Polymerase Chain Reaction (PCR) is an in vitro method for amplifying specific DNA regions using complementary oligonucleotide primers and DNA polymerase. Key parameters for designing primers include primer sequences, length, GC content, melting temperature (Tm), and annealing temperature (Ta). PCR is applied in various fields such as genotyping, cloning, mutation detection, and forensics due to its simplicity and sensitivity.

1. POLYMERASE CHAIN REACTION
(PCR)

2. PCR - Polymerase Chain Reaction
• PCR is an in vitro technique for the amplification of a region of DNA which lies
between two regions of known sequence.
• PCR amplification is achieved by using oligonucleotide primers.
– These are typically short, single stranded oligonucleotides which are
complementary to the outer regions of known sequence.
• The oligonucleotides serve as primers for DNA polymerase and the denatured
strands of the large DNA fragment serves as the template.
– This results in the synthesis of new DNA strands which are complementary
to the parent template strands.
– These new strands have defined 5' ends (the 5' ends of the oligonucleotide
primers), whereas the 3' ends are potentially ambiguous in length.

3. PCR PROCESS/STEPS

4. COMPONENTS OF PCR REACTION
The following components are require to execute the PCR reaction of a gene of
interest
• Water
• Buffer
• DNA template
• Primers
• Nucleotides
• Mg++ ions
• DNA Polymerase

5. PARAMERTERS TO DESIGN A GOOD PRIMER
Primer sequence
Primer length
GC%
GC clamp
Melting temperature (Tm)
Annealing temperature (Ta)

6. • Must be complementary to flanking sequences of target region.
• Avoid:
Complementary sequences between primers.
 Repeat(ex: ATATATAT) 
 misprime.
 Runs (ex: AGCGGGGGAT) 
 misprime.
2. Primer length:
• It is generally accepted that the optimal length of primers is 18-25 bp.
• Not too long nor too short
1. Primer Sequence:

7. 3. GC Content:
• GC% = Number of G's and C's in the primer as a percentage of the total bases.
• Should be 40-60%.
• 4. GC clamp:
• Presence of G or C bases within the last five bases from the 3' end of primers.
• Not more than 2 G's or C's .

8. 5. Melting temperature (Tm):
• What is Tm?
• Melting temperatures in the range of 50-60 °C generally produce the best results.
• Maximum difference between primer pairs is 5°C.
• The Tm of the primer can be calculated by the following formula:
Tm = [(G + C) x 4] + [(A + T) x 2]
6. Annealing Temperature (Ta):
• The primer melting temperature is the estimate of the DNA-DNA hybrid stability and
critical in determining the annealing temperature.
• Depends directly on length and GC composition of the primers.
• Too high Ta produce insufficient primer-template hybridization.
• Too low Ta 
 lead to non-specific products caused by a high number of base
pair mismatches.

9. A TYPICAL PCR REACTION
Sterile Water 38.0 ul
10X PCR Buffer 5.0 ul
MgCl2 (50mM) 2.5 ul
dNTP’s (10mM each) 1.0 ul
PrimerFWD (25 pmol/ul) 1.0 ul
PrimerREV 1.0 ul
DNA Polymerase 0.5 ul
DNA Template 1.0 ul
Total Volume 50.0 ul
Aliquot
49 ul
Add DNA
as last step

10. TYPICAL THERMOCYCLER CONDITIONS
1. Initial Denaturation 95 C 3 min
2. DNA Denaturation 95 C 1 min
3. Primer Annealing 65 C 1 min
4. Primer Extension 72 C 1 min
5. Go to step #2, repeat 39 more times
6. End
cycle
One X 25

11. RESULT VISUALIZATION
 After thermal cycling, tubes are taken
out of the PCR machine.
 Contents of tubes are loaded onto an
agarose gel.
 DNA is separated by size using an
electric field.
 DNA is then stained.
 PCR products are visible as different
“bands”.
Gel running

12. Marker
Target sequence

13. PCR ADVANTAGES
• APPLICATIONS
• Simplicity, easier methodology, sensitive, extensively validated standard operating procedure and
availability of reagents and equipment

 Genotyping.

 RT-PCR.

 Cloning.

 Mutation detection.

 Sequencing.

 Microarrays.

 Forensics.

 Paternity testing.