TRFLP SITES ON CLONE F AND G SEQUENCES

THE CLONES IN THE TABLE ARE THOSE THAT CONTAINED THE REVERSE PRIMER SEQUENCES. THE FIRST NUMBER OF EACH SERIES INDICATES DISTANCE FROM LABELED BASE.

STANDARD ENZYMES FOR 16 S ANALYSIS ARE:  HaeIII, BstUI, HhaI, MspI (Hpa II), and RsaI. We used Hae III, Msp I (Hpa II), Sau 3A1. Grey columns are cut sites for the enzymes we did not use.  Judging by the restriction site distances there is more variation in samples than would show up in TRFLP. Alu I AND Rsa I give more fragment size variation than the others. Does the cut site completely rule it out? The others would indicate there are only  2 -3 different species among these samples. Hmm.

Red numbers indicate Alu 1 cut sites in primer sequences.

Rsa I isoschizomer = Afa I and neoschizomer = Csp 61

SAMPLE BP Alu 1 ag/ct BstU1 cg/cg Hae III gg/cc Hha I gcg/c Hpa II c/cgg Rsa I gt/ac Sau 3a1 /gatc 
F-31 478 366 408 128 253 238 323 NO CUTS 40 150   264 448
F-32 493 4 365 407  75 127 252 437 480 237 388 483 77 437 39 134 149 318 336   263 448
F-35 485 365 408 75 127 252 480 237 322 388 424 483 77 39 134 149   263 370 449
F-36 482 365 407 75 127 252 435 477 237 480 77 435 39 134 149 318 336   176 263 446
F-37  495 4 366 409  75 127 252 439 481 237 389 484  77 439 39 134 149 318 336   263 450
F-41 497 144 221 365 411 127 252 484 237 427 487 75 276  39 134 149 168 263 346 370 453
F-42 333 145 76 128 254 239 78 39 150 321   265
G-31 409 199 366 127 252 237 322 348 377 39 75 379 39 134 149 392 368 396 263
G-32 461 144 199 364 406 75 127 252 435  237 440 77 435 134 149 318 336    263 337 446
G-39 301 265 73 80 132 257 242 82 71 130 154 77 268
G-40** 352 342 REDONE 75 127 253 326 342 342  350 77 342 39 150 319 324 343  322 177 265
G-41 442 4 366 406 431 75 127 252 433 237 388 77 379 433 39 134 149 318 337   263

 

REVERSE PRIMER SEQUENCES ARE IN COLOR

>F_31_sp6
CTAGCCTTGTAGTTTCAAACGCAATTCCCAGGTTCGAGCCCGGGGATTTCACATCCGACTTACAAAACCGTCTACGCACG
CTTTACGCCCAGTAATTCCGATTAACGCTCGCACCCTCTGTATTACCGCGGNTGcTGGCACAGAGTTAGCCGgTGCTTTT
TCTTTGGGTTAGGTCAAGACCCAAGGTTATTAGCCCTGGGCTTTTCATCCCCACTAAAAGTGCTTTACAACCCGCAGGCC
TTCTTCACACACGCGGCGTCGCTGGATCAGGGTTGCCCCCATTGTCCAATATTCCCCGCTGCTGCCTCCCGTAGGAGTCT
GGGCCGTGTCTCAGTCCCAGTGTGGCTGGTCGTCCTCTCAGACCAGCTACGGATTGTCGCCTTGGTGAGCCGTTACCTCA
CCAACTAGCTAATCCGACTTCGGCTCATCCAATAGTGCGAGGTCTTGCGATCCCCTACTTTNCTCCAATCGAATTCCC


