What is a primer? A primer is a short synthetic oligonucleotide which is used in many molecular techniques from PCR to DNA sequencing.
Important considerations on primer design 1. Primers should be 17-30 bases in length 2. Base composition should be 40 40-60 60 % (G (G+C) C) 3. Primers should end (3') in a G or C, or CG or GC: this increases efficiency of priming 1
4 Tm between 55-72C are preferred 4. 5. Tm difference between forward and reverse primers i should h ld be b 5C 6. 3'-ends of primers should not be complementary (ie. base pair), as otherwise primer dimers will be synthesized preferentially to any other product 7. Primer self self-complementarity complementarity (ability to form 2o structures such as hairpins) should be avoided
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Practical consideration for primer pair design
1. Amplicon Length: The amplicon length is dictated by the experimental goals. If you know the positions of each primer with respect to the template, the product is calculated as: Product length = (Position of antisense primer - Position of sense primer) + 1. 2. Product Position: Primer can be located near the 5' end, the 3' end or any where within specified length. G Generally, ll the th sequence close l t the to th 3' end d is i known k with greater confidence and hence preferred most frequently. frequently
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3. Tm of Product: Melting Temperature (Tm) is the temperature at which one half of the DNA duplex will dissociate and become single stranded. The stability of the primer-template DNA duplex can be measured by the melting temperature (Tm). 4. Tm Calculation: Tm = 4(G+C) + 2(A+T) C Where, G+C is the total GC content in the primer, A+T is the total AT content in the primer
Note: 1. The actual ( (Tm) is influenced by y the concentration of Mg2+, K+, and co-solvents 2. The formula given above for (Tm) is simplistic; there are many primer design programs which use more complex nearestneighbor thermodynamics values
5. Annealing temperature: The annealing temperature (T ) chosen (Ta) h f for a PCR depends d d directly di tl on length l th and d composition of the primer(s). Generally, an annealing temperature about 5 5C C below the lowest Tm of the pair of primers is used 6. Optimal annealing temperature calculation: The optimal annealing temperature for any given primer pair on a p p particular target g can be calculated as follows: Ta Opt = 0.3 x(Tm of primer) + 0.7 x(Tm of product) 25 Where, Tm of primer is the melting temperature of the less stable primer-template pair and Tm of product is the melting temperature of the PCR product.
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General considerations:
Primer designing for cloning purpose
1. All commonly-used cloning/expression vectors contain a multiple cloning site (MCS) with different restriction sites 2. When designing your primers, you should add one of th these restriction t i ti sites it t the to th end d of f your primer i i such in h as was as to preserve the reading frame of the target gene/PCR product at both the 5 and the 3 end. end 3. Choose a restriction site that is not present in the gene you are cloning 4. Add 3-4 extra G, C or GCs at the end of the restriction , as it is necessary y( (depending p g the restriction sites) ) sites, for restriction enzyme to cleave the restriction site.
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Example Primer design for the PCR amplification of the cut1 gene The cut1 gene from Thermobifida fusca encodes for cutinase, an enzyme involved in the hydrolysis of plant cutin. The cut1 gene: >cut1 ATGCAAAAACGGGCGATTTATCCGGGTACTTTCGATCCCATTACCAAT GGTCATATCGATATCGTGACGCGCGCCACGCAGATGTTCGATCACGTT ATTCTGGCGATTGCCGCCCCGCAGCGACGATTGCAGTCACAGCAGGC AACCGCGCATCTGGGGAACGTGGAAGTGGTCGGGTTTAGTGATTTAA TGGCGAACTTCGCCCGTAATCAACACGCTACGGTGCTGATTCGTGGC CTGCGTGCGGTGGCAGATTTTGAATATGAAATGCAGCTGGCGCATATG AATCGCCACTTAATGCCGGAACTGGAAAGTGTGTTTCTGATGCCGTCG AAAGAGTGGTCGTTTATCTCTTCATCGTTGGTGAAAGAGGTGGCGCG CCATCAGGGCGATGTCACCCATTTCCTGCCGGAGAATGTCCATCAGGC GCTGATGGCGAAGTTAGCG 8 Cont
Suppose, we decided to clone the gene into an expression vector using the restriction enzymes EcoRI (5'-end) and BamHI (3'-end). Here we show the design of both primers: 5' 5'-end d primer i The EcoRI site in the vector is in frame with the ATG in the gene to create the N-terminal methionine residue of CUT1. 5-CATGGGATCCATGCAAAAACGGGCGATTTATCC-3 5' extension (CATG)
EcoRI restriction site (GGATCC)
Start codon (ATG)
ATGCAAAAACGGGCGATTTATCCGGGTACTTTCGATCCCATTACCAA TGGTCATATCGATATCGTGACGCGCGCCACGCAGATGTTCGATCACG TTATTCTGGCGATTGCCGCCCCGCAGCGACGATTGCAGTCACAGCAG GCAACCGCGCATCTGGGGAACGTGGAAGTGGTCGGGTTTAGTGATT TAATGGCGAACTTCGCCCGTA
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3'-end primer C-terminal His tag is being used, so we have to delete stop codon (TAG, the natural stop codon for CUT1 to get a fusion of cutinase 1 protein with C-terminal His tag). p g) 5-CGGGATCCCGCTAACTTCGCCATCAGC-3 5' extension (CG) ( )
BamH I restriction site (GGATCC)
ATGCAAAAACGGGCGATTTATCCGGGTACTTTCGATCCCATTACCA ATGGTCAT ATGGTCAT .GCCATCAGGGCGATGTCACCCATT TCCTGCCGGAGAATGTCCATCAGGCGCTGATGGCGAAGTTAGCG 5' GCTGATGGCGAAGTTAGCG 3' 3' CGACTACCGCTTCAATCGC 5 5' CGCTTAACTTCGCCATGAGC 3 5 3' 10 Cont
Primer binding pattern with gene >cut1 5-CATGGGATCCATGCAAAAACGGGCGATTTATCC-3 ATGCAAAAACGGGCGATTTATCCGGGTACTTTCGATCCCATTACC AATGGTCATATCGATATCGTGACGCGCGCCACGCAGATGTTCGATCACGTTATTCTGGCGAT TGCCGCCCCGCAGCGACGATTGCAGTCACAGCAGGCAACCGCGCATCTGGGGAACGTGG AAGTGGTCGGGTTTAGTGATTTAATGGCGAACTTCGCCCGTAATCAACACGCTACGGTGCT GATTCGTGGCCTGCGTGCGGTGGCAGATTTTGAATATGAAATGCAGCTGGCGCATATGAAT CGCCACTTAATGCCGGAACTGGAAAGTGTGTTTCTGATGCCGTCGAAAGAGTGGTCGTTTA TCTCTTCATCGTTGGTGAAAGAGGTGGCGCGCCATCAGGGCGATGTCACCCATTTCCTGCC GGAGAATGTCCATCAGGCGCTGATGGCGAAGTTAGCG 3-CGACTACCGCTTCAATCGCCCTAGGGC-5
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Some useful primer designing softwares Primer Premier Primer3 P i Primer-BLAST BLAST Fast PCR
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