PCR Primer Designing

1. Primer Designing
Krishnendu Sinha
Assistant Professor in Zoology
Jhargram Raj College

2. PCR
Primers
Polymerase chain reaction (PCR) is a method, invented in 1983 by the American biochemist Kary Mullis, used widely
in molecular biology to make millions to billions of copies of a specific DNA sample rapidly, allowing scientists to take
a very small sample of DNA and amplify it to a large enough amount to study in detail (Wikipedia).
A primer is a short single-stranded nucleic acid utilized by all living organisms in the initiation of DNA synthesis as
the enzymes responsible for DNA replication, DNA polymerases, are only capable of adding nucleotides to the 3’-
end of an existing nucleic acid, requiring a primer be bound to the template before DNA polymerase can begin a
complementary strand. Taq polymerase use in PCR also have similar requirement. For effective amplification, a pair
of forward and reverse primer is required in PCR. Good primer design is one of the most important parameters in
PCR.

3. PCR
Primers
Polymerase chain reaction (PCR) is a method, invented in 1983 by the American biochemist Kary Mullis, used widely
in molecular biology to make millions to billions of copies of a specific DNA sample rapidly, allowing scientists to take
a very small sample of DNA and amplify it to a large enough amount to study in detail (Wikipedia).
A primer is a short single-stranded nucleic acid utilized by all living organisms in the initiation of DNA synthesis as
the enzymes responsible for DNA replication, DNA polymerases, are only capable of adding nucleotides to the 3’-
end of an existing nucleic acid, requiring a primer be bound to the template before DNA polymerase can begin a
complementary strand. Taq polymerase use in PCR also have similar requirement. For effective amplification, a pair
of forward and reverse primer is required in PCR. Good primer design is one of the most important parameters in
PCR.

4. Here are the keys to specific amplification with high yield
1. Primer Length: 18-25 nt and pairs should not differ by >3bp (This provides for practical annealing temperatures)
2. Melting Temperature: 52-580C (for a member pair difference should be <=50C and <= 100C from amplified product)
3. GC Content: 40-60% with even distribution of bases across the primer (Primers with high GC content can form stable imperfect hybrids)
4. GC Clamp: 3’ base should have a G or C (GC rich to enhance annealing of the end that will be extended) but not more than 3nt and never
in clump (as these can hybridize inappropriately)
5. Repeated and Self-Complimentary Sequence: not allowed over 3bp
6. Amplicon Length: should be approximately 50–150 bp (since longer products do not amplify as efficiently)
*Analyze primer pair sequences to avoid complementarity and hybridization between primers (primer-dimers)

5. Method
1. Finding cDNA sequence:
a) Go to NCBI website by clicking https://www.ncbi.nlm.nih.gov/

6. Method
1. Finding cDNA sequence:
a) Go to NCBI website by clicking https://www.ncbi.nlm.nih.gov/

7. Method
1. Finding cDNA sequence:
a) Go to NCBI website by clicking https://www.ncbi.nlm.nih.gov/
From dropdown menu
select Nucleotide instead
of all database

8. Method
1. Finding cDNA sequence:
a) Go to NCBI website by clicking https://www.ncbi.nlm.nih.gov/

9. Method
1. Finding cDNA sequence:
a) Go to NCBI website by clicking https://www.ncbi.nlm.nih.gov/
In the search box type the
name of the gene and its
species you want to copy by
PCR (e.g. FSH gene of mu i.e.
scientific species name of
mice)…

10. Method
1. Finding cDNA sequence:
a) Go to NCBI website by clicking https://www.ncbi.nlm.nih.gov/
…and type complete
CDS (i.e. complete
coding sequence of the
gene which is actually a
cDNA sequence of the
gene with start and
stop codon)…press
search

11. Method
1. Finding cDNA sequence:
a) Go to NCBI website by clicking https://www.ncbi.nlm.nih.gov/
Click on the
desirable
search result

12. Method
1. Finding cDNA sequence:
a) Go to NCBI website by clicking https://www.ncbi.nlm.nih.gov/
Will take to the
gene GenBank
database of the
gene…now click
on FASTA seq

13. Method
1. Finding cDNA sequence:
a) Go to NCBI website by clicking https://www.ncbi.nlm.nih.gov/
This is the FASTA
seq of the cDNA

