1. 중합효소연쇄반응 (PCR) 핵산 염기서열 분석(DNA SEQUENCING)

2. Polymerase Chain Reaction (PCR)
중합효소연쇄반응
Primer set와 내열성 DNA polymerase를 이용하여
짧은 시간 내에 미량의 시료에서
주형 DNA 분자의 특정 부분을 선택적으로 증폭 :
1983, Kary Mullis (1993, Nobel Prize in Chemistry)
PCR에는 무엇이 필요한가?
주형 (Template) :
contain the DNA region (target) to amplify
Primer set (forward & reverse primer) : oligonucleotide
complementary to the 3’ ends of each of the strand of the
DNA target
내열성 DNA 중합효소
with a temperature optimum at around 70°C
dNTPs
Thermal cycler (PCR machine)

3. Polymerase Chain Reaction (PCR)
중합효소연쇄반응의 단계
Initial Denaturation : strand separation
94℃, 5 min
Denaturation : strand separation
94℃, 0.5 ~ 1 min
Annealing : complementary pairing (Template – Primer)
55℃, 1 min (Temp. must be adjusted)
Extension : Enzymatic DNA replication
Heat stable DNA polymerase (Taq polymerase)
72℃, 1 min
Final Extension : 72℃, 5 min
After 10 cycles : 210 = 1,024
20 cycles : 220 = 1,048,576
30 cycles : 230 = 1,073,741,824

6. Applications of PCR 임상 (Diagnosis of dieseases)
병원균의 감염 여부 (배양이 어렵거나 불가한 병원균)
유전병의 존재 여부, 유전자 돌연 변이 여부
암의 발병 진단 또는 발병 가능성 예측
DNA 증폭 및 정량 (Amplification and quantification of DNA)
법의학 (Forensic analysis) : Genetic fingerprinting
고생물학, 고고학
계통발생학적 분류 (phylogenetic tree) : 16S rDNA,
유전자의 발현 여부 및 발현 정도 결정
(Quantitative PCR, Real time PCR)
위치지정 돌연변이 (Site-directed Mutagenesis)

7. Limitations of PCR 염기서열이 알려진 DNA 만을 증폭할 수 있음
Primer를 필요로 함
Target DNA sequence 이외에 시료에 오염된 DNA도 증폭 가능
1 분자의 DNA도 증폭 가능함
염기서열이 바뀐 산물 생성 가능
DNA polymerase is prone to error
유전자 원으로 사용할 경우 염기서열 확인 필수

8. Thermal cycler
For repeated heating and cooling

9. DNA polymerase for PCR Klenow fragment of DNA polymerase I
Heat stable DNA polymerases
Taq polymerase (Thermus aqaticus)
Relatively low replication fidelity
Lack of 3' → 5' exonuclease proofreading activity
Size limit : < 2 kb
Pfu DNA polymerase : Pyrococcus furiosus
proofreading activity
Tth DNA polymerase : Thermus thermophilus
Deep VentTM DNA polymerase : Pyrococcus sp.
Deep VentTM (exo-) DNA polymerase : Pyrococcus sp.

11. Primers for PCR Primer set : oligonucleotide
Forward primer
Reverse primer
Important design considerations
Primer Length : 17 ~ 22 bp
Primer G+C content : 40 ~ 60%
Primer Melting temperature (Tm)
Primer Annealing Temperature (Ta)
Primary Secondary Structures : Hairpin, Self dimer, Cross dimer

12. Optimization of PCR Primer Length : 17 ~ 22 bp GC Content : 40 ~ 60%
Long enough for adequate specificity
Short enough for primers to bind easily to the template
at the annealing temperature
8-mer : primer의 결합부위는 평균 48 = 65,536 bp 당 한 번
(human genome 3,2000,000 kb, 49,000개 존재)
17-mer : 417 = 17,179,869,184 bp 당 한 번
(human genome의 한 부위에서만 결합)
GC Content : 40 ~ 60%
Primer Melting Temperature (Tm) : 52 ~ 58℃
indicate the duple (DNA-DNA hybrid) stability
critical in determining the annealing temperature
Forward primer와 reverse primer의 Tm이 비슷해야 함
Tm = (4 x [G + C]) + (2 x [A + T])℃ (nucleotide의 개수)

