Primer design for the PCR amplification of the coad gene

Primer design for the PCR amplification of the coaD gene
Back to Primer Design page
The coaD gene from E. coli encodes for phosphopantetheine adenylyltransferase (PPAT), an enzyme involved in the
biosynthesis of coenzyme A.
Reference: Geerlof, A., Lewendon, A. & Shaw, W.V. (1999) J. Biol. Chem. 274, 27105-27111.
The coaD gene:
ATGCAAAAACGGGCGATTTATCCGGGTACTTTCGATCCCATTACCAATGGTC
ATAT
CGATATCGTGACGCGCGCCACGCAGATGTTCGATCACGTTATTCTGGCGATT
GCCG
CCAGCCCCAGTAAAAAACCGATGTTTACCCTGGAAGAGCGTGTGGCACTGG
CACAG
CAGGCAACCGCGCATCTGGGGAACGTGGAAGTGGTCGGGTTTAGTGATTTA
ATGGC
GAACTTCGCCCGTAATCAACACGCTACGGTGCTGATTCGTGGCCTGCGTGCG
GTGG
CAGATTTTGAATATGAAATGCAGCTGGCGCATATGAATCGCCACTTAATGCC
GGAA
CTGGAAAGTGTGTTTCTGATGCCGTCGAAAGAGTGGTCGTTTATCTCTTCAT
CGTT
GGTGAAAGAGGTGGCGCGCCATCAGGGCGATGTCACCCATTTCCTGCCGGA
GAATG TCCATCAGGCGCTGATGGCGAAGTTAGCG
We decided to clone the gene into an expression vector using the restriction enzymes Nco I (5'-end) andBamH I (3'-
end).
Here we show the design of both primers:
5'-end primer
The Nco I site in the vector is in frame with the N-terminal His
6
tag and can be used directly providing the ATG in the
site is used to create the N-terminal methionine residue of PPAT.
CATG CCATGGAAAAACGGGCGATTTATCC

1. 5' extension(CATG)
2. Nco I restriction site (CCATGG)
3. Start codon (ATG)
4. Overlap with the 5'-end of the coaD gene starting with the ATGstart codon
Estimated T
m
is 62°C [13 * (A+T) + 9 * (C+G)]
GC content is 48%
However, PCR using this 5'-end primer will not result in the amplification of the original coaD gene. The use of the
Nco I restriction site dictates what the first nucleotide of the next triplet codon must be (G). In the coaD gene,
however, the second triplet begins with a C and PCR amplification will change this codon from CAA to GAA (and the
second residue in the recombinant protein will be glutamate instead of glutamine). An alternative strategy which will
result in the amplification of the original gene is described in Utilisation of the Nco I site.
3'-end primer
No C-terminal tag is being used, so we have to add a stop codon (TAG is the natural stop codon for PPAT).
CG GGATCCCTA CGCTAACTTCGCCATCAGC
1. 5' extension (CG)
2. BamH I restriction site (GGATCC)
3. Complement of stop codon (CTA)
4. Overlap with the strand complement to the 3'-end of the coaD gene
5. Estimated T
m
of 60°C [8 * (A+T) + 11 * (C+G)]
GC content is 60%.