Sanger Method Frederick Sanger - 1977. Radioactively labelled dNTPs or radioactive/fluorescent primers  Autoradiography, laser detector Chain Termination.

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Presentation transcript:

Sanger Method Frederick Sanger

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

Automated Sequencing

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

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

Homozygote mutant normal chromatogram

304 C  T (R102C) normal Heterozygote mutant

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 ( 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.

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

Find its whole mRNA sequence using Blast (

CDSCDS

Copy all the sequence in FASTA format!

Restriction Mapping: Custom Digest (322)(4307)

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

Primer3: Add new nucleic sequence tool Copy and paste your mRNA sequence PRIMER3-Design primer pairs and probes

SIXFRAME: 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

WEB SITES USED FOR CLONING WORK Blastn: /nucleotide blastwww.ncbi.nih.gov/blast Restriction Mapping: Primer3/Sixframe: Mammalian Expression Vectors: