Table 2.
Primer Design and DHPLC Conditions
| Primers |
DHPLC |
||||
|---|---|---|---|---|---|
| Forward | Reverse | bp | %A | Melt(°C) | |
| Pm | 5′-GGTAGACAGTGGATACATAACAAATGCATG-3′ | 5′-TTCTCCGAAGGTAATTGCCTCCCAGATCTGAGTCC-3′ | 531 | 49% | 58.8 |
| 2 | 5′-TTTAATTTGGATGCCCCAAACCAG-3′ | 5′-AATGACACTATGAGAGAAATAAAACGG-3′ | 347 | 45% | 53 |
| 3 | 5′-GATAATCGTGAAAATGTATCATTGGA-3′ | 5′-CAGTTTCTGGTCTGAAATTCTACTAAGTTT-3′ | 222 | 53% | 57.6 |
| 4 | 5′-TTGTCGGTCTCCTGCTGGTCAGTG-3′ | 5′-CAAAGCCCTCACTCAAACATGAAGC-3′ | 194 | 50% | 59.9 |
| 5 | 5′-TTGCAACTAGGCATTTGGTCTCTTACC-3′ | 5′-AGATTAATGTTACCCAAAAGGAAACC-3′ | 193 | 50% | 55.3 |
| 6 | 5′-TCTATTTATCACTGAAGATCAAGGAC-3′ | 5′-TGGGGAAAAATATGTCATCAGAGTC-3′ | 344 | 50% | 57.9 |
| 7 | 5′-ACACTCAAGACTTAAGGACTATGGGC-3′ | 5′-TACCATACTAAAAGCAGTGGTAGTCCAG-3′ | 287 | 53% | 59.5 |
| 8 | 5′-ATATAGAAACCAAAAATTGATGTGTAG-3′ | 5′-ATGCATATAAAACAGAAAACATCTTG-3′ | 280 | 49% | 57.3 |
| 9 | 5′-TTCTACCATGTTGGAAAGTAGTCCT-3′ | 5′-AAGCAGTGTTAGATTATCTTGGAAGC-3′ | 283 | 50% | 59.9 |
| 10 | 5′-TTGTGCAGCATTGGAAGCTCCTGA-3′ | 5′-TAGTTTACCTCATGAGTATGAAACTGGTC-3′ | 205 | 52% | 58.8 |
| 11 | 5′-ACCACACCGATTTACCTAGAG-3′ | 5′-CACAAGCTTCCAAAACTTGTT-3′ | 292 | 49% | 57.4 |
| 12 | 5′-GATAGTGGGCTTTACTTACATCCTTC-3′ | 5′-GAAAGCACGCAACATAAGATACACCT-3′ | 332 | 48% | 56.9 |
| 13 | 5′-GCAGAAATAAATTTCACCATTTGAGAGC-3′ | 5′-ACTTCAGCTGATTATGAGTGTGTG-3′ | 362 | 48% | 54.2 |
| 14 | 5′-GATACTTTGGCAAATTATTCATGCC-3′ | 5′-CGTGTCTTTTACAGCTAGTTTCTCAC-3′ | 227 | 51% | 58.4 |
| 15 | 5′-GTGAGAAACTAGCTGTAAAAGACACG-3′ | 5′-TGGGTTTTTATAAGACCATTGAAAGC-3′ | 244 | 50% | 56.1 |
| 16 | 5′-CTATAGTGGTGTATGGAATGCAACC-3′ | 5′-TGAGATAGTCTGTAGCATGATAATTGG-3′ | 276 | 48% | 57.3 |
| 17 | 5′-GTCTGACCTCTGTTTCAATACTTCTCAC-3′ | 5′-AAGCTTGAGATGCTCTCACCTTTTCC-3′ | 225 | 50% | 58.3 |
| 18 | 5′-GTGTCAGGCAGGAGTCTCAGATTGAGA-3′ | 5′-GCACGGAGTTTACAAGCAGCACAAAATGAG-3′ | 301 | 50% | 56.4 |
| 19 | 5′-TGAATTACTCATCTTTGCTCTCATGCTG-3′ | 5′-CCCTAAGAAGATTATCTAAATCAACTCGTG-3′ | 156 | 56% | 60.1 |
| 20 | 5′-GCTTTCAGATCATTTCTTTCAGTCTG-3′ | 5′-CCAAGAAATACCTATTGATTATGCTC-3′ | 360 | 50% | 59.5 |
