Pt.

Ravishankar Shukla University, Raipur

SoS In Life Science

PCR Primer Designing

Assigned By :- Presented By :-
Dr. Manoj Kumar Patel Deepa Chouhan
M.Sc. 3rd Sem.
Microbiology
PCR (Polymerase Chain Reaction)
 Polymerase Chain Reaction (PCR ) is a technique that
result in exponential amplification of a desired region of
a DNA molecules in vitro.
 PCR is a technique used in molecular biology for DNA
amplification.
 PCR can amplify a single copies of DNA segment in
millions of copies of a particular DNA sequence.
 It is developed in 1983 by kary mullis.

Fig:- PCR – Thermal cycler

Step in PCR

 Denaturation –
93 to 95°C for 1 min.

 Annealing –
50 to 55°C for 45 sec.

 Extension or
Elongation –
70 to 75 °C for 1-2 min.
PCR Primer Designing
 Primer :- Short, single-stranded DNA sequence.
 It is about 20–25 base pair.
 It is of two type–forward and reverse.
 A pair of primer used in polymerase chain reaction
technique for specific amplification of DNA region.
 Primer are highly specific.
 Used in diagnosis of diseases.
 General recommendations on designing PCR
primers:
 15 to 30 nucleotides long.
 Tm 55 to 70°C (within 5°C, for two primers).
 40 to 60 % GC (with uniform distribution).
 One C or G at 3’ end.
Primer Length
 Length of the primers should not be very short or long.
 If the primer are too short , they might hybridize to non
target site and give undesired amplification products.
 It is generally accepted that the optimal length of PCR
primers is 18-22 bp.
 Calculation of length of primers: The longer the
primer, the higher its specificity for particular target.
 K = [ g / 2]G+C × [(1 – g) / 2]A+T.
 Where, K is the expected frequency of occurrence of
target sequence in a DNA molecules.
 G is the relative G+C content of the DNA molecules
and G, C, A and T are the number of specific nucleotide
in the primer.
Primer Melting Temperature
 This is the temperature at which 50% of the primer and
it’s complement are hybridized.
 Primer Tm is the range of 52–58 °C generally produce
the best result.
 Primer with the melting temperature above 65% have a
tendency for secondary annealing.
 The ‘ Wallace Rule’ make a quick estimate of the Tm
value.
 Formula – Tm = 2 × ( A + T ) + 4 × ( G + C ).
 For example- sequence;
 5'-AGCGGGCGCGCGTAC-3‘.
 Tm = 2 × (2 + 1) + 4 × (7 + 5)
Tm = 2 × 3 + 4 × 12
Tm = 54.
Primer Annealing Temperature
 The primer melting temperature is the estimate of
the DNA-DNA hybrid stability and critical in
determining the annealing temperature.
 Too high Ta will produce insufficient primer
template hybridization resulting in low PCR
product yield.
 Too low Ta may possibly lead to non specific
product caused by a high number of base pair
mismatches.
GC Content -The GC content (the number of G’s
and C’s in the primer as a percentage of the total base )
of primer should be 40 to 60 %.
GC Clamp – The presence of G or C bases within the
last five bases from the 3’ end of primer GC clamp) help
promote specific binding at the 3‘ end due to the
stronger bonding by G or C base.
 More than 3 G’s or C’s should be avoided in the last 5
bases at the 3’ end of the primer.

Fig :- GC Clamp
Primer Secondary Structure
 Hairpins - It is formed by intramolecular interaction
with in the primer.
 Self Dimer – It is formed by intermolecular
interaction between the two (same sense)
primer.
 Cross Dimer – It is formed by intermolecular
interaction between sense and antisense primer.

Fig:- Primer Secondary Structure.

Repeats
 A repeat is a di nucleotides occurring many times
consecutively.
 For eg. :- ATATATATAT – maximum acceptable
number of di nucleotide repeats is 4.
Runs
 Primer with long runs of a single base should generally
be avoided .
 For example – AGCGGGGGATGGGG has runs of base
‘G’ of value is 5 and 4.
3’ End Stability
 It is the maximum ∆G value of the five bases from the
3’end.
 An unstable 3’ end (less negative ∆G) will result in less
false priming.
Application
 Selective DNA isolation.
 Infectious disease detection ( HIV, Meningitis).
 Forensic application (crime report).
 Detection of mutation(genetic disease).
 Gene cloning.

Advantages of PCR
 Easy to use.
 Good sensitivity.
 Speed.

Limitation
 Short size and limiting amount of PCR product.
 Cannot amplify gene > 100 Kb.
Primer Design Using Software
 A number of primer design tools are available that can
assist in PCR primer design for new and experienced
users alike.
 These tools may reduce the cost and time involved in
experimentation by lowering the chance of failed
experimentation.
 Software such as Alleles D and Beacon Designer can
design primer and oligonucleotide probes for complex
detection assays such as multiplex assays, cross species
primer design.
 Primer BLAST was developed at NCBI to help the
users make primers that are specific to the input PCR
template. It use primer 3 to design PCR primer and
then submit them to BLAST search against user
selected database.
 The BLAST result are then automatically analyzed to
avoid primer pair that can cause amplification of target
other than the input template.
Current Research
Researchers :- Sumnil Marwaha, Basanti Brar, Vinod
Kumar Jain, Rachna Poonia, Minakshi Prasad.
Affiliation :- Theileriosis is a tick borne disease where
there are several species of Theileria that can infect
bovines. The differentiation of these species may not be
possible by microscopic examination or serological test.
Therefore, multiplex PCR assay was standardized and
evaluated for rapid and simultaneous differential detected
of two species of Theileria viz, Theileria annulata and
Theileria orientalis. This is first report of T. orientalis from
Haryana, India.

Research Area :- Veterinary Medicine, Lala Lajpat Rai

University of Veterinary and Animal Science, Hisar, India.
References
 Marwaha, S., Brar, B., Jain, V.K. et al. Multiplex PCR for
rapid differential diagnosis of co-prevalent species of
Theileria (Theileria annulata and Theileria orientalis) in
cattle. Parasitol Res (2023).
https://doi.org/10.1007/s00436-023-07819.

 Pranav Kumar, @2020, Biophysics and Molecular

Biology, Fundamentals and Techniques, 3rd Edition,
Pathfinder Publication, New Delhi, India.

THANK YOU….