a lecture on polymerase chain reaction (PCR). PCR is a technique used to amplify a specific segment of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. The key components required for PCR are a DNA template, primers, DNA polymerase, dNTPs, buffer and magnesium chloride. The process involves repeated cycles of heating and cooling of the reaction to denature and extend DNA. Applications of PCR include DNA fingerprinting, prenatal diagnosis of genetic diseases, diagnosis of viral infections, and studying ancient DNA samples.

1. Series of Lectures
Course Title: Methods in Molecular Biology
Course code: BCH-613
Credit Hours: 3(1-2)
By
Dr.Sumera Shaheen
Assistant Professor
Dept. of Biochemistry
Govt.College Women University of Faisalabad

2. Lecture : Polymerase Chain Reaction (PCR)

3. PCR
 PCR was invented by Kary Mullis and his colleagues in the 1980s.
 PCR is a means to amplify a particular piece of DNA
Amplify= making numerous copies of a segment of DNA
 PCR can make billions of copies of a target sequence of DNA in short time
 It is a laboratory version of DNA Replication in cells
 The amplified DNA can be cloned directly or used in a variety of analytical
procedures.

4. PCR
 Why “Polymerase”?
Because the only enzyme used in the reaction is DNA polymerase.
 Why “Chain” ?
Because the products of the first reaction become the substrates of the
following one and so on.

5. Requirements of PCR
1. Target DNA (sequence of DNA required to be amplified)
2. Pair of primers (short oligonucleotide to start the amplification reaction)
3. dNTPs (deoxy Nucleotide Triphosphates)
4. Thermostable DNA Polymerase
5. Taq buffer
6. MgCl2
7. Nuclease Free water

6. Steps and conditions of PCR reaction
 Initial Denaturation
1.Denaturation
2. Annealing
3. Extension
Steps 1-3 repeated 25-35 times
 Final Extension:

8. Thermal cycler

9. Initial Denaturation: 94⁰C
 Double-stranded template DNA is denatured by heating, typically to 94°C
 To separate all the double stranded DNA molecules in the reaction
 Duration 2-3 mins

10. 1. Denaturation: 94⁰C
 This is the first step of amplification cycle
 DNA template is denatured by heating, typically to 94°C, to separate the
double stranded DNA
 It is necessary for binding of Primer
 Usually for 45 Sec

11. 2. Annealing (temp 50-65⁰C)
 The reaction is rapidly cooled to an annealing temperature
 It allow the oligonucleotide primers to hybridize to the template
 It anneal only to sequences that are complementary to Primers.

12. 3. Extension/Elongation
 The reaction is heated to a temperature, typically 72°C for efficient DNA
synthesis by the thermostable DNA polymerase.
 This means the optimum temperature of amplification is 72°C
 Time depends upon length of the amplicon 1min/1kb

13. Final Extension
 When all PCR cycles are completed
 Temperature is maintained at 72°C to perform final extension
 It completes the polymerization at the ends of amplified fragments
 For 7-10 mins
 Time depends on the size of the amplicon

15. DNA template
 A purified DNA molecule containing desired GENE sequence.

16. dNTPs s(deoxy Nucleotide Triphosphates)
Four types of nucleotides which are
component of DNA molecule
These include;
1.deoxy Adenine Triphosphate (dATP)
2.deoxy Guanine Triphosphate (dGTP)
3.deoxy Cytosine Triphosphate (dCTP)
4.deoxy Thymine Triphosphate (dTTP)

17. Enzymes used in PCR
Several types of thermostable DNA polymerases are available for use in PCR
 Taq DNA polymerase,
 Isolated from the eubacterium Thermus aquaticus
 Most commonly used enzyme for standard end-point PCR.
 The robustness of this enzyme allows its use in many different PCR assays.
 However, as this enzyme is active at room temperature,
 It is necessary to perform reaction setup on ice to avoid nonspecific amplification

18. Hot-start DNA polymerase
 When amplification reaction setup is performed at room temperature, primers can bind
nonspecifically to each other, forming primer–dimers.
 During amplification cycles, primer–dimers can be extended to produce nonspecific
products, which reduces specific product yield.
 For more challenging PCR applications, the use of hot-start PCR is crucial for successful
specific results.
 To produce hot-start DNA polymerases, Taq DNA polymerase activity can be inhibited at
lower temperatures with antibodies or, more effectively, with chemical modifiers that
form covalent bonds with amino acids in the polymerase.
 The chemical modification leads to complete inactivation of the polymerase until the
covalent bonds are broken during the initial heat activation step.

