DNA Sequencing

1. Two Methods: • chemical cleavage xxx (Maxim and Gilbert) • synthetic oligonucleotides • GC-rich DNA • dideoxy (Sanger) • based on 2’3’-dideoxynucleotides as chain terminators ì H DNA Sequencing

2. Dideoxy Chain Termination

3. Semi-Automated Sequencing • thermal cycler • fluorescent ddNTPs • unique spectra • measure intensity at 4 wavelengths of DNA products on gel è

4. Co-termination (aka stops) • band in all 4 lanes • 2o structure in template will lead to false stop • Compression • gel-related problem • 2o structure in product results in anomalous migration

5. Alleviating Compressions and Stops • sequence other strand • formamide in gel • nucleotide analogs (dITP, 7-deaza-dGTP) • Taq polymerase, 72o • formamide or DMSO in reaction • terminal transferase chase

6. Extended Sequencing Strategies • restriction digests and subcloning • nested deletions • primer walking • shotgun • easy to carry out • easy to assemble contiguous sequence • requires convenient restriction sites

7. Unidirectional Nested Deletions • preparation of deletion series is labor-intensive • sequencing and contig assembly is fast and easy  î

8. Extended Sequencing Strategies • restriction digests and subcloning • nested deletions • primer walking • shotgun • easy to carry out • easy to assemble contiguous sequence • slow and expensive

9. Extended Sequencing Strategies • restriction digests and subcloning • primer walking • nested deletions • shotgun • contig assembly is inefficient • gaps • repetitive • easy to automate

