1. 핵산 염기서열 분석(DNA SEQUENCING)

2. 핵산의 염기서열 분석 (Nucleotide sequencing)
RNA sequencing
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)
Automatic sequencer
Next-generation (Next-Gen) sequencing methods

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

4. NTP, dNTP, ddNTP

5. DNA replication 3’-OH 말단에 다음 nucleotide의 5’-PO4가 phosphodiester 결합
dNTP (deoxyribonucleoside triphosphate)
3’-OH 있음
다음 nucleotide 결합 가능
복제 진행
ddNTP (dideoxyribonucleoside triphosphate)
3’-OH 없음
다음 nucleotide 결합 불가
복제 (사슬) 종결

6. Sanger method의 특징 DNA 복제 중 특정 위치에서 사슬 종결 부분적 종결 유도 dNTP와 ddNTP의 비율 조절
4 Rxs : ddATP, ddTTP, ddGTP, ddCTP
종결 위치에 따라 복제된 DNA 사슬의 길이가 다름 : 전기영동으로 분리
부분적 종결 유도
dNTP와 ddNTP의 비율 조절
ex) (dATP : ddATP) 조절
dTTP, dGTP, dCTP는 충분히

7. 사슬 종결법에 필요한 것들 Target DNA ssDNA dsDNA : 가닥 분리(변성) 후 primer 부착 Primer
Klenow fragment
dNTP (dATP, dTTP, dGTP, dCTP)
ddNTP (ddATP, ddTTP, ddGTP,ddCTP)
labeled with fluorescent material (different colors)

8. Dideoxyribonucleotide Chain Termination
1. Denaturation of Target DNA : dsDNA
2. Primer annealing
3. Radio-labeling by DNA replication
Klenow fragment
35S-labeled dCTP*
4. Dideoxyribonuclotide Chain Termination
Divide into FOUR (ddATP, ddTTP, ddGTP, ddCTP)
Chain elongation & Chain termination
5. Electrophoresis & Autoradiography
Polyacrylamide gel electrophoresis (Capillary)
X-ray film (Fluorescent, Laser)
6. Reading : from the short one

10. 핵산 염기서열 분석의 이용 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, …

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

15. Next-Gen Sequencing methods
Massively parallel signature sequencing (MPSS)
Polony sequencing
454 pyrosequencing
Illumina (Solexa) sequencing
SOLiD 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 …