PCR primer design

Methee Sriprapun, PhD

(Principle and how to design primers)

Sriprapun.m@gmail.com

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/

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

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

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 !

• 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)

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

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 - 5http://www.gravitywaves.com/chemistry/CHEMXL153/NucleotidesCompandStruc.htm

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)

AnnealingTemp. (C)

5’ GAT TAC TTG GGC AAG GCC GA 3’ 62 575’ ATG GGC AAT AAT TTG GGA 3’ 50 45

5’ ATT GGC AAG TTG AAG GCG GGG 3’ 64 595’ TTG TTG AAG AGC CCC GGA C 3’ 60 55

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

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

Multiple sequence alignment forprimers and probe design

Universal primers for PCR assay

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

Program for primer design

(Abd-Elsalam KA, Afr J Biotechnol 2003)

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

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

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)

Example: Design primer for human -actin detection (Tm = 60C 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

Set parameters: product size and Tm of primers

Change to “nr”

Fill in or not

Press “Get Primers”

Result

You obtained 5 pairs of primer from this program.

What can we retrieve from the program?1. Primer characteristics and data2. Size of PCR product 3. Primer specificity using BLAST algorithm

How to check secondary structure

OligoCalc

http://www.basic.northwestern.edu/biotools/oligocalc.html

Place each primer sequence here

Choose “ssDNA”

Click “calculate”

Example of Oligo Calc Program

Result

reverse complement sequenceYou can getsome parametersFrom this program

Click to checksecondary structure

Result

Primer design with Primer3

• Online program: “Primer3” and “Primer3Plus”• Primer3Plus is improved in program efficiency but the

used parameters in Primer3 = Primer3Plus

http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/

Place sequence of interest here

Click to generateprimers

Click to set parameters

Choose -actin (human) sequence from NCBI

Place sequence or upload FASTA file of interested sequence

Example: Design primer for human -actin detection (Tm = 60C PCR product 100-300 bp)

Set parameter: Size of PCR product and Tm of primers

Click “Pick Primers”

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

You can find position of eachprimer on your sequence of interest

Other details of designed primers

You can “CLICK” to order primer synhesis

You can blast eachprimer with GenBank

You can use “primer-Blast” to check your designed primers from Primer3 or Primer3Plus

Put each primerhere

Change to “nr”

Can fill it or Ignore

Click “Get Primers”

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

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 !!!!!!

Primer design at a glance

Choose organisms or gene of interest from database such asGenBank, 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 designed primers from primer design program

Consider primer characterization such as length, Tm, %GC, secondary structure of primers, etc.

Final checking of primer sequences and order primer synthesis

Use the primers in PCR or RT-PCR and optimization with reagents and PCR machineas well as genome template