>F_32_sp6 Alu CUT SITE 
CTAG/CTTTGTAGTTTCAAACGCAGTTCCCAGGTTGAGCCCGGGGATTTCACATCTGACTTACAAAACCGTCTACGCGCGC
TTTACGCCCAGTAATTCCGATTAACGCTCGCACCCTCTGTATTACCGCGGCTGCcGGCACAGAGTTAGCCGGgGCTTTTT
CTTTGGGTTAAATCAAGACCATCAAGTGTTAGTCAATGGCTTTTTGTCCCCACTAAAAGTGCTTCACAACCCGCAGGCCT
TCTTCACACACGCGGCGTCGCTGGATCAGGGTTTCCCCCATTGTCCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCCG
GACCGTGTCTCAGTTCCGGTGTGGCTGGTCGTCCTCTCAGACCAGCTACGGATTGTCGCCTTGGTAGGCCATTACCCCAC
CAACTAGCTAATCCGACTTTGGGCTCATCCAATAGCGCGAGGTCTTACGATCCCCTACTTTCCTCCAATCGAATTCCCCG
CGGCCCGCCATGG


>F_35_sp6
CTAGCCTTGTAGTTTCAAACGCAGTTCCCAGGTTGAGCCCGGGGATTTCACATCTGACTTACAAAACCGTCTACGCGCGC
TTTACGCCCAGTAATTCCGATTAACGCTCGCACCCTCTGTATTACCGCGGCTGCcGGCACAGAGTTAGCCGGgGCTTTTT
CTTTGGGTTAAATCAAGACCATCAAGTATTTGTCAATGGCTTTTTGTCCCCACTAAAAGTGCTTTACAACCCGCAGGCCT
TCTTCACACACGCGGCGTCGCTGGATCAGGGTTTCCCCCATTGTCCAAAATTCCCCACTGCTGCCTCCCGTAGGAGTCTG
GGCCGTGTCTCAGTCCCAGTGTGGCTGGTCGTCCTCTCAGACCAGCTACAGATCGTCGCCTTGGTAGGCCTTTACCCCAC
CAACTAAGCTAATCTTGCCATCGGCCGCCCCTGTAGCGGGAGGTCTTGCGATCCCCTACTTTCCTCCAATCGAATTCCCG
CGGCC


>F_36_sp6
CTAGCCTTGTAGTTTCAAACGCAGTTCCCAGGTTGAGCCCGGGGATTTCACATCTGACTTACAAAACCGTCTACGCGCGC
TTTACGCCCAGTAATTCCGATTAACGCTCGCACCCTCTGTATTACCGCGGcTGCcGGCACAGAGTTAGCCGGgGCTTTTT
CTGTGAGTTAAATCAAGATCACAGAGTATTAATCCGTGACTTTTTGTCCCCACTAAAAGTGCTTTACAACCCGCAGGCCT
TCTTCACACACGCGGCGTCGCTGGATCAGGGTTTCCCCCATTGTCCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCCG
GACCGTGTCTCAGTTCCGGTGTGGCTGGTCGTCCTCTCAGACCAGCTACGGATTGTCGCCTTGGTGAGCCATTACCTCAC
CAACTAGCTAATCCGACTTTGGCTCATCCATAGCGCGAGGTCTTACGATCCCCTACTTTNCTCCAATCNAATTCCCGCGG
CC


>F_37_sp6  has Alu CUT SITE
CTAG/CTTTGTAGTTTCAAACGC  AGTTCCCAGGTTGAGCCCGGGGATTTCTCATCTGACTTACAAAACCGTCTACGCGCGC
TTTACGCCCAGTAATTCCGATTAACGCTCGCACCCTCTGTATTACCGCGGCTGCcGGCACAGAGTTAGCCGGgGCTTTTT
CTTTGGGTTAAATCAAGACCATCAAGTATTCGTCAATGGCTTTTTGTCCCCACTAAAAGTGCTTTACAACCCGCAGGCCT
TCTTCACACACGCGGCGTCGCTGGATCAGGGTTTCCCCCATTGTCCAATATTCCCCACTGCTGCCTCCCGTAGGGGTCCG
GACCGTGTCTCAGTTCCGGTGTGGCTGGTCGTCCTCTCAGACCAAGCTACGGATTGTCGCCTTGGTAGGCCATTACCCCA
CCAACTAAGCTAATCCGACTTTGGCTCATCCAATAAGCGCGAGGTCTTGCGATCCCCTACTTTCCTCCAATCGAATTCCC
GCGGCCGCCATTGGC


