A Quick Guide for Primer Design

A quick & basic guide for primer design for amplification of Genomic DNA:

Good primer length ~24 bp.

Annealing temperature ~600C.

GC content usually less than 50%.

A good internet site for designing primers:

http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi

For primers to be a good primer pair, they need to have similar qualities (Tm, GC etc)

Increased length increases their specificity.

They should not anneal to each other and should have minimal secondary structure. These things can be checked manually or by feeding the primers sequence into a program such as 'GeneRunner' – can be downloaded from http://www.generunner.com/ or by letting a program like primer3 design your primers for you.

Primer3

This is the program's main window

Step1 – paste the sequence you want to amplify in PCR into the window.

Step2 – add '<' and '>' and the beginning and end of the region you want to be amplified (sequence that should later be sequenced or just for limiting the sequence for a minimal length of an amplified fragment).

For example:

If the sequence is

TGGTAGAGACATGCCCCTACCGCCCTGATTGGCTGAAGCTGCGTGCCTGGTGAGGTGACGTGGCTTGCTGTGAGTGGTTGGGGGCTGAGAGTATATAAGAGTGAGAGATCCGGTGATCGAGGGAGAAAAAGATGAAGCGAGAGAGATGAAGATTGAAGCTTGCTGACTAAACTGCTGTTAGAACTGGTGGTCGTGTCGTTCTTGCTGGTTGAGAGCAGACACGACAGTTGCCTACGAGAATGCGATAGTAAGACTGCATTGCTGAAGACAGCACACATCTTGGTCACAGAATATAAAGAAATAAAACTGAAACTGGCCTGGGAGCGTCCTCCCCATTAGCTAGCCATCATGACGCATCAATACAACTGACTACTCAGGCATAATATAGTGCAAGAGTGGCCAAGTTTGCCGGGCGTGGTGGCACACGCCTTTAATCCGAGCACTCGGGAGGCAGAGGAAGGCGAATTTCTGAGTTCAAGGCCAGCCTGGTCTACAAAGTGAGTTCCAGGACAGCCAGGGCTACACAGAGAAACCCTGTCTCCAAAAACCAAAAAAAAAAAAAAAAATAGTGGCCAAGTTTGTTCCAGAGGTAAGCAACCACTTTCTGATTGGATTTGAAGCCAATCCTATGAAGGGATTCCTCTCTGATACTGTAAATCTGGTCAAATCCCATGGTTGGAAAGGCTATAGGCCCTAGTTGGGAAGCTACGCTGTTCTTTTGGTAAATAGACATCATGTACCCATCAAATTGAAATTAATTAAGTGGGGCTTTTATAGGCCTATGTCTCTCCTTGCTGCTGGATTAATGCTCCTAAGATAACAGGGGGGAAAAAAACTCTCTGCAGGATTGTTGAACTTCGGAACCTCCCTGTCCCAGACAGATTCCTCCTTGGAAGTGGATCTTTTGCTGCAACTCTGTGTCTGATCGATTCCCTTTTAAGGGATAAAAGGACTTGGAATCCCTCTAGGGGGAATGCTCTGAACTGCAGTATCAACTGATAACAA

And I'm interested in the sequence in Red. Then '<' and '>' should be added before and after this sequence – for example –

Now this sequence can be pasted to the programs window. It's enough to paste it with 2-4 lines of sequence before and after the sequence of interest.

Step 3 – setting parameters

The program has default parameters that are better to change. This is the parameter window:

Product size – choose the size you need. If the sequence you have entered between the brackets is 300 bp in length, you set product size to minimum 320, maximum 450.

Primer size - Min 23

Opt 24

Max 26

Primer Tm - Min 58

Opt 60

Max 60

Primer GC - Min 20

Max 50

Step 4 –

Picking primers – click on the 'pick primers' button.

The following output will show

the first primer pair is usually the best one.

The program calculates also the product size.

By scrolling down additional primer pairs will show.

· If no primer pairs show, try less stringent conditions in the setting box.

· If the sequence amplified doesn't include your sequence of interest – limit the extra sequence before and after the amplified fragment.

PCR program:

A basic PCR program that works very well (on which many modifications can be made) is the following one –

PCR mix –

X1 (quantities in microliters) / X____
DNA – 50ng final / 2 ul
PCR buffer 10X no Mg++ / 2.5 ul
Mg++ 25mM (to a final concentration of 2mM)[1] / 2 ul (1.5-1.75)
dNTPs 10X solution[2] / 2.5 ul
Primers[3] / 0.5F + 0.5R
Enzyme
ddH2O
Total / 25ul

PCR program –

I 950c 3min

II 940c 45sec

III 600c 45sec

IV 720c 45sec

V goto II X5

VI 940c 45sec

VII 550c 45sec

VIII 720c 45sec[4]

IX goto VI X 34

X 720c 10min

XI END

Calculating Tm for the annealing Temperature –

Calculate 20C for every A or T and 40C for every G or C.

Choose use the annealing temperature of the lower one of the primers as the annealing temperature for step III in the PCR reaction and that temperature –(4-5)0C for the annealing temperature in step VII of the PCR reaction.

If there is no product the temps at III and VII can be lowered in a couple of Degs.

[1] Can use 2mM concentration of Mg++ for less stringent conditions or 1.5 or 1 for higher stringency = reduce background.

[2] Each dNTP is at 2mM concentration in the 10X solution and in the PCR the final concentration should be 200micromolar each.

[3] In the PCR reaction should be in 0.2µM each, I use a 10µM stock solution.

[4] This step can be longer – the rule of thumb is 1 min per 1000 bp.