3 주차 molecular and biological chemistry 2 -...
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Reading frame
Frame
• 모든 진핵생물은 + strand에만 정보 갖고 있음• 원핵생물은 – strand에도 정보를 갖고 있지만 +strand와 서로 겹치지 않음• Virus는 +, - 모두를 사용하며, 사용구역이 겹치기도 하고, 다른 frame을 사
용하기도 함.
Magnolia kobus DC.chloroplast
genome159,245bp
Magnolia chloroplast uses + and – strand without overlap
Polymerase Chain Reaction (PCR)
• A technique for the in vitro amplification of specific DNA sequences by the simultaneous primer extension of complementary strands of DNA (>105).
• PCR method was devised and named by Mullis and colleagues at the Cetus Corporation (Mullis and Faloona, 1987)
• The principle had been described in detail by Khorana et al. (Kleppe et al., 1971) over a decade earlier.
• Kary Mullis received the Novel prize (1993).
PCR
3) DNA POLYMERASE
GT T T TC CA A G G G
2) PRIMER3’5’
AC TA C G TA CG TA C GT G T C CT AC G G TT CT T CT T A G T TG T A C CA CA G TA C TCG TA C T CG TA C T AAA
1) TEMPLETE3’ 5’
…
4) dNTP’s pool
G
C
A
T
dATP
dCTP
dGTP
dTTP
5) Mg++
G
CA
T
A
A
A
A
C
C
C
C
G
T
G
T
G
T G
T
GT T T TC CA A G G G
AC TA C G TA CG TA C GT G T C CT AC G G TT CT T CT T A G T TG T A C CA CA G TA C TCG TA C T CG TA C T AAA
3’
5’
5’
…
Newly synthesised strand
C A A G G A CG T C CA A A T G C A A G A A A A A A AG G G G GT T T T T TC C C CA AG G
DNA polymerization: DNA polymerase extends a primer by using a complementary strand as a template.
Schematic diagram of PCR
… …Denature
94 ℃
3’
5’
5’… …
3’
3’
5’
5’
…
…
…
…
Anneal primers
ca. 50 ℃5’3’
3’
5’…3’
GenomicDNA
…5’
3’
… …
…5’ 3’
…3’ 5’
…
5’ 3’
…3’ 5’
…Synthesize
Newstrand72 ℃
…
3’
5’
5’
…
…
…
…
5’3’
3’
5’
…5’ 3’
Anneal primers
ca. 50 ℃
5’
…3’
5’
5’3’5’ 3’
3’
5’
5’
SynthesizeNew
strand72 ℃
3’ 5’
…
3’
…5’ 3’
… …
5’ 3’
3’ 5’
……
5’
…3’ …
3’ 5’
3’ 5’… …
5’… …3’
3’ 5’
…5’ 3’Denature
94 ℃
…
…
… …
……
…
…
…
Denature94 ℃
…
… …
…
…
…
… …
Anneal primers
ca. 50 ℃
…
… …
…
…
…
… …
…
…
SynthesizeNew
strand72 ℃
…
Taq. DNA Polymerase:
• Most commonly used DNA polymerase for PCRIsolated from Thermus aquaticusHeat stable polymerase
• PCR technique put to practical use by finding of Taq.
• The use of a heat stable enzyme has two major advantages:
1) replenishment after each heating step is not required, thus simplifying the process
2) the enzyme is active at high temperatures, where annealing of the oligonucleotide primers is more specific and DNA synthesis more rapid.
Thermophilic DNA polymerases
Thermus aquaticus (Taq)Thermus thermophilus (Tth)Bacillus stearothermophilus (Bst)Pyrococcus furiosis (Pfu)
Reaction components
1) Thermophilic DNA polymerases
• Thermus aquaticus (Taq)Thermus thermophilus (Tth)Bacillus stearothermophilus (Bst)Pyrococcus furiosis (Pfu)
• Genetically modified for improving proof reading function
(3’ to 5’ exonuclease function)and for hot start PCR.
Reaction components
1) Thermophilic DNA polymerases
• Thermus aquaticus (Taq)Thermus thermophilus (Tth)Bacillus stearothermophilus (Bst)Pyrococcus furiosis (Pfu)
• Genetically modified for improving proof reading function
(3’ to 5’ exonuclease function)and for hot start PCR.