| 21 | 5′-CTTGCCTTACTGCTTTTTAATACCTTC-3′ | 5′-TTATTGTTTCATGTTAGTACCTTCTGG-3′ | 360 | 47% | 57.3 |
| 22 | 5′-GAGTTTGCTGACAATTTAGGAAAACATGGC-3′ | 5′-GATAAGCGTGCTTTATTGTTTTGAC-3′ | 270 | 52% | 60.1 |
| 23 | 5′-GTTTGAATCATATAGATTTCAAGTACAG-3′ | 5′-AACAAGTAAATAAAAATGAGGGTAG-3′ | 357 | 50% | 57.6 |
| 24 | 5′-ACCAGTAATGCCTTATAACGGGTCTCG-3′ | 5′-ATCCACCCCAGCTGTAAAACACTGATC-3′ | 233 | 52% | 57.4 |
| 25 | 5′-ATCCAATATGCAATGCCATCAGTTCCC-3′ | 5′-CTTAGTTAAGTACGTTGAGGCAAGC-3′ | 315 | 50% | 58.1 |
| 26a | 5′-GTCTATGCCAGAAAGGAGGCCTTGA-3′ | 5′-ACCAGGAAAGAGCAGACTGTATACGAC-3′ | 274 | 51% | 56.2 |
| 26b | 5′-TCTAAGCTTTCTGTTATTTACATACTGATG-3′ | 5′-TTCAACTGCTTTCTGTAATTCATCTGGAG-3′ | 258 | 51% | 56.4 |
| 27 | 5′-CTCATTCTAACTGGATGTTGTGAGAAAG-3′ | 5′-CACTATGCCTCACATATGACCATG-3′ | 355 | 50% | 58.6 |
| 28 | 5′-CTGTCTGCTGCATTTTGAATTACCTGC-3′ | 5′-TTCTATTTGGTACTTGACCTCTTTTA-3′ | 356 | 50% | 55.8 |
| 29 | 5′-TCAGAAGATACTGAGCATTTGCTGATAATCC-3′ | 5′-CTGAGAGCTGTATCTGCTATACATTAATGC-3′ | 300 | 51% | 58.4 |
| 30 | 5′-CAGGATTACAGAAAAGCTATCAAGAGT-3′ | 5′-AAGAATGGAAGCTGATTCCCAGATGTAC-3′ | 259 | 51% | 59.6 |
| 31 | 5′-GTTGTTCTTTGTAGAGCATGCTGACT-3′ | 5′-TGCCCAACGAAAACACGTTCCTTAG-3′ | 203 | 50% | 56.3 |
| 32 | 5′-GACCAGTTATTGTTTGAAAGGCAAA-3′ | 5′-GTACCTGCGTATTTGCCACCAGAAAT-3′ | 265 | 49% | 58.2 |
| 33 | 5′-CAAACATGGAATAGCAATTAAGGGGATCTC-3′ | 5′-GAAGTGTTTGTGGTCTCAGCATGC-3′ | 293 | 50% | 57 |
| 34 | 5′-ACAGAAATATAAAAGTTCCAAATAAGT-3′ | 5′-ACGTATGTTCAAAATAACCTTCAGTG-3′ | 299 | 49% | 55 |
| 35 | 5′-ACAAGACATTACTTGAAGGTCAATGC-3′ | 5′-AAGCTTCTAGCCTTTTCTCTTACC-3′ | 243 | 50% | 58.3 |
| 36 | 5′-CCAATAATGCCATGGTATGTCTCTG-3′ | 5′-GGACAAAGATGATTGAAGTAACTGGTG-3′ | 229 | 52% | 57.7 |
| 37 | 5′-CTTCAAGTCCTATCTCTTGCTCATGG-3′ | 5′-CACAAGTTTCCACCTTGGAGTAGATC-3′ | 237 | 52% | 60.6 |
| 38 | 5′-GCATGTGATTAGTTTAGCAACAGGAGG-3′ | 5′-CAGTTGGAGACTTATCTAAGTTCTTTCC-3′ | 311 | 50% | 55.7 |
| 39 | 5′-TGAAGACTGTACTTGTTGTTTTTGATCAG-3′ | 5′-GTTTCTGATGACTAAGAGTCTGAAGCAG-3′ | 276 | 51% | 56.3 |
