MOLECULAR BIOLOGY AND DIAGNOSTICS LABORATORY PRIMER DEVELOPMENT OUTLINE • Primer Development (Canvas) o Introduction o Principle o Materials o Procedure: Primer Design Tips • Primer Development o Important Design Considerations o Parameters for Primer Pair Design o Primer Design Using Software o Designing Primer Software o Designing Primer Using NCBI PRIMER DEVELOPMENT (CANVAS) INTRODUCTION • DNA replication, can only initiate the replication process by adding nucleotides to primers. In order to produce the desired DNA sequence, you must start with the right primer. Thus, proper primer design is necessary for successful DNA amplification. PRINCIPLE • Designing oligonucleotides and making sure that you have the right parameters for your oligo is an important step in securing results, especially in PCR Primer Design. In order to achieve successful DNA amplification, it’s important to start off with the right primer. MATERIALS • Commercially available (Genome compiler) • Requires a software PROCEDURE: PRIMER DESIGN TIPS 1. Aim for the GC content to be between 40 and 60% with the 3’ of a primer ending in G or C to promote binding. This is known as a GC Clamp. The G and C bases have stronger hydrogen bonding and help with the stability of the primer. Be mindful not to have too many repeating G or C bases, as this can cause primer-dimer formation. 2. A good length for PCR primers is generally around 18-30 bases. Specificity usually is dependent on length and annealing temperature. The shorter the primers are, the more efficiently they will bind or anneal to the target. 3. Try to make the melting temperature (Tm) of the primers between 65°C and 75°C, andwithin 5°C of each other. Because the Tm is dependent on the length, it’s important to keep primers on the shorter end. The bases also impact the Tm, G and C result in higher melting temperatures than A and T. If the Tm of your primer is very low, try to find a sequence with more GC content, or extend the length of the primer a little. 4. Typically, 3 to 4 nucleotides are added 5 ’of the restriction enzyme site in the primer to allow for efficient cutting. 5. Try to avoid regions of secondary structure and have a balanced distribution of GC-rich and AT- rich domains. 6. Try to avoid runs of 4 or more of one base, or dinucleotide repeats (for example, ACCCC or ATATATAT). 7. Avoid intra-primer homology (more than 3 bases that complement within the primer) or interprimer homology (forward and reverse primers having complementary sequences). These circumstances can lead to self-dimers or primer-dimers instead of annealing to the desired DNA sequences. 8. If you are using the primers for cloning, we recommend cartridge purification as a minimum level of purification. 9. If you are using the primers for mutagenesis, try to have the mismatched bases towards the middle of the primer. 10. If you are using the primers for a PCR reaction to be used in Invitrogen TOPO cloning, the primers should not have a phosphate modification. PRIMER DEVELOPMENT (PPT) IMPORTANT DESIGN CONSIDERATIONS • Primer Length o It is generally accepted that the optimal length of PCR primers is 18-22 bp. o his length is long enough for adequate specificity and short enough for primers to bind easily to the template at the annealing temperature. • Primer Melting Temperature o This is the temperature at which 50% of the primer and its complement are hybridized. o Primers with T m in the range of 52-58 oC generally produce the best results. o Primers with melting temperatures above 65oC have a tendency for secondary annealing. o The GC content of the sequence gives a fair indication of the primer T m . o “Itakura's empirical rule” makes a quick and dirty estimate of the Tm of an oligonucleotide: ▪ Tm = 2 (A+T) + 4 (G+C) • Primer Annealing Temperature o The primer melting temperature is the estimate of the DNA-DNA hybrid stability and critical in determining the annealing temperature. o Too high T a will produce insufficient primer-template hybridization resulting in low PCR product yield. o Too low T a may possibly lead to non-specific products caused by a high number of base pair mismatches. • GC Content o The GC content (the number of G's and C's in the primer as a percentage of the total bases) of primer should be 40-60%. • GC Clamp o The presence of G or C bases within the last five bases from the 3' end of primers (GC clamp) helps promote specific binding at the 3' end due to the stronger bonding of G and C bases. o More than 3 G's or C's should be avoided in the last 5 bases at the 3' end of the primer. • Primer Secondary Structures o Hairpins : It is formed by intramolecular interaction within the primer.
o Self Dimer ▪ A primer self-dimer is formed by intermolecular interactions between the two (same sense) primers. ▪ Optimally a 3' end self dimer with a ΔG of -5 kcal/mol and an internal self dimer with a ΔG of -6 kcal/mol is tolerated generally. o Cross Dimer ▪ Primer cross dimers are formed by intermolecular interaction between sense and antisense primers. ▪ Optimally a 3' end cross dimer with a ΔG of -5 kcal/mol and an internal cross dimer with a ΔG of -6 kcal/mol is tolerated generally. • Repeats o A repeat is a di-nucleotide occurring many times consecutively. For example: ATATATAT. o maximum acceptable number of di-nucleotide repeats is 4. • Runs o Primers with long runs of a single base should generally be avoided. For example, AGCGGGGGATGGGG has runs of base 'G' of value 5 and 4. o maximum number of runs accepted is 4 bp. • 3' End Stability o It is the maximum ΔG value of the five bases from the 3' end. o An unstable 3' end (less negative ΔG) will result in less false priming. • Avoid Template Secondary Structure o The stability of the template secondary structures depends largely on their free energy and melting temperatures(T m ). o Consideration of template secondary structures is important in designing primers, especially in qPCR. o If primers are designed on a secondary structures which is stable even above the annealing temperatures, the primers are unable to bind to the template. o Hence, it is important to design primers in the regions of the templates that do not form stable secondary structures. • Avoid Cross Homology o Primers designed for a sequence must not amplify other genes in the mixture. o Commonly, primers are designed and then BLASTed to test the specificity. PARAMETERS FOR PRIMER PAIR DESIGN • Amplicon Length o The amplicon length is dictated by the experimental goals. For qPCR, the target length is closer to 100 bp and for standard PCR, it is near 500 bp. o Product length = (Position of antisense primer-Position of sense primer) + 1. • Product Position o Primer can be located near the 5' end, the 3' end or any where within specified length. Generally, the sequence close to the 3' end is known with greater confidence and hence preferred most frequently. • T m of Product o Melting Temperature (Tm) is the temperature at which one half of the DNA duplex will dissociate and become single stranded • Optimum Annealing Temperature (T a Opt) o The formula of Rychlik is most respected. o T a Opt = 0.3 x(T m of primer) + 0.7 x(T m of product) - 14.9 o Where: o T m of primer: the melting temperature of primer- template pair o Tm of product: the melting temperature of the PCR product. • Primer Pair T m Mismatch Calculation o The two primers of a primer pair should have closely matched melting temperatures for maximizing PCR product yield. o The difference of 5oC or more can lead no amplification. PRIMER DESIGN USING SOFTWARE • A number of primer design tools are available that can assist in PCR primer design for new and experienced users alike. • These tools may reduce the cost and time involved in experimentation by lowering the chances of failed experimentation. DESIGNING PRIMER SOFTWARES
DESIGNING PRIMER USING NCBI • Sequence Retrieval • Input Data