Primer design is important for PCR, cDNA synthesis, and DNA sequencing. There are general guidelines for primer design such as 18-30 base pairs in length, 40-60% GC content, melting temperature between 55-66°C. Primers should avoid secondary structures and mismatches at the 3' end. Common programs for primer design are Primer-BLAST and Primer3, which check specificity and secondary structures. Manual design is used for universal primers from multiple sequence alignments. The primers are then checked and optimized before being ordered and used in PCR experiments.

1. PCR primer design
Methee Sriprapun, PhD
(Principle and how to design primers)
Sriprapun.m@gmail.com

2. Objective of primer usage
•To generate cDNA for PCR assay
•To use as the origin of new DNA synthesis
•To use as the origin of DNA sequencing
http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html http://kem-en-tec-nordic.com/pcr-page/

3. Types of primer
•Oligo dT (16-20 T primer)  primes at the poly-A tail of mRNA
• Random hexamers: Total RNA template for cDNA
•Gene specific primer: usually uses reverse primer
http://www.thermoscientificbio.com/general-reagents-and-
accessories/primers-for-cdna-synthesis/
http://www.bio.davidson.edu/genomics/method/cDNAproduction.html

4. General guidelines for primer design
• Primer sizes: 18-30 bp
• 40-60% of GC contents in each primer
• Optimum temperature (Tm) of each primer should be 55-66 C and the
difference of forward and reverse primers should not more than 2 C
• 3’ end of each primer contains G or C (Last 5 bases: only 2G, 2C or GC)
• Avoid repetitive bases
• Avoid mismatch between primers and template at 3’end

5. General guidelines for primer
design (II)
•Avoid secondary structures:
- Hairpin
- Self-complementary
- Primer-dimer
3’ 5’
DNA template
5’ 3’ 3’ end of primer is important !

6. •Check primer specificity by blasting with database such as
GenBank
•Designed F primer: ready for use (primer synthesis)
•Designed R primer: reverse complement before Blast or
primer synthesis
•Primer-BLAST: not do reverse complement for R primer
•Design primers for qPCR: PCR product < 300 bp
General guidelines for primer
design (III)

7. Avoid secondary structure
•Hairpin loop
•Self-complementary
•Primer-dimer
https://bioweb.uwlax.edu/GenWeb/Molecular/seq_anal/primer_design/primer_design.htm
http://www.nature.com/nprot/journal
/v1/n3/fig_tab/nprot.2006.247_F2.html

8. Calculation of Tm and annealing
temperature
•Tm: temperature that makes dsDNA  ssDNA 50%
Tm= 2 x (no. of [A+T]) + 4 x (no. of [G+C])
Annealing temp. = Tm of primer - 5
http://www.gravitywaves.com/chemistry/CHEMXL153/NucleotidesCompandStruc.htm

9. Tm calculation quiz
Tm= 2 x (no. of [A+T]) + 4 x (no. of [G+C])
Annealing temp. = Tm of primer - 5
Sequence Tm
(C)
Annealing
Temp. (C)
5’ GAT TAC TTG GGC AAG GCC GA 3’ 62 57
5’ ATG GGC AAT AAT TTG GGA 3’ 50 45
5’ ATT GGC AAG TTG AAG GCG GGG 3’ 64 59
5’ TTG TTG AAG AGC CCC GGA C 3’ 60 55

10. Reverse complement
• Reverse: change nt. sequence from (5’3’) to (3’5’)
• Complement with the reversed sequence
5’CTCCAAGCTCCAAGCTCCAG 3’
Reverse: 5’GACCTCGAACCTCGAACCTC 3’
Complement: 5’CTGGAGCTTGGAGCTTGGAG 3’
Courier New: suitable font for
typing DNA sequence

11. How to design primers
1. Manual design
- Download all interested sequences from GenBank and manually
select the position for primer design
- Multiple sequence alignment of all interested sequences from
and find the conserve position to generate “universal primers”
- Primer checking: length , %GC, Tm, specificity to DNA target,
etc.
- Advantage in primer design for multiplex PCR or probe design
in some specific work such as in situ hybridization