19. High-fidelity DNA polymerase
 Unlike standard DNA polymerases (such as Taq DNA polymerase), high-fidelity
PCR enzymes generally provide a 3' to 5' exonuclease activity for removing
incorrectly incorporated bases.
 High-fidelity PCR enzymes are ideally suited to applications requiring a low
error rate, such as cloning, sequencing, and site-directed mutagenesis.
 However, if the enzyme is not provided in a hot-start version, the 3' to 5'
exonuclease activity can degrade primers during PCR setup and the early stages
of PCR.
 Nonspecific priming caused by shortened primers can result in smearing on a gel
or amplification failure — especially when using low amounts of template.
 It should be noted that the proofreading function often causes high-fidelity
enzymes to work more slowly than other DNA polymerases.

20. Primers
 A short sequence of Oligonucleotides required to start the amplification.
 Two synthetic oligonucleotides are prepared, complementary to sequences on
opposite strands of the target DNA at positions just beyond the ends of the
segment to be amplified.
 The oligonucleotides serve as replication primers that can be extended by DNA
polymerase.
 The 3´ ends of the hybridized probes are oriented toward each other and
positioned to prime DNA synthesis across the desired DNA segment

21. PCR Primer Design Guidelines
Primers must be complementary to flanking sequences of target region
Primer Length: It is generally accepted that the optimal length of PCR primers is
18-22 bp.

22.  Primer Melting Temperature (Tm):
 Primers with Tm in the range of 52-58 oC generally produce the best results.
 The GC content of the sequence gives a fair indication of the primer Tm.
 Tm = (G + C)  4 + (A + T)  2
 The difference of Tm should be < 5oC of the forward and reverse primers.
 GC Content: The GC content (the number of G's and C's in the primer as a
percentage of the total bases) of primer should be 40-60%.
 GC Clamp: The presence of G or C bases within the last five bases from the 3'
end of primers (GC clamp)

23. Primer Annealing Temperature (Ta): The primer melting temperature is the
estimate of the DNA-DNA hybrid stability and critical in determining the
annealing temperature.
(Ta = Tm – 5)
 -Too high Ta will produce insufficient primer-template hybridization
resulting in low PCR product yield.
 -Too low Ta may possibly lead to non-specific products caused by a high
number of base pair mismatches

24. Primer pairs should not have complementary regions
 Should avoid Self Dimer
 Primer Secondary Structures:
 Presence of the primer secondary structures produced by intermolecular or
intramolecular interactions can lead to poor or no yield of the product.
 Di-nucleotide repeats (e.g., GCGCGCGCGC or ATATATATAT) or single base
runs (e.g., AAAAA or CCCCC) should be avoided.

25. Software to design Primers
1 Primer-BLAST
2 Primer3
3 Primer3Plus
4 PrimerQuest
5 OligoPerfect
6 PerlPrimer
7 OLIGO
8 GenScript Real-time PCR (TaqMan) Primer Design
9 GenScript Online PCR Primers Designs Tool
10 AutoPrime
11 RExPrimer
12 BatchPrimer3
13 Eurofins Genomics’ Primer Design Tools

26. Designing of Primers
Steps for Designing of Primers
 Need desired DNA sequence
 Sequence can be collected from NCBI
 Copy the sequence and paste in the primer designing software
 Get Primer sequence
 Carefully check the properties of primer
 Get primer synthesized from company

27. The segment that you want to amplified is in the blue square
5’
3’
Design the primers using Primer3, then send them to any companey who will synthesize them
Make sure that the area that you want to study is between the primers
The region to be studied should be between the forward and reverse
Example : you want to study a mutation in a DLG3 gene and how it relate to
memory,
Find you’re required region from any website, eg.

28. Applications of PCR
 This technology is highly sensitive
 PCR can detect and amplify as little as one DNA molecule in almost any type of
sample

29. cont
 Epidemiologists can use PCR-enhanced DNA samples from human remains to
trace the evolution of human pathogenic viruses.
 It is also being used for detection of viral infections before they cause symptoms

30.  DNA fingerprinting; to know genetic makeup of a person or other living things.
It is used to identify individual, to establish parenthood, track down blood relatives,
cures for disease, in forensic science to identify potential crime suspect etc

31. cont
 Prenatal diagnosis (detection of abnormalities before birth) of a wide array of
genetic diseases.
 Diagnosis of retroviral disease, used for detection of HIV infection

32.  Although DNA degrades over time, PCR has allowed successful cloning of DNA
from samples more than 40,000 years old.
 Investigators have used the technique to clone DNA fragments from the
mummified remains of humans and extinct animals such as the woolly mammoth,
creating the new fields of molecular archaeology and molecular paleontology.
 Nucleic Acid from burial sites has been amplified by PCR and used to trace
ancient human migrations.