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Table 2 Oligonucleotide primer sequences

From: Expression of urease by Haemophilus influenzaeduring human respiratory tract infection and role in survival in an acid environment

Primer Gene Direction Sequence1
Construction of ureCmutant
539 frag1 5' ureC upstream Forward 5'-GACCTTTACCCACAGCTAAT-3'
539 frag1 3' ureC upstream Reverse 5'-TAGTTAGTCACTTGAAATTGTTA
ATGCCAT-3'
539 frag2 5' Kanamycin cassette Forward 5'-CAATTTCAAGTGACTAACTAGGA
GGAATAA-3'
539frag2 3' Kanamycin cassette Reverse 5'-TGACCCAATGCATTATTCCCTCC
AGGTACT-3'
539 frag3 5' ureC downstream Forward 5'-GGGAATAATGCATTGGGTCAGC
GATA-3'
539 frag3 3' ureC downstream Reverse 5'-ATCGCACACCGAGTTTG-3'
Cloning of ureC into pSPEC1 to complement ureC mutation
539promoter
F1
ureA upstream Forward 5'-GAGAGGATCCGTAAAATTCGCTG
ACTTTCG-3'
539C R1 ureC downstream Reverse 5'-ATATGAATTCGCTACTTCACGCC
CCGTA-3'
Construction of urease operon mutant
539 Op frag1 F1 ureA upstream Forward 5'-TACACCTTCCTTGCCCAC-3'
539 Op frag1 R1 ureA upstream Reverse 5'-TAGTTAGTCAATTTTCATTCCTTAAT-3'
539 Op frag2 F1 Kanamycin cassette Forward 5'-GAATGAAAATTGACTAACTAG
GAGGAATAA-3'
539 Op frag2 R1 Kanamycin cassette Reverse 5'-ACCAATTTTCCATTATTCCCTCCA
GGTACT-3'
539Op frag3 F1 ureA downstream Forward 5'-GGGAATAATGGAAAATTGGTAG
GCTAT-3'
539Op frag3 R1 ureA downstream Reverse 5'-CAGATGTTGCTTCAATTAAG-3'
Subjecting multiple strains to PCR to assess presence of urease operon
UreaseA F1 ureA Forward 5'-ATGCACTTAACTTCCAGAG-3'
UreaseA R1 ureA Reverse 5'-TTATCTGATTGGATTATGC-3'
539 F1 ureC Forward 5'-CAACATGGCATTAACAATTTCAAG-3'
539 R1 ureC Reverse 5'-TTAGAATAGGAAATATCGCTG-3'
UreaseH F1 ureH Forward 5'-ATGAACAGTAAATTATCC-3'
UreaseHR1 ureH Reverse 5'-GAATTTGCTCTGCACGACA-3'
Cloning of ureC gene to express recombinant protein
539 F1 ureC Forward 5'-CAACATGGCATTAACAATTTCAAG-3'
539 R1 ureC Reverse 5'-TTAGAATAGGAAATATCGCTG-3'
Performing RT PCR
UreA-UreB F1 ureA, ureB Forward 5'-ATGAGTGTAGCGGAAGTG-3'
UreA-UreB R1 ureA, ureB Reverse 5'-ACGTTTAAACGCATTCCGC-3'
UreB-UreC F1 ureB, ureC Forward 5'-TGAAACCAATAATGCCCT-3'
UreB-UreC R1 ureB, ureC Reverse 5'-TTGCGTGCGTACCATCAGC-3'
UreC-UreE F1 ureC, ureE Forward 5'-GGTATTGCGGAGCATATTGG-3'
UreC-UreE R1 ureC, ureE Reverse 5'-GAATGACTGTGAGAATGCC-3'
UreE-UreF F1 ureE, ureF Forward 5'-GCTTGAAACACGATGATG-3'
UreE-UreF R1 ureE, ureF Reverse 5'-TGATTGCACCGACTGCTG-3'
UreF-UreG F1 ureF, ureG Forward 5'-TGCCTTCGATGCAATGAAC-3'
UreF-UreG R1 ureF, ureG Reverse 5'-AGCACCAACGAATGGAGC-3'
UreG-UreH F1 ureG, ureH Forward 5'-AGTTTGCTTCCACCAGAGC-3'
UreG-UreH R1 ureG, ureH Reverse 5'-GTTCGTCATTCAACACCC-3'
  1. 1underlined sequence denotes restriction enzyme site or cloning site