Final-extend72 ℃
An example of a PCR method
0
20
40
60
80
Temp.℃
100
Holdprogram
Pre-denature95 ℃
Anneal55 ℃
Extend72 ℃
Cycleprogram
Holdprogram
Cycle 1 Cycle 2 • • • Cycle 30
Denature95 ℃
Soak4 ℃
Reaction components
2) Deoxynucleotide Triphosphates
• Low dNTP concentrations minimize mispriming at nontarget sitesand reduce the likelihood of extending misincorporatednucleotides (Innis et al. 1988)
Reaction components
3) Primers
• Generally, use over 18 nucleotides
• Genome size of higher plants:5X108-6X109
• Check list for primer design:1) Similar Tm values are recommended for two primers2) Avoid self-complement sequences3) Avoid primer dimer formation
Probability of presence of same nucleotide sequences
6 mer 1/46= 1 / 4X103
9 mer 1/49= 1 / 2.6X105
12 mer 1/412= 1 / 1.7X107
15 mer 1/415= 1 / 1.1X109
18 mer 1/418= 1 / 6.9X1010
21 mer 1/421= 1 / 4.4X1012
Q: 왜 PCR을 할 때 사용하는 primer는 주로18bp 이상을 사용해야 할까?
Reaction components
4) Reaction buffer
• Mainly modified from Saiki et al. (1988)
• Low conc. of Mg++: reaction failedHigh conc. of Mg++: mis-pairing
pseudo bands
Components of PCR buffer
Saiki et al. (1988): Suh lab:Tris pH 8.4 10mM 20mMKCl 50mM 50mMMgCl2 1.5mM 1.5mMgelatin 0.01% -NP40 0.01% -Tween-20 0.01% 0.001%
Reaction components
5) Template DNA
• General amount of template DNA: 105-106 target molecules
• 1 ug of human DNA10 ng of yeast DNA ≒ 3X105 molecules of single copy gene1 ng of E.coli DNA
• Quantification of extracted DNA:1) spectrophotometer: OD260
2) spot test in agarose gel
An Example of spot test
• Marker: PCR Marker (Promega G3161)
- Marker concentration: 5 ul contains 30-40 ng of each DNA fragments
- Sample DNA concentration:6-8 ng/ul
∴ dilute to 1/10 use 2 ul of DNA
for 100ul reaction (1.2-1.6 ng)
Thermal condition and cycle number
e.g. PCR amplification of ndhF gene in Magnoliaceae (Kim et al., 2001)
Pre-denaturation: 95 ℃ 10 min ( AmpliTaq. Gold)
Denaturation: 95 ℃ 1 minPrimer annealing: 55 ℃ 1 min 30 cyclesPrimer extension: 72 ℃ 1 min
Final extension: 72℃ 7 min
PCR 온도 조건의 예
Final-extend72 ℃
An example of a PCR method
0
20
40
60
80
Temp.℃
100
Holdprogram
Pre-denature95 ℃
Anneal55 ℃
Extend72 ℃
Cycleprogram
Holdprogram
Cycle 1 Cycle 2 • • • Cycle 30
Denature95 ℃
Soak4 ℃
Depending on reaction conditions and thermal cycling, one or more of the following may influence plateau:
1) Utilization of substrates (dNTPs or primers)2) Stability of reactants (dNTPs or enzymes)3) End-product inhibition (pyrophosphate, duplex DNA)
…
• RCA (Rolling circle amplificaion)
used for small amount of template such as DNA in herbarium sheets or even fossils.
Krause et al. 2006. Multiplification of the mammoth mitochondial genome and the evolution of Elephantidae. Nature 439: 724-727.
Poinar et al. 2006 Metagenomics to paleogenomics: large-scale sequencing of mammoth DNA. Science 311: 392-394.
