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Table 3 PCR primers used for site-directed mutagenesis.

From: Cloning and analysis of a bifunctional methyltransferase/restriction endonuclease TspGWI, the prototype of a Thermus sp. enzyme family

Primer sequence

Selection

Mutant

5'-ACCGGCCTGGGCCGGCCCG(CA)G ATGGCGGTCTACCAC-3'

BstUI

(mutant D58A)

D 58 A

D 58 E

5'-GGTGGTAGACCGCCATC(TG)CG GGCCGGCCCAGGCCGG-3'

  

5'-TCCTGGTGGGCTTCGTG(ACGT)(ACGT)(ACGT) CTCAAGGCTCCGGGCAAG-3'

SacI

restriction site disappears

E 72 X

5'-CTTGCCCGGAGCCTTGAG(ACGT)(ACGT)(ACGT) CACGAAGCCCACCAGGA-3'

  

5'-CTGGTGGGCTTCGTGGAGCTC(GC)(GC) GGCCCCG GGCAGG-3'

SmaI/ApaI

(K74G, K74R)

K 74 G

K 74 A

K 74 R

K 74 P

5'-CTTGCCCGGGGCC(GC)(GC) GAGCTCCACGAAGCCCACAGG-3'

  

5'-GTTACCGTGATAGACCCGGCCA TGGGCACGGGGACCTT-3'

NcoI

(V356M)

V 356 M

5'-AAGGTCCCCGTGCCCAT GGCCGGGTCTATCACGGTAAC-3'

  

5'-TGGTGATCCTTGGCGCC CCCCCCTACGACCGGGTG-3'

NarI

(N473A)

N 473 A

5'-CACCCGGTCGTAGGGGGGGGC GCCAAGGATCACCA-3'

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