>F_41_sp6
CTAGCCTTGTAGTTTCAAACGCAGTTCCCAGGTTAAGCCCGGGGCTTTCACATCTGACTTAAGAAACCGCCTGCGCACGC
TTTACGCCCAGTAATTCCGATTAACGCCTGCACCCTACGTATTACCGCGGcTGCcGGCACGTAGNTAGCCGGgGCTTCTT
CTTCAGGTACCGTCATCAATCCGTGGTATTAGCACGAACCATTTCGTTCCTGCCGAAAGAGCTTTACAACCCGAAGGCCT
TCTTCACTCACGCGGCATGGCTGGATCAGGCTTGCGCCCATTGTCCAAAATTCCCCACTGCTGCCTCCCGTAGGAGTCTG
GACCGTGTCTCAGTTCCAGTGTGGCTGATCATCCTCTCAGACCAGCTACGGATCGTCCGCCTTGGGTGGGGCTTTTACCC
CGCCAACTAAGCTAATCCCGATATCGGCCACTCCTATAGTGCGTGGTCTTGGCGATCCCTACTTTCCTCCAATCGAATTC
CCCGCGGCCGCCATGGC


>F_42_sp6
CTAGCCTTGTAGTTTCAAACGCAGTTCCCAGGTTGAGCCCGGGGATTTCACATCTGGACTTACAAAACCGTCTACGCGCG
CTTTACGCCCAGTAATTCCGATTAACGCTCGCACCCTCTGTATTACCGCGGcTGcTGGCACAGAGNTAGCCGgTGCTTTT
TCTTTGGGTTAAATCAAGACCATCGTGTGTTAAGTCAATGGCTTTTTGTCCCCACTAAAAGTGCTTTACAACCCGCAGGC
CTTCTTCACACACGCGGCGTCGCTGGATCAGGGTTTCCCCCATTGTCCAATATTCCCCACTGCTTGCCTCCCGTAGGAGT
CCGGACCGTGTCT


>G-31_sp6
CTAGCCTTGTAGTTTCAAACGCAATTCCCAGGTTGAGCCCGGGGCTCTCACATCTGACTTACAAAACCGCCTGCGCACGC
TCTACGCCCAGTAATTCCGATTAACGCTCGCACCCTCTGTATTACCGCGGCTGCcGGCACAGAGTTAGCCGGgGCTTTTT
CTTTGGGTTAGGTCAAGACTCAGAGTTATTAGCCCTGAGCTTCTCATCCCCACTAAAAGTGCCTTACAACCCGCAGGCCT
TCTTCACACACGCGGCGTCGCTGGATCAGGGTTGCCCCCATTGTCCAATATTCCCTACTGCTGCCTCCCGTAGGAGTCTG
GGCCGTGTCTCAGTCCCAGTGTGGCTGGNCGTCCTCTCAGACCAAGNTACGGATTGNCNCCTTGGTAAGGCCCGNTACCC
TACCAACTA


>G-32_sp6
CTAGCCTTGTAGTTTCAAACGCAGTTCCCAGGTTGAGCCCAGGGATTTCTCATCTGACTTACAAAACCGTCTACGCGCGC
TTTACGCCCAGTAATTCCGATTAACGCTCGCACCCTCTGTATTACCGCGGCTGCcGGCACAGAGNTAGCCGGgGCTTTTT
CTTTGGGTTAAATCAAGACCGCAGAGTATTAATCCGTAGCTTTTTGTCCCCACTAAAAGTGCTTTACAACCCGCAGGCCT
TCTTCACACACGCGGCGTCGCTGGATCAGGGTTTCCCCCATTGTCCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCCG
GACCGTGTCTCAGTTCCGNTCTNCTGGTCGTCCACTCAGACCAGCTACGGATTGTCGCCTTGGTGAGCCATTACCTCACC
AACTAGCTAATCCGACTTTGGCTCATNCAATANCGCGAGGNCTTACNATCCCCTTGTTTCC


