3 주차Molecular and

Biological Chemistry 2

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.

Amborella (eukaryotes) uses only + strand without overlap!

http://amborella.org

Magnolia kobus DC.chloroplast

genome159,245bp

Magnolia chloroplast uses + and – strand without overlap

Cyanobacterium also uses both strand without overlap

Cyanobacteriumcompletegenome

Herpes simplex virus

DNA virus uses both strand and multiple frame!!!

Ethidiumbromide (EtBr) causes frame shift mutation (carcinogen)!

Frame shift mutation

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’

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Anneal primers

ca. 50 ℃5’3’

3’

5’…3’

GenomicDNA

…5’

3’

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…5’ 3’

…3’ 5’

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5’ 3’

…3’ 5’

…Synthesize

Newstrand72 ℃

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3’

5’

5’

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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 ℃

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Denature94 ℃

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Anneal primers

ca. 50 ℃

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SynthesizeNew

strand72 ℃

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심해 열수구(hydrothermal vents)

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.

TA cloning

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?

프라이머디자인사이트

Primer order 의 예

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

UV-spec vs. Nanodrop

An A260 of 1.0 = 50 mg/ml concentration of DNA.

Gel electrophoresis

침강제+Dye+DNA

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)

…

Non-PCR amplification

• Cloning

• RCA (Rolling circle amplificaion)

• Cloning

• 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.

1993

PCR의 발전과 Pyro-sequencing 같은 최신기술은 화석에서의 DNA 추출 및 염기서열 결정도 가능하게 함

Magnolia latahensis (Berry) Brown

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

Maxam-Gilbert method

Sanger method

Manual method: 1. radio-isotope S352. Silver staining

Automated 1. Plate type2. Capillary type

i. one capillaryii. Multiple capillaries

Sanger method

One-dye (or isotope) four-lane system

Four-dye one-lane system

ABI 377: Gel-type Automatic sequencer

ABI 3730: Capillary-type Automatic sequencer

ABI 3100

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

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

Restrictionenzyme

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

Blotting:Southern Hybridization

Blotting:Southern Hybridization

Southern Hybridization

Microarray

http://www.youtube.com/watch?v=ePFE7yg7LvM&feature=related

Shot-gun sequencing

gDNA librarycDNA libraryEST: expressed sequencing tagBAC library

(bacterial artificial chromosome) http://www.youtube.com/watch?v=vg7Y5EeZsjk