| 40 | 5′-ATAACTGCAGCCAGAAGTGCACTATAC-3′ | 5′-GTATAATAAAATCTGGTATTGACATTC-3′ | 261 | 50% | 56.2 |
| 41 | 5′-ATGTGGTTAGCTAACTGCCCTGGGC-3′ | 5′-CATACGTGGGTTTGCCAGTAACAACTC-3′ | 260 | 54% | 63.2 |
| 42 | 5′-GGAGGAGGTTTCACTGTTAGGAAGC-3′ | 5′-ATGATCACCTTGTAAAATACGAATG-3′ | 297 | 47% | 56.4 |
| 43 | 5′-GCAACACCATTTGCTACCTTTGGGA-3′ | 5′-CCTGAAAACAAATCATTTCTGCAAG-3′ | 331 | 48% | 54.8 |
| 44 | 5′-CTTGATCCATATGCTTTTACCTGCA-3′ | 5′-TCCATCACCCTTCAGAACCTGATCT-3′ | 268 | 48% | 56 |
| 45 | 5′-AGTACAACTGCATGTGGTAGCACACTG-3′ | 5′-CATTCCTATTAGATCTGTCGCCCTAC-3′ | 296 | 48% | 58.2 |
| 46 | 5′-ATTGCCATGTTTGTGTCCCAGTTTGC-3′ | 5′-TAACCTAATGGGCAGAAAACCAATG-3′ | 336 | 47% | 55 |
| 47 | 5′-AAAGACAAGGTAGTTGGAATTGTGCTG-3′ | 5′-TTAACACATGTGACGGAAGAGATGG-3′ | 252 | 49% | 57.9 |
| 48 | 5′-GCTTATGCCTTGAGAATTATTTACCT-3′ | 5′-TCCTGAATAAAGTCTTCCTTACCACACT-3′ | 372 | 48% | 55.6 |
| 49 | 5′-TTGCTAACTGTGAAGTTAATCTGCAC-3′ | 5′-TGATTATAAATAGTCCACGTCAATGG-3′ | 243 | 49% | 57.4 |
| 50 | 5′-CACCAAATGGATTAAGATGTTCATGAAT-3′ | 5′-TCTCTCTCACCCAGTCATCACTTCATAG-3′ | 271 | 51% | 59.3 |
| 51 | 5′-GAAATTGGCTCTTTAGCTTGTGTTTC-3′ | 5′-GGAGAGTAAAGTGATTGGTGGAAAATC-3′ | 388 | 49% | 58.8 |
| 52 | 5′-GTAAAAGGAATACACAACGCTGAAG-3′ | 5′-AAATGTGAGGGGGATATATGAACTTAAG-3′ | 265 | 50% | 58.3 |
| 53 | 5′-TTTAAAATGTCTCCTCCAGACTAGC-3′ | 5′-GTCTACTGTTCATTTCAGCTTTAACGTG-3′ | 410 | 47% | 54.3 |
| 54 | 5′-GACCTGAGGATTCAGAAGCTGTTTACGA-3′ | 5′-CACCACCCCATTATTACAGCCAACAG-3′ | 312 | 49% | 57.2 |
| 55 | 5′-TGAGTTCACTAGGTGCACCATTCTGA-3′ | 5′-CACAAGAGTGCTAAAGCGGAAATGCC-3′ | 288 | 48% | 59.3 |
| 56 | 5′-GCACATATTCTTCTTCCTGCTGTCCTG-3′ | 5′-GTGGCCTTTTTGCTCCACATCTTTTCC-3′ | 233 | 49% | 58.2 |
| 57 | 5′-ACTTCTAGATATTCTGACATGGATCGC-3′ | 5′-TGTGCTTAACATGTGCAAGGCACGAG-3′ | 243 | 49% | 60 |
| 58 | 5′-GAATGCCACAAGCCTTTCTTAGCACTTC-3′ | 5′-TGCTCCGTCACCACTGATCCTTCTATC-3′ | 225 | 50% | 57.4 |
| 59 | 5′-ATGTGGCCTAAAACCTTGTCATATTGCC-3′ | 5′-TTGTGGGAAGATAACACTGCACTCAAG-3′ | 392 | 47% | 60.9 |
| 60 | 5′-CCTAAAGAGAATAAGCCCAGGTATC-3′ | 5′-TCCTATCCTCACAAATATTACCATGA-3′ | 353 | 49% | 57.4 |
