In Class Activity
PIPE Mutagenesis Primer Design
1.
Choose the mutation you wish to introduce at the protein level (amino acid change)
2.
Isolate the site of mutation (the codon) and the codons for the twelve amino acids on each
side (total of 25 codons) (find the DNA sequence on the JCSG page and use ExPASy
translator to help; http://web.expasy.org/translate/). For DNA sequences, use Courier
New font so that all letters are equal width. This makes alignments much easier.
3.
Determine what the mutation will be at the DNA level. Consult a DNA codon table to
determine this (e.g., http://en.wikipedia.org/wiki/DNA_codon_table).
a. Choose your mutation with the goal of conserving as much of the wild type DNA
sequence as possible. Example: to change from Arg to Asp, choose to change from CGT
to GAT (T is conserved) not GAC (no conservation).
b. Make that change in your DNA sequence; be sure you label that as the mutant. You may
want to highlight the mutation to track it.
4.
Formulate your forward primer first. You will need a pair or primers for PIPE mutagenesis;
primers must fit these constraints:
a. Two primers (one forward, one reverse), each 40-45 nucleotides (nt) long
b. The two primers share an overlap of 15 base pairs (5 codons) with the middle codon
being the codon for mutation
c. Primers should start and end with G or C (most important at 3’ end)
d. Melting temperature (Tm) should be 70-75 °C
e. Primers should have GC content in the 50-60 percent range
f. Check the primers for GC content, Tm and secondary structure using an online oligo
analyzer (e.g., http://www.geneinfinity.org/sms/sms_primanalysis.html#)
5.
Generate the complementary strand of DNA using an online tool (e.g.,
http://arep.med.harvard.edu/labgc/adnan/projects/Utilities/revcomp.html)
6.
Follow step 4 again, this time to generate your reverse primer.
7.
Write primers in the 5’ → 3’ direction for ordering.
Primer configurations for
substitution mutations
using PIPE technology.
Example:
Wild type TM1131 DNA sequence (and amino acid translation):
gcc gcg gac aaa gac gcc att tct ttc gga ggg gga gta ccc gat cct gag act ttc ccg aga aaa gaa ctc gca
ala ala asp lys asp ala ile ser phe gly gly gly val pro asp pro glu thr phe pro arg lys glu leu ala
TM1131 DNA sequence (and amino acid translation) with mutation V39Q:
gcc gcg gac aaa gac gcc att tct ttc gga ggg gga caa ccc gat cct gag act ttc ccg aga aaa gaa ctc gca
ala ala asp lys asp ala ile ser phe gly gly gly gln pro asp pro glu thr phe pro arg lys glu leu ala
The forward primer* will be identical to the coding sequence and will anneal to the complementary
strand.
TM1131 V39Q Forward Primer:
5’ ggg gga caa ccc gat cct gag act ttc ccg aga aaa gaa ctc gc
3’
The reverse primer* will be identical to the complementary strand.
Generate the complementary strand:
cgg cgc ctg ttt ctg cgg taa aga aag cct ccc cct gtt ggg cta gga ctc tga aag ggc tct ttt ctt gag cgt
TM1131 V39Q Reverse Primer:
3’ gg cgc ctg ttt ctg cgg taa aga aag cct ccc cct gtt ggg cta 5’
Re-write the reverse primer in the 5’ → 3’ direction.
5’ atc ggg ttg tcc ccc tcc gaa aga aat ggc gtc ttt gtc cgc gg 3’
Finally….
1. Confirm that the primers anneal/make sense:
5’ ggg gga caa ccc gat cct gag act ttc ccg aga aaa gaa ctc gc 3’
3’ gg cgc ctg ttt ctg cgg taa aga aag cct ccc cct gtt ggg cta 5’
2. Run the primers you design through the oligo calculator to verify that they meet the criteria.
Make small changes as needed.
*Which primer is labeled forward and which is labeled reverse is arbitrary. They must be complementary and
antiparallel.