T1OM-M: Chemical Biology

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Markus Müller T1OM-M: Chemical Biology “Basics of Cloning , Genomics and Proteomics“ Part 10: Analysis of Nucleic Acids

Transcript of T1OM-M: Chemical Biology

Page 1: T1OM-M: Chemical Biology

Markus Müller

T1OM-M: Chemical Biology“Basics of Cloning , Genomics and Proteomics“

Part 10: Analysis of Nucleic Acids

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Detection and Quantificationof Nucleic Acids

Global Quantification/Detection

• UV-Vis

• Fluorescent dyes

Sequence specific quantification/detection

• Hybridization

• PCR

• Quantitative PCR (qPCR)

Sequencing

• Sanger or Maxam-Gilbert Sequencing

• NGS, deep sequencing

• TGS, single molecule sequencing

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Detection and Analysis of Nucleic Acids

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Oligomer Quantification

# 418.01.2016TriLink Biotechnologies website: http://www.trilinkbiotech.com/tech/extinction_intro.asp

E = ε ∙ c ∙ d

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Polymer Quantification

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Source: "Optical density of ribosome sample" by Vossman - Own work. Licensed under CC BY-SA 4.0 via Commons https://commons.wikimedia.org/wiki/File:Optical_density_of_ribosome_sample.svg

Sample ε OD1

dsDNA 0.020 (μg/ml)−1 cm−1 50 µg/ml

ssDNA 0.027 (μg/ml)−1 cm−1 37 µg/ml

RNA 0.025 (μg/ml)−1 cm−1 40 µg/ml

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Detection and Analysis of Nucleic Acids

Nucleic acid specific dyes

• Ethidium bromide, Propidium iodide

• SYBR green etc.

• Acridine Orange

• DAPI

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Molecular Probes Handbook, Life Technologies, Chapter 8

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Ethidium and Propidium

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• Intercalating dyes (every 4-5 base pairs)• Large Stokes shift• Not sequence specific• Not cell-permeant• Mutagenic

(bound to DNA)

• Used to influence supercoiling, because one intercalation unwinds DNA by −26°

• About 10 fold increase in fluorescence upon DNA binding• Bind both DNA and RNA

Molecular Probes Handbook, Life Technologies, Chapter 8

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SYBR Green

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• Excitable by both UV and visible light (UV Exmax at ~250 nm)• Extinction coefficient ε495 = 75000 M-1 cm-1

• Intercalation, minor groove binding and ionic interaction• Strong fluorescence increase upon DNA binding (~100x)• Lower increase on ssDNA or RNA• Mutagenic

Molecular Probes Handbook, Life Technologies, Chapter 8Dragan et al., 2012, J. Fluoresc., 22:1189-1199

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Acridine Orange

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Dual binding mode: intercalation as monomer, ionic binding as polymer.

Distinguishes RNA and DNA: Intercalation in dsDNA enforces green fluorescent monomer (low dye/DNA ratio required), while RNA and ssDNA bind the red fluorescent polymer

Molecular Probes Handbook, Life Technologies, Chapter 8

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Minor groove binder

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DAPI

Hoechst 33258

• Minor groove binder• Prefer AT-rich regions• UV-excitation • Not or badly cell permeant

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Hybridization

DNA Hybridization (Southern Blot)

• Nick translation

• PCR amplification

RNA Hybridization (Northern Blot)

• Nick translation

• In vitro transcription

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Dyes for nick translation

# 1218.01.2016http://www.thermofisher.com/de/de/home/references/molecular-probes-the-handbook/reagents-for-modifying-groups-other-than-thiols-or-amines/click-chemistry.html

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Quantitative PCR

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0

500000

1000000

1500000

2000000

2500000

3000000

0 5 10 15 20 25

Sig

nal in

ten

sity

Cycle

exponential amplification

one copy

two copies

five copies

ten copies

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Calibration

Absolute Quantification

• Dilution series of reference material

Relative Quantification

• Use of „housekeeping“ genes.

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Relative Quantification

# 1518.01.2016© 2004 Bio-Rad Laboratories

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Probe Types

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Molecular Beacon• Hairpin opens up upon hybridization to the probe sequence• Fluorescence increases with distance from quencher

TaqMan Probe• Fluorophore is released from probe by exonuclease• Fluorescence increases with distance from quencher• Limited to Taq polymerase

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More Probes

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http://www.sigmaaldrich.com/content/dam/sigma-aldrich/life-science/custom-dna-probes/scorpions-probes.jpg

Scorpion Probe:• Signal is produced by hybridization

to the newly synthesized strand

Hybridization Probe:• Signal is produced by FRET between

two neighboring probes.

http://wiki.biomine.skelleftea.se/biomine/molecular/index_25.htm

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Genome Size Quantification

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Jochen Wilhelm et al. Nucl. Acids Res. 2003;31:e56 ©2003 by Oxford University Press

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Digital PCR

# 1918.01.2016http://strategy.gene-quantification.info/

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# 2018.01.2016http://strategy.gene-quantification.info/

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Pyrosequencing

# 2118.01.2016https://www.qiagen.com/de/resources/technologies/pyrosequencing-resource-center/technology-overview/

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Next Generation Sequencing

Sequencing by Synthesis

• Solexa/Illumina

• Bridge amplification

• cleavable dye terminator

• SOLiD

• Emulsion PCR amplification

• Sequencing by Ligation

• Random octamer probes

• Pyrosequencing

• Emulsion PCR amplification

• Semiconductor sequencing

• Emulsion PCR amplification

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Bridge Amplification

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Sequencing by Synthesis

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Emulsion PCR

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SOLiD Sequencing by Ligation

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http://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/solid-next-generation-sequencing/next-generation-systems/solid-sequencing-chemistry.html

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SOLiD data assembly

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http://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/solid-next-generation-sequencing/next-generation-systems/solid-sequencing-chemistry.html

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Quantification from read numbers

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