Table I.
Primer sequences used in the study.
| Primer name | Sequence 5′-3′ | Product length |
|---|---|---|
| I. PCR amplification for genotyping p.Arg8Trp, p.Val56Leu and p.Pro73Arg mutations by RFLP | ||
| Amplification of CGB5 gene | ||
| CGB5_F | CAGGAAAGCCTCAAGTAGAGGAG | 1757 bp |
| CGB5_R | CGCTCGACGATGTTTTCTATTTT | |
| Amplification of CGB8 gene | ||
| CGB8_F | CACGCCTGTAATTGTCGGAGGCTGT | 8384 bp |
| CGB8_R | GAAAAGAGAGTGAAGATGGGGGACGAC | |
| CGB8nested_F | CCCGGATAACTTTTCGTATTTTTA | 2544 bp |
| CGB8nested_R | TCCTCAGATCAACTCTCATGGAT | |
| II. PCR amplification and site-directed mutagenesis for hCGβ plasmid construction | ||
| Amplification of hCGβ coding region | ||
| CGB_coding_F | CACCAAGGATGGAGATGTTCC | 523 bp |
| CGB_coding_R | TGCGGATTGAGAAGCCTTTA | |
| Mutagenesisa | ||
| Mut_CGB5_V56L_F | GCCCTGCCTCAGGTGCTGTGCAACTACCGCG | |
| Mut_CGB5_V56L_R | CGCGGTAGTTGCACAGCACCTGAGGCAGGGC | |
| Mut_CGB8_R8W_F | GCCGCTTCGGCCATGGTGCCGCCCCATC | |
| Mut_CGB8_R8W_R | GATGGGGCGGCACCATGGCCGAAGCGGC | |
| Mut_CGB8_P73R_F | CTCCCTGGCTGCCGGCGCGGCGTGAAC | |
| Mut_CGB8_P73R_R | GTTCACGCCGCGCCGGCAGCCAGGGAG | |
| III. PCR amplification for the hCGα + β joint plasmid construction and production of ‘high yield’ hCGb | ||
| Amplification of hCGα coding region | ||
| CGA_coding_F | CGTACGAGCGCCATGGATTA (Pfl23II) | 369 bp |
| CGA_coding_R | ACCGGTTTAAGATTTGTGATAATA (BshTI) | |
| Amplification of FLAG-tagged hCGβ coding region | ||
| CGB_plasmid_F | TTCGAACACCAAGGATGGA (Bsp119I) | 551 bp |
| CGB_plasmid_R | ATTAATTTACTTATCATCATCATCT (VspI) | |
aMutagenesis site is underlined.
bRestriction sites are indicated in italics and respective restriction enzymes given in brackets.