>G-39_sp6
CTAGCCTTGTAGTTTCAAACGCAGTTCCCAGGTTGAGCCCAggaGaGCATTTCACATCTGACTTACAAAACCGNGGTACG
CGCGCTTTACGACCAGTAATTCCGATTAACGCTCGCACCCTCTGTATTACCGNNGTTcTTGGCACAGAGTTAGCCgGTGC
TTTTTNTTTGGGTTAAATCAAgACCATCGAGTGTTAATCAATGGCTTTtTGTCCCCACTAAAAGTGCTTTACAACCCGCA
GGCCTTCTTNACACANGCGGGGTNGCTGGATNAGGGTTTCCCCCATTGTCCAATATTCCCC


>G-40_sp6
CTAGCCTTGTAGTTTCAAACGCAGTTCCCAGGTTGAGCCCGGGGATTTCACATCTGACTTACAAAACCGTCTACGCGCGC
TTTACGCCCAGTAATTCCGATTAACGCTCGCACCCTCTGTATTACCGCGGCTTGcTGGCACAGAGTTAGCCGgTGCTTTT
TCAGTGAGTTAAATCAAGATCACAGAGTATTAATCCGTGACTTTTTGTCCCCACTAAAAGTGCTTTACAACCCGCAGGTC
TCCTTCACACACGCGGCGTCCGCTGGATCAGGGTTTCCCCCCATTGTCCAATATTCCCCACTGTGCTNCCGTAGNAGTCC
GNACCGNGTCTNANTTCCAGNGNNGGNTGGCC


>G-41_sp6  Alu cut site
CTAG/CTTTGTAGTTTCAAACGCAGTTCCCAGATTGAGCCCGGGGATTTCACATCTGACTTACAAAACCGTCTACGCGCGC
TTTACGCCCCGTAATTCCGATTAACGCTCGCACCCTCTGTATTACCGCGGCTGCcGGCACAGAGTTAGCCGGgGCTTTTT
CTTTGGGTTAAATCAAGACCATCGAGTATTCGTCAATGGCTTTTTGTCCCCTCTAAAAGTGCTTTACTACCCGCAGGCCT
TCTTCACACACGCGGCGTCGCTGGATCAGGGTTTCCCCCATTGTCCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCCG
GACCGTGTCTCAAGTTCCGGTGAGGCTGGTCGTCCTCTCAGACCAGCTACGGATTGNCGCTTGGTAGGCCATTACCCACC
AACTAGCTAATCCGACTTTGGTCATCCATAGCNCGAGGCTTG

THE FOLLOWING CLONES HAVE FORWARD PRIMER (COLORED) SEQUENCES WITH NOTHING RESEMBLING THE REVERSE PRIMER OR ITS COMPLEMENTARY BASES THAT I COULD FIND. 

>F_33_sp6
GGA GGA AAG TAG GGG ATC GTA AGA CCT CGCGCTATTGGATGAGCCAAAGTCGGATTAGCTAGTTGGTGAGGTAATGGCTC
ACCAAGGCGACAATCCGTAGCTGGTCTGAGAGGACGACCAGCCACACCGGAACTGAGACACGGTCCGGACTCCTACGGGA
GGCAGCAGTGGGGAATATTGGACAATGGGGGAAACCCTGATCCAGCGACGCCGCGTGTGTGAAGAAGGCCTGCGGGTTGT
AAAGCACTTTTAGTGGGGACAAAAAGTCACGGATTAATACTCTGTGATCTTGATTTAACTCACAGAAAAAGCACCGGCTA
ACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGACGGT
TTTGTAAGTCAANATGTGAAATCCCCGG


