Primer design for the PCR amplification of the coad gene

Primer design for the PCR amplification of the coaD geneBack to Primer Design page The coaD gene from E.. The coaD gene: ATGCAAAAACGGGCGATTTATCCGGGTACTTTCGATCCCATTACCAATGGTC ATAT CGATATC

Primer design for the PCR amplification of the coaD gene

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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 His6 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 Tm 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 Tm of 60°C [8 * (A+T) + 11 * (C+G)]

GC content is 60%.