| 61 | 5′-GAGAACATAATTTCTCTCCTTTTCCTCCC-3′ | 5′-CAAGATGCAATAAAGTTAAGTGATAAAAGC-3′ | 154 | 55% | 58 |
| 62 | 5′-TGGAGATTAATGTTGTCTTTCCTGTTTGCGA-3′ | 5′-TACTCACTTGTGAATATACAGGTTAGTCAC-3′ | 207 | 52% | 57.1 |
| 63 | 5′-TCCTGTTTTCTTGACTACTCATGGTAAATGC-3′ | 5′-TAACTTGGAGGAAACATGGCCATGTCC-3′ | 154 | 53% | 56.6 |
| 64 | 5′-TATTTCTGATGGAATAACAAATGCTC-3′ | 5′-TAGTATCAAGATCTTCAAATACTGGCCAATAC-3′ | 157 | 53% | 56.9 |
| 65 | 5′-GAGTCCTAGCTAGGATTCTCAGAGG-3′ | 5′-CTAAGCCTCCTGTGACAGAGCCC-3′ | 341 | 49% | 59.7 |
| 66 | 5′-AGAAGTGTTTACCCTCTAGGAAAGGGTC-3′ | 5′-TCCCATCTAGAACTAGGGTAATTAGCCAAC-3′ | 216 | 51% | 56.4 |
| 67 | 5′-CCACTACTGTGGAAATACTGGCTACTC-3′ | 5′-CCTACTGCCTACTGAAGAGCTAATATGAG-3′ | 391 | 48% | 59.6 |
| 68 | 5′-GATATACACCTCCTTTGCCATCTTGCC-3′ | 5′-AACTAACAGCAACTGGCACAGGAGA-3′ | 342 | 53% | 62.5 |
| 69 | 5′-TGGTAGAAGGTTTATTAAAGAGTGTTCTTTGGG-3′ | 5′-TGAACTAACTCTCACGTCAGGCTGGCGTC-3′ | 230 | 51% | 58 |
| 70 | 5′-CATCCTGTCCTAAATCTGATCTCACC-3′ | 5′-TGGGAGTGAAAGGAGGGTGTTCAGCT-3′ | 262 | 50% | 59.8 |
| 71 | 5′-TGCGTGTGTCTCCTTCACCACCTCA-3′ | 5′-GCGAGCGAATGTGTTGGTGGTAGCAGCACCC-3′ | 131 | 54% | 58.2 |
| 72 | 5′-CATAACTGTGTGGTGGGTTTTTTCTCCA-3′ | 5′-TATTTGCCTGGCATACAACTAGCCTCA-3′ | 168 | 54% | 59.6 |
| 73 | 5′-TTTCAGGAATGTTCGATTAGGTCTTGAA-3′ | 5′-TCCTGTGCTATCCTACCTCTAAATCCCTC-3′ | 226 | 50% | 56.4 |
| 74 | 5′-CTGAGTCCCTAACCCCCAAAGCA-3′ | 5′-GTGCAAGTGTATGCACTCTGCATACC-3′ | 280 | 50% | 59 |
| 75 | 5′-CCATGGTATATAAAATTTGGTGATGA-3′ | 5′-GCACCTATAAAAAGTGCTCTCTGAGG-3′ | 429 | 50% | 60.8 |
| 76 | 5′-TAATTCTGTTTTCTTTTGGATGACTTAGCC-3′ | 5′-GGCCAAATATTCATGTCCCTGTAATACG-3′ | 230 | 54% | 61.9 |
| 77 | 5′-GCTTGAGGGTTTTCTTTGTTATTTATGAGCAAG-3′ | 5′-TGATCCCAGCAAATCTGAGTCCCAC-3′ | 269 | 50% | 55 |
| 78 | 5′-TCCCTTTCTGATATCTCTGCCTCTTCC-3′ | 5′-AGCAGGATGAGACAGACAGAAGCCAT-3′ | 127 | 55% | 56.5 |
| 79 | 5′-AACAGAGTGATGCTATCTATCTGCACC-3′ | 5′-TCTGCTCCTTCTTCATCTGTCATGACTG-3′ | 159 | 54% | 58 |
%A indicates the starting concentration of buffer A (without acetonitrile) used to load samples. Melt(°C) indicates the preferred temperature of analysis.