DNA Sequencing

Two Methods:

• chemical cleavage xxx (Maxim and Gilbert)• synthetic oligonucleotides•GC-rich DNA

• dideoxy (Sanger)• based on 2’3’-dideoxynucleotides

as chain terminators

H

Dideoxy Chain Termination

Semi-Automated Sequencing

• thermal cycler• fluorescent ddNTPs•unique spectra

•measure intensity at 4 wavelengths of DNA products on gel

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

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

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

Unidirectional Nested Deletions• preparation of deletion

series is labor-intensive• sequencing and contig

assembly is fast and easy

Extended Sequencing Strategies

• restriction digests and subcloning• nested deletions• primer walking• shotgun

• easy to carry out • easy to assemble

contiguous sequence• slow and expensive

Extended Sequencing Strategies

• restriction digests and subcloning• primer walking• nested deletions• shotgun

• contig assembly is inefficient • gaps • repetitive

• easy to automate

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 cagaacccgc1021 tagtacagga acacctagtt caggtgaagt tagtacagga actagtacag gaggagctag1081 tgcaggcgtc actaatacag gagcagctac tacaggaact actggtacag gagcagctac1141 tacaggaact actggtgcag aagcagttac tacaggaaat actggtgcag aagcagctac1201 tacaggaaat actaatacag aagtaactca agtgcaaacc gttccaactc ttacaccaga1261 agaaaaaaag aaaaaaatgg acggacttta tgctcaaatt aaagaaattg caaaaactat1321 aaaattcaac ttagacggaa tatttgtaaa tccaatcgaa ttagaatatt taaaaaaaga1381 aaaaaaaaaa gaaagctgca atttatcaac ttcatcctgt aaaaaaaata aaacatctga1441 aactataata ccattaaatg tacgttatcc aaatggtatt ggttatccat tacctgaaaa1501 tgatgtttac aataaaattg ctaataatgc cgctgaaaca acatatggtg atttaacaaa1561 tcctgataat acaccaataa cagaagattt agctacaaat gaacaagcaa gaaaaaattt1621 aataaaagct attaaaaaga aaatagaagc agaagaaaaa aacttagaat cattaaaaac1681 taattatgat aataaacttg catcatttaa tcaacaaaaa gctccattca aagaagcagc1741 taaactattt tatgaatcaa aatttggaaa taaattgact tctgatattt ttgaaaaatt1801 caaaacacaa agaactgaat atatgaacaa gaaaaccgaa ttagaaaatg gtttatatgg1861 aaatactaaa cagttaatta gtaaattaaa taaacaactt aattacttac aagattattc1921 attaagaaaa gatataatta gtgatgaaat tgaatatttt tcaaataaaa aaaaaggatt1981 acaatataat attaatagat tagcagaagc tgttcaagca aaacaaaata tattagttgc2041 atcaaaagat gtaccacttt caacacttgt agaattacaa atacaaaaat ctttattaac2101 aaaacaaatt gagcaattaa ataaaactga actatcttta agaaaagctc aattaaaaga2161 caaagtatac gttcccaaat catacggtaa tggtggaaaa ccagaaccat actatttaat2221 agctgtaaaa agggaagttg acagacttgc ccaatttatt ccaaaaatcg aaaatatgat2281 tgctaaagag aaggaaaaaa cggaacaagt gcctgtagtt actggagaat ctgaagaaac2341 atctagcgtt agtactgaag tatcagtaca agcctcatca caatctggaa catcttcaac2401 cgtaccagca gcgggggcaa catcaccaac agtaacccct gtcacagaag aagcacaatc2461 ctcacaaaat gcaccaccca caacagcagc gacaccggca acaacaccag aagcagcaac2521 aacagcagcg acaccggcaa caacaccaga agcagcaaca acatcaacga caacatcaac2581 gacaacatca acgacaacat caacgacaac atcaacgaca acaccagtta tgacaaaatt2641 atattatctt gaaaaattac aaaagttttt agtattttca tattcatgcc ataaatatct2701 tttactacaa aactctacca taaacaaaga tgctttaagc aaatatgctc ttacaactga2761 ggaagataaa ataagaacat taaagagatg cagtgaatta gatatattat tagctattca2821 aaataatatg cctactatgt attcacttta tgaaagtata gttgatggtt tacaaaacat2881 ttatgctgaa ttatatgaaa aagaaatgat gtatcatata tataaattaa aagacgaaaa2941 tccatctatt aaatctatat tggtaaaagc tggcgtcatt gatccagaac cagtagcatc3001 accaccaaca ccaccaacac caccaacacc accaacacca ccaacactac caacaacccc3061 aacaccagta actccagcag caccatccga acaaacaaca acacctgaag cagcaacagc3121 agcatctaat ccaggcgcgt cagcctcaga aacaccagca tctaatccag gcgcatcagc3181 ctcatcaaca ccagcagcag catctaaccc aggcgcatca gcctcatcaa caccatcaac3241 agcatctaac ccaagcgcac cagcctcatc aacagtacaa ccaacacaag tacaaccggc3301 agcaccagcg gcaccagcag cagtaccagc acaaccagca aattctaatg gaagtattac3361 cacacgagca gaaagtgaag aagatattcc tgccgatgat tttgaattag acaatttata3421 caaatcttac ttgcaacgaa ttgatggaaa taatactgaa ttcataaatt ttataaaatc3481 taaaaaagaa ttaataaaag cattaacacc tgaaaaagtt aatcaattat atcttgaaat3541 cgcacactta aaggaattat cagagcatta ttatgatcgt tattataaat ataaattaaa3601 attagaaaga ttatatgaaa agcatgaaca aattcaagta agcaatcgac aaattagaga3661 acttagtata ttaaaagcac gattattaaa aagaaaacaa aatattaatg gaatatttta3721 tatattaagt ggttatgtaa atttctttaa caaaagaaga gaagctgata aacagtatgt3781 agataatgca ttaaaaaata atgatatgtt attgaaatac tacaaagctc gtattaaata3841 ctttacctct gaagctgttc ctttaaaaac attaactaaa gcatcacttg acagagaaac3901 caactacttg aaaatcgaaa aattcagagc atacagtcga ttagaattaa gatttaaaaa3961 aaatattaat ttaggaaagg aaaaaatttc atatgtatca ggaggtttgt atcacgtatt4021 tgaagaattt aaggaacttt taaaaaataa aaattatacc ggaaaaacca accctgatac4081 tgttcctgaa gttaccaatg cattcgaaca atataaagaa ttgcttccaa agggagtaac4141 agcttcagct tcacctgctg cagctacaac acccacatca gctgacgcag caacacaacg4201 agcaacacca gaatcgggat caggatcggg atcaggatca gtagtatcat cgacaccaga4261 agaagtagca agatcaggat caggagaaaa tgcagtggta agtggaagca gtgttgatga

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)

Software Sources

• purchase packages• collect various programs via ftp• internet sites that carry out analyses

• cut/paste sequence• submit to e-mail servers