10. Sequence Analysis • compiling and editing sequences • manipulating sequences (eg, complement, reverse, etc.) • identify specific sites (eg, restric-tion, promoters, etc.) • translate and identify coding regions (gene finder) • analyze proteins (eg, 2o structure, motifs. etc) 1 aagctttaat atttattttt acacaaatta gtactattat aaaatgcaaa agtaatgtac 61 atttgtgtgt attaatttta gcattataat ttattccatt ctgtatatta gttaagttta 121 gttgaaaatg aaggtgatcg gacttttgtt ttctttcgtt ttttttgcta taaaatgcaa 181 atctgaaaca attgaagttt ataatgatat cattcaaaag ttagaaaaat tagaatcatt 241 gtcagtagaa ggattagaac tatttcaaaa aagtcaagta attataaatg catcaccacc 301 aagtgaaact attaatccat tttcagataa tacctttgca ccaaaattac aaggatttat 361 tacaaaattt gaagaactgg gatttacaga acaaacagaa ttagtaaatt taataaaaac 421 attagtcccc aataaatatg gactaaaata tttaattgaa agtaaagaag aatttaatgg 481 attaatgcac gcaataaatt tttattatga tgtatttaga gacaaattaa atgatatgtg 541 tgcaaataat tattgtgaaa ttcctgaaca tcttaaaatt agtgaagaag aaacagaaat 601 gcttaaaaaa gtaattttag gttatagaaa accaatagaa aatattcaag acgatattga 661 aaagttagaa atttacataa caaaaaataa agaaactgtt acagctttaa acactcttat 721 tgctgaagaa acaaaaaaaa taacacctga aaacgaagca gattgcaatg acaatacttg 781 tgatgaaagt aaatatagta aaaaaaaaat aatatatcaa gctatgtata atgttatatt 841 ttacaaaaag caattagctg aaatacaaaa ggttattgaa accttagaaa aacgagtttc 901 tgcattaaag aaaaatgatg tcataaagcc attattgcaa caaattgaag atatcaaggc 961 tgcccctgtt actaccgaag gacaaataac tacatcaggg caatctagta cagaacccgc 1021 tagtacagga acacctagtt caggtgaagt tagtacagga actagtacag gaggagctag 1081 tgcaggcgtc actaatacag gagcagctac tacaggaact actggtacag gagcagctac 1141 tacaggaact actggtgcag aagcagttac tacaggaaat actggtgcag aagcagctac 1201 tacaggaaat actaatacag aagtaactca agtgcaaacc gttccaactc ttacaccaga 1261 agaaaaaaag aaaaaaatgg acggacttta tgctcaaatt aaagaaattg caaaaactat 1321 aaaattcaac ttagacggaa tatttgtaaa tccaatcgaa ttagaatatt taaaaaaaga 1381 aaaaaaaaaa gaaagctgca atttatcaac ttcatcctgt aaaaaaaata aaacatctga 1441 aactataata ccattaaatg tacgttatcc aaatggtatt ggttatccat tacctgaaaa 1501 tgatgtttac aataaaattg ctaataatgc cgctgaaaca acatatggtg atttaacaaa 1561 tcctgataat acaccaataa cagaagattt agctacaaat gaacaagcaa gaaaaaattt 1621 aataaaagct attaaaaaga aaatagaagc agaagaaaaa aacttagaat cattaaaaac 1681 taattatgat aataaacttg catcatttaa tcaacaaaaa gctccattca aagaagcagc 1741 taaactattt tatgaatcaa aatttggaaa taaattgact tctgatattt ttgaaaaatt 1801 caaaacacaa agaactgaat atatgaacaa gaaaaccgaa ttagaaaatg gtttatatgg 1861 aaatactaaa cagttaatta gtaaattaaa taaacaactt aattacttac aagattattc 1921 attaagaaaa gatataatta gtgatgaaat tgaatatttt tcaaataaaa aaaaaggatt 1981 acaatataat attaatagat tagcagaagc tgttcaagca aaacaaaata tattagttgc 2041 atcaaaagat gtaccacttt caacacttgt agaattacaa atacaaaaat ctttattaac 2101 aaaacaaatt gagcaattaa ataaaactga actatcttta agaaaagctc aattaaaaga 2161 caaagtatac gttcccaaat catacggtaa tggtggaaaa ccagaaccat actatttaat 2221 agctgtaaaa agggaagttg acagacttgc ccaatttatt ccaaaaatcg aaaatatgat 2281 tgctaaagag aaggaaaaaa cggaacaagt gcctgtagtt actggagaat ctgaagaaac 2341 atctagcgtt agtactgaag tatcagtaca agcctcatca caatctggaa catcttcaac 2401 cgtaccagca gcgggggcaa catcaccaac agtaacccct gtcacagaag aagcacaatc 2461 ctcacaaaat gcaccaccca caacagcagc gacaccggca acaacaccag aagcagcaac 2521 aacagcagcg acaccggcaa caacaccaga agcagcaaca acatcaacga caacatcaac 2581 gacaacatca acgacaacat caacgacaac atcaacgaca acaccagtta tgacaaaatt 2641 atattatctt gaaaaattac aaaagttttt agtattttca tattcatgcc ataaatatct 2701 tttactacaa aactctacca taaacaaaga tgctttaagc aaatatgctc ttacaactga 2761 ggaagataaa ataagaacat taaagagatg cagtgaatta gatatattat tagctattca 2821 aaataatatg cctactatgt attcacttta tgaaagtata gttgatggtt tacaaaacat 2881 ttatgctgaa ttatatgaaa aagaaatgat gtatcatata tataaattaa aagacgaaaa 2941 tccatctatt aaatctatat tggtaaaagc tggcgtcatt gatccagaac cagtagcatc 3001 accaccaaca ccaccaacac caccaacacc accaacacca ccaacactac caacaacccc 3061 aacaccagta actccagcag caccatccga acaaacaaca acacctgaag cagcaacagc 3121 agcatctaat ccaggcgcgt cagcctcaga aacaccagca tctaatccag gcgcatcagc 3181 ctcatcaaca ccagcagcag catctaaccc aggcgcatca gcctcatcaa caccatcaac 3241 agcatctaac ccaagcgcac cagcctcatc aacagtacaa ccaacacaag tacaaccggc 3301 agcaccagcg gcaccagcag cagtaccagc acaaccagca aattctaatg gaagtattac 3361 cacacgagca gaaagtgaag aagatattcc tgccgatgat tttgaattag acaatttata 3421 caaatcttac ttgcaacgaa ttgatggaaa taatactgaa ttcataaatt ttataaaatc 3481 taaaaaagaa ttaataaaag cattaacacc tgaaaaagtt aatcaattat atcttgaaat 3541 cgcacactta aaggaattat cagagcatta ttatgatcgt tattataaat ataaattaaa 3601 attagaaaga ttatatgaaa agcatgaaca aattcaagta agcaatcgac aaattagaga 3661 acttagtata ttaaaagcac gattattaaa aagaaaacaa aatattaatg gaatatttta 3721 tatattaagt ggttatgtaa atttctttaa caaaagaaga gaagctgata aacagtatgt 3781 agataatgca ttaaaaaata atgatatgtt attgaaatac tacaaagctc gtattaaata 3841 ctttacctct gaagctgttc ctttaaaaac attaactaaa gcatcacttg acagagaaac 3901 caactacttg aaaatcgaaa aattcagagc atacagtcga ttagaattaa gatttaaaaa 3961 aaatattaat ttaggaaagg aaaaaatttc atatgtatca ggaggtttgt atcacgtatt 4021 tgaagaattt aaggaacttt taaaaaataa aaattatacc ggaaaaacca accctgatac 4081 tgttcctgaa gttaccaatg cattcgaaca atataaagaa ttgcttccaa agggagtaac 4141 agcttcagct tcacctgctg cagctacaac acccacatca gctgacgcag caacacaacg 4201 agcaacacca gaatcgggat caggatcggg atcaggatca gtagtatcat cgacaccaga 4261 agaagtagca agatcaggat caggagaaaa tgcagtggta agtggaagca gtgttgatga • Software Sources • purchase packages • collect various programs via ftp • internet sites that carry out analyses • cut/paste sequence • submit to e-mail servers