Sanger Sequencing Steve Harris SPASH Biotech Instructor

Sanger Sequencing Steve Harris SPASH – Biotech Instructor

• DNTP’s (d. ATP, d. TTP, d. CTP, d. GTP) • Deoxynucleotide – Extends DNA strand. • DDNTP’s (dd. ATP, dd. TTP, dd. CTP, dd. GTP) • Dideoxynucleotide – Terminates DNA synthesis – Color coded D Y E

Unknown Sequence: TCAGCTAG A (T) T C G (A) A T C G A T (C) C G A T C (G) G T A C G SIZE • AGT (3) • AGTCGAT (7) • AGTCGATC (8) • A (1) • AGTCGA (6) • AGTCGATC (8) • AGTC (4) • AGTCGATC (8) • AG (2) • AGTCG (5) • AGTCGATC (8) GTCGATC (T)CAGCTAG

Unknown Sequence: TCAGCTAG SIZE *notes page on sequencing.

Sequence from the human genome project

Sanger Sequencing Lab Researchers at UW-Madison’s department of Ophthanmology and Vision Sciences have discovered a gene [“switch”] that turns off or on the development of the optic nerve of cave fish found in the Mammoth cave system in Kentucky. They are optimistic that understanding how this gene functions can be used to slow or even cure degenerative blindness in humans. Before human trials can begin, researchers must first test this gene in mice. In order to maintain this gene in the lab researchers want to ligate the gene into the p. UC 19 vector at it’s multi-cloning site [MCS]. Then clone the recombinant vector in E-coli for storage. Your role is to sequence the MCS so researchers can identify possible compatible restriction sites used in the isolation of the vision gene in the first place.

p. UC 19 – 2, 686 bp Forward Primer Reverse Primer

Sanger Sequencing N’s___________________ D__________ H__________ primer-template mix for p. UC 19 MCS *Initial after each reagent is added Pipette tips in recycling bin dd. NTP's Tube Label 10 x Sequencing Buffer Mix (1 tube) d. ATP d. TTP d. GTP (4 tubes) dd. ATP dd. TTP 10 x Forward Reverse N-H 20 Primer Taq DNA p. UC 19 dd. CTP Polymerase Add LAST! Located in my rack up front dd. GTP A 5μl 10μl A=10μl 5μl 10μl 3μl 2μl C 5 10 C=10 5 5 10 3 2 T 5 10 T=10 5 5 10 3 2 G 5 10 G=10 5 5 10 3 2 + 5 10 - 5 5 20 3 2 - 5 10 - 5 5 23 - 2

Sequencing Machine

14 -DEC-2015 DEFINITION Cloning vector p. UC 19, complete sequence. 2686 bp DNA circular ACCESSION M 77789 M 11662 VERSION M 77789. 2 GI: 6691170 SOURCE Cloning vector p. UC 19 Stevens Point Area Senior High – Steve Harris ORGANISM Cloning vector p. UC 19 1201 North Point Dr. Stevens Point, WI 54423, USA REMARK Sequence update by submitter COMMENT this sequence version replaced gi: 209214. FEATURES Location/Qualifiers source 1. . 2686 /organism="Cloning vector p. UC 19" /mol_type="other DNA" /strain="ATCC 37254" /culture_collection="ATCC: 37254" /db_xref="taxon: 31851" misc_feature 1. . 447 /note="M 13 mp 19" misc_feature 1. . 230 /note="Lac-operon" misc_feature 233. . 289 /note="polylinker of M 13 mp 19; Hind. III-Sph. I-Pst. I-Sal. I-Xba. I-Bam. HISma. I-Kpn. I-Sac. I-Eco. RI" misc_feature 290. . 447 /note="Lac-Operon" misc_feature complement(448. . 547) /note="p. BR 322" misc_feature complement(548. . 684) /note="p. BR 322" misc_feature complement(685. . 2686) /note="p. BR 322"

ORIGIN 1 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 61 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct 121 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat 181 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttgc 241 atgcctgcag gtcgactcta gaggatcccc gggtaccgag ctcgaattca ctggccgtcg 301 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac 361 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac 421 agttgcgcag cctgaatggcgcc tgatgcggta ttttctcctt acgcatctgt 481 gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt 541 taagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc 601 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt 661 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg 721 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc 781 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac 841 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt 901 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 961 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg 1021 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa 1081 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc 1141 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag 1201 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa 1261 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc

1321 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg 1381 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa 1441 caacgttgcg caaactatta actggcgaac tactct agcttcccgg caacaattaa 1501 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg 1561 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag 1621 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg 1681 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt 1741 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt 1801 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac 1861 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 1921 atcctttttt tctgcgcgta atctgctgct tgcaaa aaaaccaccg ctaccagcgg 1981 tggttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 2041 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga 2101 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 2161 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 2221 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 2281 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa 2341 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 2401 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 2461 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 2521 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat 2581 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca 2641 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaaga //

p. UC 19 Multi-Cloning Site

A T C G Hyperlink Sequencing

Sequencing Assignment Key Blue=C Black=A Green=G Red=T Name Date Hour

Name_____ Date______ Hour______ Sequencing of Autoradiographs Band Number Analysis (Your Answer) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Wild Type Sequence (Actual Answer) Band Number Your Answer 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 Wild Type Sequence (Actual Answer)

Name_____ Date______ Hour______ Sequencing of Autoradiographs Number Your Answer Actual Anwer 1 2 3 A G C 26 27 28 C C G 4 5 6 7 8 9 T T G G C T 29 30 31 32 33 34 G G A A T T 10 11 12 13 G C A G 35 36 37 38 C? G? T? A? 14 15 16 17 18 19 20 21 22 23 24 25 G T C G A C G G A T C C 39 40 41 42 43 44 45 46 A? T? C? A? T? G? G? T?

Name_______ Date________ Hour______ Reading Questions 1. What are the two approaches to DNA sequence analysis? Which one are we going to be looking at? 2. What is the difference between the four enzymatic reactions that are performed? 3. What method is replacing the traditional isotopic method? Why? 4. What is the “nested set”? 5. How is the image from the gel transferred to the x-ray? What component makes this possible?

Name_______ Date________ Hour______ Reading Questions 1. What are the two approaches to DNA sequence analysis? Which one are we going to be looking at? One is a set of organic chemical reactions the other uses enzymes. The chemical method is tedious. We will be looking at autoradiographs produced from enzymatic reactions. 2. What is the difference between the four enzymatic reactions that are performed? “G” contains dideoxy. GTP , “C” contains dideoxy. CTP, “A” contains dideoxy ATP, “T” contains dideoxy TTP 3. What method is replacing the traditional isotopic method? Why? Non-isotopic methods of using fluorescent dyes and automated DNA sequencing machines. Why? These methods are better, more efficient. Radiation is SCARY! 4. What is the “nested set”? The products of the reaction. 5. How is the image from the gel transferred to the x-ray? What component makes this possible? The gel is placed directly on the x-ray film. DNA fragments have been labeled with 32 P.