Nucleotide sequencing v RNA sequencing v DNA sequencing

핵산의 염기서열 분석 (Nucleotide sequencing) v RNA sequencing v DNA sequencing methods § 1977 Sanger & Coulson at MRC Centre, UK DNA sequencing with chain-terminating inhibitors (효소작용과 사슬 종결법) § 1977 Gilbert & Maxam at Harvard Univ. DNA sequencing by chemical degradation (말단 표지와 화학적 분해법) v Automatic sequencer v Next-generation (Next-Gen) sequencing methods

Sanger method v Chain termination method (사슬 종결법) § Based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication

NTP, dd. NTP

v DNA replication § 3’-OH 말단에 다음 nucleotide의 5’-PO 4가 phosphodiester 결합 § d. NTP (deoxyribonucleoside triphosphate) 3’-OH 있음 다음 nucleotide 결합 가능 복제 진행 § dd. NTP (dideoxyribonucleoside triphosphate) 3’-OH 없음 다음 nucleotide 결합 불가 복제 (사슬) 종결

v 사슬종결법에 의한 염기서열 결정에 필요한 것들 § Target DNA • ss. DNA • ds. DNA : 가닥 분리(변성) 후 primer 부착 § Primer § Klenow fragment (DNA polymerase) § d. NTP (d. ATP, d. TTP, d. GTP, d. CTP) § dd. NTP (dd. ATP, dd. TTP, dd. GTP, dd. CTP) labeled with fluorescent material (different colors)

Dideoxyribonucleotide Chain Termination 1. Denaturation of Target DNA : ds. DNA 2. Primer annealing 3. Radio-labeling by DNA replication Klenow fragment 35 S-labeled d. CTP* 4. Dideoxyribonuclotide Chain Termination Divide into FOUR (dd. ATP, dd. TTP, dd. GTP, dd. CTP) Chain elongation & Chain termination 5. Electrophoresis & Autoradiography Polyacrylamide gel electrophoresis (Capillary) X-ray film (Fluorescent, Laser) 6. Reading : from the short one

핵산 염기서열 분석의 이용 v v v Molecular biology study genomes and the proteins they encode identify new genes, associations with diseases & phenotypes Medicine determine the risk of genetic diseases identify potential drug targets Forensics forensic identification, parental testing Evolutionary biology how different organisms are related how different organisms evolved Metagenomics identification of unculturable organisms important in ecology, epidemiology, microbiology, …

Automated DNA Sequencing v Automated DNA Sequencer § fluorescent labeling § capillary electrophoresis § general automation (computer, etc) § used the Sanger sequencing method § Human Genome Project (2001)

차세대 염기서열 분석법 (NGS, Next-Generation Sequencing) • Massively parallel signature sequencing (MPSS) • Polony sequencing 454 pyrosequencing Illumina (Solexa) sequencing SOLi. D sequencing Ion Torrent semiconductor sequencing DNA nanoball sequencing Heliscope single molecule sequencing Single molecule real time (SMRT) sequencing Nanopore DNA sequencing Tunneling currents DNA sequencing Sequencing by hybridization in vitro virus high-throughput sequencing … • • •

https: //www. youtube. com/watch? v=j. FCD 8 Q 6 q STM