13. Primer Annealing Temperature (Ta) :
Too high Ta : insufficient primer-template hybridization
low PCR product yield
Too low Ta : high number of base pair mismatches
non-specific products (low PCR specificity)
Determined experimentally
Tm - (1 ~ 5℃)
Opt. Ta = 0.3 x Tm(primer) Tm (product) – 14.9

14. Primer Secondary Structures
by intermolecular or intramolecular interactions
Hairpin : intramolecular
Self dimer : intermolecular, F-F, R-R
Cross dimer : intermolecular, F-R
adversely affect primer template annealing
can lead to poor or no yield of the product

16. Cross-Dimer

17. Monovalent cation : potassium ion (K+)
Length of the Target
0.1 kb ~ 3 kb (~ 1 kb)
~ 10 kb, ~ 40 kb
Buffer solution
Bivalent cations :
generally Mg2+ is used
Mn2+ can be used for PCR-mediated DNA mutagenesis
Monovalent cation : potassium ion (K+)

19. Cloning of PCR products
제한효소 자리가 있는 primer를 사용하는 방법
Primer의 5’ 방향에
restriction site를 가진 oligonucletide가 결합된 primer
PCR product를 RE 처리 후 vector에 삽입
TA cloning
Taq, Tth : PCR product의 3’- 말단에 A 첨가
T vector 사용
Topoisomerase와 연결된 vector를 이용하는 방법
Vaccinia virus topoisomerase
특정 염기서열[5’-(C/T)CCTT-3’]을 인식하고
이 서열의 3’ 말단의 T에 공유결합을 통해 결합
비접착성 말단 PCR product의 cloning
3’-T 말단 PCR product의 cloning

21. 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, …

22. 핵산의 염기서열 분석 (Nucleotide sequencing)
RNA sequencing
Early DNA sequencing methods
1970 Ray Wu at Cornell Univ.
1977 Sanger & Coulson at MRC Centre, UK
"DNA sequencing with chain-terminating inhibitors"
1973 Gilbert & Maxam at Harvard the sequence of 24 bp
1977 Gilbert & Maxam at Harvard Univ.
"DNA sequencing by chemical degradation "
Automatic sequencer
Next-generation (Next-Gen) sequencing methods

23. NTP, dNTP, ddNTP NTP (ribonucleoside triphosphate)
dNTP (deoxyribonucleoside triphophate)
ddNTP (dideoxyribonucleoside triphosphate)

24. DNA replication : Template DNA DNA polymerase Primer dNTP
* 3’-OH 말단에 다음 nucleotide의 5’-PO4가 phosphodiester 결합
dNTP : 3’-OH 있음 : 다음 nucleotide 결합 가능 – 복제 진행
ddNTP : 3’-OH 없음 : 다음 nucleotide 결합 불가 – 복제 (사슬) 종결

25. Sanger method 1977 F. Sanger & coworkers
Based on the selective incorporation of chain-terminating
dideoxynucleotides by DNA polymerase during in vitro DNA
replication
Prevailed from the 80's until the mid-2000s
The method used in the first generation of DNA sequencers
Human Genome Project
Recently, supplanted by "Next-Gen" sequencing methods
Remains in wide use,
smaller-scale projects, validation of Next-Gen results, …

26. 사슬종결법 (Chain Termination Method)
Template DNA
Klenow fragment
Primer
dNTP
ddNTP (labeled with fluorescent material : different colors)
특정 위치에서 DNA 사슬 종결
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), dTTP, dGTP, dCTP는 충분히
Target DNA
ssDNA
dsDNA : 가닥 분리(변성) 후 primer 부착

27. 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

30. DNA Sequencer Automated DNA Sequencing
fluorescent labeling
capillary electrophoresis
general automation (computer, etc)
The first automated DNA sequencer
invented by Lloyd M. Smith
introduced by Applied Biosystems in 1987
used the Sanger sequencing method
enabled the completion of the Human Genome Project (2001)
Next Generation & Third-generation sequencers
Applied Biosystems (Life Technologies)
Beckman Coulter
Illumina
Pacific Biosciences
Roche

34. 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 …