Table 2 Primers for site directed mutagenesis.

From: Structural and functional role of disulphide bonds and substrate binding residues of the human beta-galactoside alpha-2,3-sialyltransferase 1 (hST3Gal1)

Position in pST3Gal1

Mutation in hST3Gal1a

Primers sequence (5′to 3′)

N150

N147S

Forward: GCTGTAGTAGGCTCTAGCGGGAATCTG

Reverse: CAGATTCCCGCTAGAGCCTACTACAGC

S151

S148A

Forward: GTAGTAGGCAATGCAGGGAATCTGCGTG

Reverse: CACGCAGATTCCCTGCATTGCCTACTAC

N173

N170A

Forward: GTCCTGCGTATGGCAAAAGCCCCGACTG

Reverse: CAGTCGGGGCTTTTGCCATACGCAGGAC

Y194

Y191A

Forward: CCATCATCTGGTTGCGCCTGAGAGCTTTC

Reverse: GAAAGCTCTCAGGCGCAACCAGATGATGG

Y233

Y230A

Forward: CATCTCACACACTGCGATCCCTGTGCCGG

Reverse: CCGGCACAGGGATCGCAGTGTGTGAGATG

Y230F

Forward: CATCTCACACACTTTTATCCCTGTGCCGG

Reverse: CCGGCACAGGGATAAAAGTGTGTGAGATG

V318

V315A

Forward: CGTAAAACCGGCGCGCACGATGCTG

Reverse: CAGCATCGTGCGCGCCGGTTTTACG

V315Y

Forward: CTTTTCGTAAAACCGGCTATCACGATGCTGATTTC

Reverse: GAAATCAGCATCGTGATAGCCGGTTTTACGAAAAG

C62-C67

C59S-C64Sb

Forward: CCTTCTACGTGTACTCACTCTATTGGTCAGCGTAAACTG

Reverse: ACGGTGTTTAATCAGACGTTTCAGGTTCTCGCTCAG

C64-C142

C61S-C139S

Forward C61S: CCGTCCTTGTACGTCTACTCACTGTATTGGTC

Forward C139S: CGTTCTGTGGGTTCCCGCCGTTGTGC

C145-C284

C142S-C281S

Forward C142S: GGTTGCCGCCGTTCTGCTGTAGTAGGC

Forward C281S: CTCCATGCATGTCTCTGACGAGGTGGATCTG

  1. aNumbers in model are based on the full-length sequence. bWhole plasmid amplification was performed by inverse PCR with phosphorylated primers; the forward primer contains the double mutation.
Back to article page