>F_34_sp6
GGA GGA AAG TAG GGG ATC G CA AGACCTCGCGCTATTGGATCGAGCCAAAGTCGGATTAGCTAGTTGGTGGGGTAATGGCC
TACCAAGGCGACAATCCGTAGCTGGTCTGAGAGGACGACCAGCCACACCGNTANCTGAGACACGGTCCGGACTCCTACGG
GAGGCAGCAGTGGGGAATATTGGACAATGGGGGAAACCCTGATCCAGCGACGCCGCGTGTGTGAAGAAGGCCTGCGGGTT
GTAAAGCACTTTTAGTGGGGACAAAAAGTCACGGATTAGTACTCTGTGATCTTGATTTAACTCACAGAAAAAGCACC


>F_38_sp6
GGA GGA AAG CAG GGGATCGCAAGACCTCGCGCTATTGGATGAGCCAAAGTCGGATTAGCTAGTTGGTGGGGTAATGGCCT
ACCAAGGCGACAATCCGTAGCTGGTCTGAGAGGACGACCAGCCACACCGGAACTGAGACACGGNCCGGACTCCTACGGGA
GGCAACAGTGGGGAATATTGGACAATGGGGGAAACCCTGATCCAGCGACGCCGCGTGTGTGAAGAAGGCCTGCGGGTTGC
AAAGCACTTTTAGTGGGGACAAAAAGTCACGGGTTAATACCCGGTGATCTTGATTTAACTCACAGAAAAAGCGCCGGCTA
ACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGCAAAGCGCGCGTAGACGGT
TTTGTAAGTCANATGTGAAATCCCCGG


>F_39_sp6
GGA GGA AAG TAG GGGATCGTAAGACCTCGCGCTATTGGATGAGCCAAAGTCGGATTAGCTAGTTGGTGAGGTAATGGCTC
ACCAAGGCGACAATCCGTAGCTGGTCTGAGAGGACGACCAGCCACACCNNTNNCTGAGACACGGNCCGGACTCCTACGGG
AGGCAGCAGTGGANAATATTGGACAATGGGGGAAACCCTGATCCAGCGACGCCGCGTGTGTGAAGAAGGCCTGCGGGTTG
TAAAGCACTTTTAGTGGGGACAAAAAGTCACGGATTAATACTCTGTGATCTTGATTTAACTCACAGAAAAAGCACCGGCT
AACTCTGTGCCAGCAGCCGCGGTAATACA


>G-42_sp6
GGA GGA AAG CAG GGG ATC G T AAGACCTCGCGCTATTGGGATGAGCCAAAGTCGGATTAGCTAGTTGGTCGAGGTAATGGC
TCACCAAGGCGACAATCCGTAGCTGGTCTGAGAGGACGACCAGCCACACCGGAACTGAGACACGGTCCGGACTCCTACGG
GAGGCAGCAGTGGGGAATATTGGACAATGGGGGAAACCCTGATCCAGCGACGCCGCGTGTGTGAAGAAGGCCTGCGGGTT
GTAAAGCACTTTTAGTGGGGACAAAAAGTCACGGATTAATACTCTGTGATCTTGATTTAACTCACAGAAAAAGCACC---


>F_40_sp6 NO FORWARD, NO REVERSE?
GGA GGA AAG AGG GGA TCG C AAGACCTCGCGCTATTGGATGAGCCAAAGTCGGATTAGCTAGTTGGTGGGGTAATGGCCT
ACCAAGGCGACAATCCGTAGCTGGTCTGAGAGGACGACCAGCCACACCGGAACTGAGACACGGTCCGGACTCCTACGGGA
GGCAGCAGTGGGGAATATTGGACAATGGGGGNAACCCTGATCCAGCGACGCCGCGTGTGTGAAGAAGGCCTGCGGGTTGT
AAAGCACTTTTAGTGGGGANNAAAAGNTNANGGCTAANACNTNTGGATCTTGACNTAACCCAAAGAAAAAGCACCGGCTA
ACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCGAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGACGG
TTTTGTAAGTCAGATGTGAAATCCCCGG