14. Method
1. Finding cDNA sequence:
a) Go to NCBI website by clicking https://www.ncbi.nlm.nih.gov/
Selects a particular region
of you choice (blue block
here) and copy it
(remember to leave first
and last 100-200 nt as at
the time of mRNA
isolation both the end of
the RNA got damaged
and hence the cDNA it
will give have the chance
of not having this initial
and terminal sequences)

15. Method
2. Designing the Primer:
a) Go to NCBI website by clicking https://www.ncbi.nlm.nih.gov/
Now click on https://www.idtdna.com/site/account/login?returnurl=%2FPrimerquest%2FHome%2FIndex and
login with the user name and password provided in the comment

16. 2. Designing the Primer:
a) Go to NCBI website by clicking https://www.ncbi.nlm.nih.gov/
Now click on https://www.idtdna.com/site/account/login?returnurl=%2FPrimerquest%2FHome%2FIndex and
login with the user name and password provided in the comment
Method
Here paste the copied FASTA seq as shown here

17. 2. Designing the Primer:
a) Go to NCBI website by clicking https://www.ncbi.nlm.nih.gov/
Now click on https://www.idtdna.com/site/account/login?returnurl=%2FPrimerquest%2FHome%2FIndex and
login with the user name and password provided in the comment
Method

18. 2. Designing the Primer:
a) Go to NCBI website by clicking https://www.ncbi.nlm.nih.gov/
Now click on https://www.idtdna.com/site/account/login?returnurl=%2FPrimerquest%2FHome%2FIndex and
login with the user name and password provided in the comment
Method
>
Is the FASTA format and you have to put it

19. 2. Designing the Primer:
a) Go to NCBI website by clicking https://www.ncbi.nlm.nih.gov/
Now click on https://www.idtdna.com/site/account/login?returnurl=%2FPrimerquest%2FHome%2FIndex and
login with the user name and password provided in the comment
Method

20. 2. Designing the Primer:
a) Go to NCBI website by clicking https://www.ncbi.nlm.nih.gov/
Now click on https://www.idtdna.com/site/account/login?returnurl=%2FPrimerquest%2FHome%2FIndex and
login with the user name and password provided in the comment
Method
Click here to get primer
pair on predefined
parameters
Or get primer pair on
you customized
parameters

21. 2. Designing the Primer:
a) Go to NCBI website by clicking https://www.ncbi.nlm.nih.gov/
Now click on https://www.idtdna.com/site/account/login?returnurl=%2FPrimerquest%2FHome%2FIndex and
login with the user name and password provided in the comment
Method
By default this
programme
will give five
primer set
(green lines
showing the
length of the
replicon
based on the
primer pair)

22. 2. Designing the Primer:
a) Go to NCBI website by clicking https://www.ncbi.nlm.nih.gov/
Now click on https://www.idtdna.com/site/account/login?returnurl=%2FPrimerquest%2FHome%2FIndex and
login with the user name and password provided in the comment
Method

23. 2. Designing the Primer:
a) Go to NCBI website by clicking https://www.ncbi.nlm.nih.gov/
Now click on https://www.idtdna.com/site/account/login?returnurl=%2FPrimerquest%2FHome%2FIndex and
login with the user name and password provided in the comment
Method
Select one as
per your need
or given
instruction by
the teacher
and click on
assay details

24. 2. Designing the Primer:
a) Go to NCBI website by clicking https://www.ncbi.nlm.nih.gov/
Now click on https://www.idtdna.com/site/account/login?returnurl=%2FPrimerquest%2FHome%2FIndex and
login with the user name and password provided in the comment
Method
Select one as
per your need
or given
instruction by
the teacher
and click on
assay details

25. 2. Designing the Primer:
a) Go to NCBI website by clicking https://www.ncbi.nlm.nih.gov/
Now click on https://www.idtdna.com/site/account/login?returnurl=%2FPrimerquest%2FHome%2FIndex and
login with the user name and password provided in the comment
Method
Here is the forward primer and reverse primer !!!
Copy in a text file for future reference

26. References
1. https://fire.biol.wwu.edu/cmoyer/zztemp_fire/biol324_W08/PCR_lect/GoodPCRdesign.pdf
2. Wikipedia
3. https://www.ncbi.nlm.nih.gov/
4. https://www.idtdna.com/pages
5. PrimerQuest Tool (IDT)