12. Multiple sequence
alignment for
primers and probe
design

13. Universal primers for PCR assay

14. How to design primers (II)
1. Program design
- Using programs for primer design
- Fast, convenience, easy to adjust some parameters
- Not suitable for primer design in multiplex PCR
- Probe design for qPCR
- Limitation in modified primer design at 5’ end

15. Program for primer design
(Abd-Elsalam KA, Afr J Biotechnol 2003)

16. Primer design with Primer-BLAST
http://www.ncbi.nlm.nih.gov/tools/primer-blast/
• Developed by NCBI
• Primer3 to design PCR primers + BLAST and
global alignment algorithm to screen primers

17. Primer-BLAST
• Primer design & BLAST primers with sequences in
database
• Can predict PCR product size from your own designed
primers
• Check primer specificity of your own designed primers
• Cons: Take time due to blast algorithm in primer design
program

19. How to use primer-BLAST
• Choose gene of interest and avoid selecting gene location in intron
• Take gene sequence in the form of FASTA format, only sequence or
Accession No. in program
• Parameter setting and Click at “Get primers”
• Consider possibly designed primer by checking Tm, self-
complementary, GC content, etc.
• Ready to use designed primers ( no reverse complement of R primer)

20. Example: Design primer for human -actin detection (Tm = 60C
PCR product 100-300 bp)
Choose -actin (human) from NCBI
 Take sequence or Accession No. into PCR template as mentioned in red arrow
You can either fill in or not

21.  Set parameters: product size and Tm of primers
Change to “nr”
Fill in or not
 Press “Get Primers”

22. Result
You obtained 5 pairs of primer from this program.
What can we retrieve from the program?
1. Primer characteristics and data
2. Size of PCR product
3. Primer specificity using BLAST algorithm

23. How to check secondary structure
OligoCalc
http://www.basic.northwestern.edu/biotools/oligocalc.html

24.  Place each primer sequence here
Choose “ssDNA”
Click “calculate”

25. ExampleofOligoCalcProgram
Result
reverse complement sequence
You can get
some
parameters
From this
program

26. Click to check
secondary structure
Result

27. Primer design with Primer3
• Online program: “Primer3” and “Primer3Plus”
• Primer3Plus is improved in program efficiency but the
used parameters in Primer3 = Primer3Plus

28. http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/
Place sequence of interest here
Click to generate
primers
Click to set parameters

29. Choose -actin (human) sequence from NCBI
 Place sequence or upload FASTA file of interested sequence
Example: Design primer for human -actin detection (Tm = 60C
PCR product 100-300 bp)

30.  Set parameter: Size of PCR product and Tm of primers
 Click “Pick Primers”

31. Details of designed primers: Tm, %GC, secondary structure, product size

32. You can find position of each
primer on your sequence of interest
Other details of designed primers

33. You can “CLICK” to order primer synhesis
You can blast each
primer with GenBank

34. You can use “primer-Blast” to check your designed primers from
Primer3 or Primer3Plus
Put each primer
here
Change to “nr”
Can fill it or Ignore
Click “Get Primers”

35.  If you want to check secondary structure  Program OligoCalc
 No reverse complement in R primer
 Pros: Fast and do not have a lot of time to run the program
 Cons: Cannot blast both primers simultaneously and may require
other program to check self-complementary or secondary structure

36. Tips for consideration
•Program for primer design  tool for prediction of
primers
•Designed primers may not 100% efficiency in the real situation
•The best designed Primers: Usually the first designed
sequences by each primer design program
•Program for primer design: Compass and tool for guiding your
work about how to design and optimize primers in PCR
•PRIMER Optimization in PCR should be
considered in the real situation !!!!!!

37. Primer design at a glance
Choose organisms or gene of interest from database such as
GenBank, EMBL, DDBJ, etc.
Download sequence in FASTA format or using Accession No. of interested gene
Primer design with appropriate program such as primer-BLAST, Primer3, Primer3Plus, etc.
Or manual design (universal primer  multiple sequence alignment Choose conserve sequence)
Select suitable designedprimers from primer design program
Consider primer characterization such aslength, Tm, %GC, secondarystructure of primers, etc.
Final checkingof primer sequencesand order primer synthesis
Use the primers in PCR or RT-PCR and optimizationwith reagentsand PCR machine
as well as genome template