1. Sanger Method Frederick Sanger - 1977

2. Radioactively labelled dNTPs or radioactive/fluorescent primers  Autoradiography, laser detector Chain Termination Only one primer

4. Automated Sequencing

5. Labelling of the chain terminator ddNTPs Sequencing in a single reaction, rather than four reactions as in the labelled-primer / dNTP method. Labels: Fluorescent dyes, each of which with different wavelengths of fluorescence and emission

6. Template: X Genomic? Not good: high amount is needed PCR product / plasmid Single primer Forward? Reverse? dNTP ddNTP Polymerase Capillary electrophoresis

7. Homozygote mutant normal chromatogram

8. 304 C  T (R102C) normal Heterozygote mutant

9. Do the following steps in your homework! Read 50 nucleotides from chromatogram. (T-red, C-blue, G-black, A-green) Find its whole mRNA sequence using Blast (www.ncbi.nih.gov). Make restriction map of your sequence. Choose appropriate restriction enzymes for cloning your coding sequence. Choose appropriate primer set using primer3 program. Choose a mammalian expression vector compatible with your restriction sites. Show your coded aminoacid sequence using sixframe program.

10. Read your sequence T-red, C-blue, G-black, A-green GCCCTGGGGAGCCAGAGGAGGGTGCCAGGCACCAGCATCTTCGTCTCTCTTC

11. Find its whole mRNA sequence using Blast (www.ncbi.nih.gov/Blast.cgi)

12. CDSCDS 589..4296

13. Copy all the sequence in FASTA format!

14. Restriction Mapping: http://tools.neb.com/NEBcutter2/index.php http://tools.neb.com/NEBcutter2/index.php Custom Digest (322)(4307)

15. Custom Digest with Two RE’s (322) (4307)

16. Primer3: http://workbench.sdsc.eduhttp://workbench.sdsc.edu Add new nucleic sequence tool Copy and paste your mRNA sequence PRIMER3-Design primer pairs and probes

18. SIXFRAME: http://workbench.sdsc.edu http://workbench.sdsc.edu SIXFRAME-Generate&import 6 frame translations on a NS MASPPDTDGFSDVRKVGYLRKPKSMHKRFFVLRAASEAGGPARLEYYENEKKWRHKSSAPKRSIPLESCF NINKRADSKNKHLVALYTRDEHFAIAADSEAEQDSWYQALLQLHNRAKAHHDGAGGGCGGSCSGSSGVGE AGEDLSYDTGPGPAFKEVWQVILKPKGLGQTKNLIGIYRLCLTSKTISFVKLNSEAAAVVLQLMNIRRCG HSENFFFIEVGRSAVTGPGEFWMQVDDSVVAQNMHETILEAMRAMSDEFRPRTKSQSSSSCSNPISVPLR RHHLNNPPPSQVGLTRRSRTESITATSPASMVGGKPGSFRVRASSDGEGTMSRPASVDGSPVSPSTNRTH AHRHRGSSRLHPPLNHSRSIPMPSSRCSPSATSPVSLSSSSTSGHGSTSDCLFPRRSSASVSGSPSDGGF ISSDEYGSSPCDFRSSFRSVTPDSLGHTPPARGEEELSNYICMGGKGASTLTAPNGHYILSRGGNGHRYI PGATMGTSPALTGDEAAGAADLDNRFRKRTHSAGTSPTISHQKTPSQSSVVSIEEYTEMMPAAYPPGGGS GGRLPGYRHSAFVPTHSYPEEGLEMHHLERRGGHHRPDSSNLHTDDGYMPMSPGVAPVPSNRKGNGDYMP MSPKSVSAPQQIINPIRRHPQRVDPNGYMMMSPSGSCSPDIGGGSCSSSSISAAPSGSSYGKPWTNGVGG HHTHALPHAKPPVESGGGKLLPCTGDYMNMSPVGDSNTSSPSECYYGPEDPQHKPVLSYYSLPRSFKHTQ RPGEPEEGARHQHLRLSSSSGRLRYTATAEDSSSSTSSDSLGGGYCGARPESSVTHPHHHALQPHLPRKV DTAAQTNSRLARPTRLSLGDPKASTLPRVREQQQQQQQQQQSSLHPPEPKSPGEYVNIEFGSGQPGYLAG PATSRSSPSVRCLPQLHPAPREETGSEEYMNMDLGPGRRATWQESGGVELGRVGPAPPGAASICRPTRSV PNSRGDYMTMQIGCPRQSYVDTSPVAPVSYADMRTGIAAEKVSLPRTTGAAPPPSSTASASASVTPQGAA EQAAHSSLLGGPQGPGGMSAFTRVNLSPNHNQSAKVIRADTQGCRRRHSSETFSAPTRAANTVSFGAGAA GGGSGGGSEDVKRHSSASFENVWLRPGDLGGASKESAPGCGAAGGLEKSLNYIDLDLVKDVKQHPQDCPS QQQSLPPPPPHQPLGSNEGSSPRRSSEDLSTYASINFQKQPEDRQ Frame 1 [Longest ORF], 0 stop codons

19. WEB SITES USED FOR CLONING WORK Blastn: www.ncbi.nih.gov/blast /nucleotide blastwww.ncbi.nih.gov/blast Restriction Mapping: http://tools.neb.com/NEBcutter2/index.phphttp://tools.neb.com/NEBcutter2/index.php Primer3/Sixframe: http://workbench.sdsc.eduhttp://workbench.sdsc.edu Mammalian Expression Vectors: www.stratagene.comwww.stratagene.com www.invitrogen.com www.promega.com