Sequencing
1) DNA sequencing: Maxam-Bilbert method
http://en.wikipedia.org/wiki/DNA_sequencing
2) DNA sequencing: Sanger methodhttp://www.bio.davidson.edu/Courses/Bio111/seq.html
3) Highthroughput sequencing (Pyro-sequencing)http://en.wikipedia.org/wiki/Pyrosequencing
Sanger method
Manual method: 1. radio-isotope S352. Silver staining
Automated 1. Plate type2. Capillary type
i. one capillaryii. Multiple capillaries
Next Generation Sequencing (NGS) Technologies
‐ Next (or Current) generation sequencing technologies have accelerated the
speed of genome sequencing projects and have broaden application range of
genome sequences.
Solexa; Illumina
SOLiD; ABI
GS‐Titanium; Roche 454
SMRT; Pacific Bioscience Helicos; Helicos Bioscience
• Sanger sequencing과 대응되는 말로 pyro-sequencing 이라 불림
454 technique1) Sequencing by synthesis2) Emulsion technique3) Microtiter plate
NGS 1): 454 Technology
Capacity of Next Generation Sequencers
Solexa GA2; Illumina
SOLiD 4; ABI
GS‐Titanium; Roche 454
ABI 3730; ABI
96 x 1,000 bp = 96,000 bp = 100Kb
950,000 x 450 bp = 405,000,000 bp = 405Mb
30,000,000 x 7 x (101 x 2) bp = 42,420,000,000 bp = 42.5Gb
940,000,000 x 75 bp (50+25) = 70,500,000,000 bp = 70.5Gb HiSeq2000; Illumina
30,000,000 x 7 x (101 x 2) x 4 bp = 169,680,000,000 bp = 169.7Gb
A Huge Number of Sequence Data in NCBI
‐ NCBI, which is the major sequence repository, presents the rapid growth of
sequences.
http://www.ncbi.nlm.nih.gov/Genbank/genbankstats.html
99,116,431,942 bp
미래 사회를 장악하고 있는 DNA 염기 서열정보에 대한 내용. 우주선을 발사하는 회사 <가타카>를출입하기 위해 본인 확인 및 유전자상태를 검사하려고 혈액을 뽑아내면 순간적 분석이 이루어진다.
개인 유전체 시대의 시작:각자의 전체 유전체를 밝혀 개인식별, 개인적 유전병 치료, 궁극적으로는 클로닝에 이용될 수 있음
Enzyme Source Recognition Sequence Cut
EcoRI Escherichia coli 5'GAATTC 5'---G/AATTC---3'
EcoRII Escherichia coli 5'CCWGG 5'---/CCWGG---3'
BamHI Bacillus amyloliquefaciens 5'GGATCC 5'---G/GATCC---3'
HindIII Haemophilus influenzae 5'AAGCTT 5'---A/AGCTT---3'
TaqI Thermus aquaticus 5'TCGA 5'---T/CGA---3'
NotI Nocardia otitidis 5'GCGGCCGC 5'---GC/GGCCGC---3'
HinfI Haemophilus influenzae 5'GANTC 5'---G/ANTC---3'
Sau3 Staphylococcus aureus 5'GATC 5'---/GATC---3'
PovII Proteus vulgaris 5'CAGCTG 5'---CAG/CTG---3'
SmaI Serratia marcescens 5'CCCGGG 5'---CCC/GGG---3’
HaeIII Haemophilus aegyptius 5'GGCC 5'---GG/CC---3’
AluI Arthrobacter luteus 5'AGCT 5'---AG/CT---3’
EcoR Escherichia coli 5'GATATC 5'---GAT/ATC---3’
KpnI Klebsiella pneumoniae 5'GGTACC 5'---GGTAC/C---3’
PstI Providencia stuartii 5'CTGCAG 5'---CTGCA/G---3’
SacI Streptomyces achroogenes 5'GAGCTC 5'---GAGCT/C---3’
SalI Streptomyces albus 5'GTCGAC 5'---G/TCGAC---3’
ScaI Streptomyces caespitosus 5'AGTACT 5'---AGT/ACT---3’
SphIStreptomyces phaeochromogenes
5'GCATGC 5'---G/CATGC---3’
StuI Streptomyces tubercidicus 5‘AGGCCT 5'---AGG/CCT---3’
XbaI Xanthomonas badrii 5'TCTAGA 5'---T/CTAGA---3’
N = C or G or T or A
W = A or T
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