Production of a human single-chain variable fragment antibody against esophageal carcinoma

Table 1 Oligonucleotide primers used for PCR of human immunoglobulin genes

A. Primary PCRs
Human VH back primers (sense)
HuVHlaBACK	5’-CAGGTGCAGCTGGTGCAGTCTGG-3’
HuVH2aBACK	5’-CAGGTCAACTTAAGGGAGTCTGG-3’
HuVH3aBACK	5’-GAGGTGCAGCTGGTGGAGTCTGG-3’
HuVH4aBACk	5’-CAGGTGCAGCTGCAGGAGTCGGG-3’
HuVH5aBACK	5’-GAGGTGCAGCTGTTGCAGTCTGC-3’
HuVH6aBACK	5’-CAGGTACAGCTGCAGCAGTCAGG-3’
JH forward primers (anti-sense)
HuJHl-2FOR	5’-TGAGGAGACGGTGACCAGGGTGCC-3’
HuJH3FOR	5’-TGAAGAGACGGTGACCATTGTCCC-3’
HuJH4-5FOR	5’-TGAGGAGACGGTGACCAGGGTTCC-3’
HuJH6FOR	5’-TGAGGAGACGGTGACCGTGGTCCC-3’
Human V kappa back primers (sense)
HuV κ laBack	5’-GACATCCAGATGACCCAGTCTCC-3’
HuV κ 2aBack	5’-GATGTTGTGATGACTCAGTCTCC-3’
HuV κ 3aBack	5’-GAAATTGTGTTGACGCAGTCTCC-3’
HuV κ 4aBack	5’-GACATCGTGATGACCCAGTCTCC-3’
HuV κ 5aBack	5’-GAAACGACACTCACGCAGTCTCC-3’
HuV κ 6aBack	5’-GAAATTGTGCTGACTCAGTCTCC-3’
Human J kappa forward primer (anti-sense)
HuJ κ 1FOR	5’-ACGTTTGATTTCCACCTTGGTCCC-3’
HuJ κ 2FOR	5’-ACGTTTGATCTCCAGCTTGGTCCC-3’
HuJ κ 3FOR	5’-ACGTTTGATATCCACTTTGGTCCC-3’
HuJ κ 4FOR	5’-ACGTTTGATCTCCACCTTGGTCCC-3’
HuJ κ 5FOR	5’-ACGTTTAATCTCCAGTCGTGTCCC-3’
Human V lambda back primers (sense)
HuV λ 1BACK	5’-CAGTCTGTGTTGACGCAGCCGCC-3’
HuV λ 2BACK	5’-CAGTCTGCCCTGACTCAGCCTGC-3’
HuV λ 3aBACK	5’-TCCTATGTGCTGACTCAGCCACC-3’
HuV λ 3bBACK	5’-TCTTCTGAGCTGACTCAGGACCC-3’
HuV λ 4BACK	5’-CACGTTATACTGACTCAACCGCC-3’
HuV λ 5BACK	5’-CAGGCTGTGCTCACTCAGCCGTC-3’
HuV λ 6BACK	5’-AATTTTATGCTGACTCAGCCCCA-3’
Human J lambda forward primers (anti-sense)
HuJ λ 1FOR	5’-ACCTAGGACGGTGACCTTGGTCCC-3’
HuJ λ 2-3FOR	5’-ACCTAGGACGGTCAGCTTGGTCCC-3’
HuJ λ 4-5FOR	5’-ACCTAAAACGGTGAGCTGGGTCCC-3’
B. PCR assembly
Hu JH-Linker primers
HuJH1-2Linker	5’-AGAGCCACCTCCGCCTGAACCGCCTCCACCTGAGGAGACGGTGACCAGGGTGCC-3’
HuJH3Linker	5’-AGAGCCACCTCCGCCTGAACCGCCTCCACCTGAAGAGACGGTGACCATTGTCCC-3’
HuJH4-5Linker	5’-AGAGCCACCTCCGCCTGAACCGCCTCCACCTGAGGAGACGGTGACCAGGGTTCC-3’
HuJH6Linker	5’-AGAGCCACCTCCGCCTGAACCGCCTCCACCTGAGGAGACGGTGACCGTGGTCCC-3’
Linker-Hu V κ primers
Linker-HuV κ 2-3-6 BACK	5’-GTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGAWRTTGTGHTGACKCAGTCTCC-3’
Linker-HuV κ 1-4 BACK	5’-gTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGACATCSWGATGACCCAGTCTCC-3’
Linker-HuV κ 5 BACK	5’-GTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGAAACGACACTCACGCAGTCTCC-3’
Linker-Hu V λ primers
Linker-HuV λ 1-2BACK	5’-GTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGCAGTCTGYSYTGACKCAGCCKSC-3’
Linker-HuV λ 3BACK	5’-GTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGTCYTMTGWGCTGACTCAGSMMCC-3’
Linker-HuV λ 4-5BACK	5’-GTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGCASGYTRTRCTSACTCARCCGYC-3’
Linker-HuV λ 6BACK	5’-GTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGAATTTTATGCTGACTCAGCCCCA-3’
C. Reamplification with primers containing restriction sites
Human VH back (Sfi) primers (sense)
HuVH1aBACKSfi	5’-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGGTGCAGTCTGG-3’
HuVH2aBACKSfi	5’-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGGTCAACTTAAGGGAGTCTGG-3’
HuVH3aBACKSfi	5’-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCGAGGTGCAGCTGGTGGAGTCTGG-3’
HuVH4aBACKSfi	5’-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGCAGGAGTCGGG-3’
HuVH5aBACKSfi	5’-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGTTGCAGTCTGC-3’
HuVH6aBACKSfi	5’-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGGTACAGCTGCAGCAGTCAGG-3’
Human J kappa forward (Not) primers (anti-sense)
HuJ κ 1FORNot	5’-GAGTCATTCTCGACTTGCGGCCGCACGTTTGATTTCCACCTTGGTCCC-3’
HuJ κ 2FORNot	5’-GAGTCATTCTCGACTTGCGGCCGCACGTTTGATCTCCAGCTTGGTCCC-3’
HuJ κ 3FORNot	5’-GAGTCATTCTCGACTTGCGGCCGCACGTTTGATATCCACTTTGGTCCC-3’
HuJ κ 4FORNot	5’-GAGTCATTCTCGACTTGCGGCCGCACGTTTGATCTCCACCTTGGTCCC-3’
HuJ κ 5FORNot	5’-GAGTCATTCTCGACTTGCGGCCGCACGTTTAATCTCCAGTCGTGTCCC-3’
Human J lambda forward (Not) primers (anti-sense)
HuJ λ 1FORNot	5’-GAGTCATTCTCGACTTGCGGCCGCACCTAGGACGGTGACCTTGGTCCC-3’
HuJ λ 2-3FORNot	5’-GAGTCATTCTCGACTTGCGGCCGCACCTAGGACGGTCAGCTTGGTCCC-3’
HuJ λ 4-5FORNot	5’-GAGTCATTCTCGACTTGCGGCCGCACCTAAAACGGTGAGCTGGGTCCC-3’