3.4: APPENDIX C1- Primer design for site-directed mutagenesis.
- Page ID
- 26472
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Primers may also be designed using a primer design program (http://www.stratagene.com/qcprimerdesign), or checked by that method following design. The Tm must be calculated using the formula below.
C1: Example of step-by-step design of primers for the Abl H396P mutation 53
Design of the forward (5’ to 3’) primer:
- Determine the DNA code that corresponds to His396. To do this, multiply the residue number by 3, and then subtract 2 to get the first bp in the 3 bp codon. Add 3 bases to this number (see Appendix B2 for an explanation of why) to convert to the numbering to correspond to Genbank entry NM_005157. (396*3) – 2 + 3 = 1189 His396 is encoded by base pairs 1189-1191, which are CAT.
- Check that the codon corresponds to the correct amino acid using a DNA to protein translatation tool (http://www.expasy.ch/tools/dna.html). If provided in Appendix B3, confirm that the numbering of the nucleotide substitution falls within the codon determined above (here 1189-1191), and use a translator tool to ensure that the bp change results in the expected amino acid substitution. If the DNA change is not listed, determine which nucleotide substitution gives the desired amino acid change. For H396P, the A1190C mutation results in a CCT (Pro) codon, as expected.
- Write out the desired mutation (shown in bold italics below) with 12 flanking bases on each side.
CC TAC ACA GCC CCT GCT GGA GCC AA - The first and last residues of your primer should be a G or a C. Add bases as needed to each end of your primer so that each end terminates with a G or C.
CC TAC ACA GCC CCT GCT GGA GCC AAG TTC - Check that your primer has at least 40% GC content. If needed, added more bases to one or both ends to achieve a higher GC%. % GC content = ((# of G/C bases)/(total # of bases)) * 100% = (18/29) X 100 = 62 % GC
- Calculate the % mismatch of your primer. % mismatch = 1/29 * 100% = 3 %
- Calculate the melting temperature (Tm) of your primer. In the equation below, N is the primer length in bases, and %GC and % mismatch should be written in whole numbers.
Tm = 81.5 + 0.41(%GC) – 675/N - % mismatch = 81.5 – (0.41)(62) – 675/29 -3 = 80 .6 ºC - The Tm of your primer should be greater than or equal to 78 ºC. If the Tm is less than 78 ºC, increase the length of your primer or increase the % G/C content to increase the Tm. However, make sure your primer does not exceed 45 bases.
Forward primer: 5’ CC TAC ACA GCC CCT GCT GGA GCC AAG TTC 3’
Design of the antisense primer, which is the reverse complement of the forward primer:
- Write the complement of the forward primer. Forward: 5’ CC TAC ACA GCC CCT GCT GGA GCC AAG TTC 3’ Complement: 3’ GG ATG TGT CGG GGA CGA CCT CGG TTC AAG 5’
- Rewrite the complement primer from 5’ to 3’. 5’ GAA CTT GGC TCC AGC AGG GGC TGT GTA GG 3’
- Double check everything. Then triple check everything.
- Reverse primer: 5’ GAA CTT GGC TCC AGC AGG GGC TGT GTA GG 3’