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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
# 218.01.2016Markus Müller
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Detection and Analysis of Nucleic Acids
# 318.01.2016Markus Müller
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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
# 518.01.2016
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
# 618.01.2016Markus Müller
Molecular Probes Handbook, Life Technologies, Chapter 8
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Ethidium and Propidium
# 71/18/2016
• 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
# 818.01.2016
• 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
# 918.01.2016
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
# 1018.01.2016Markus Müller
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
# 1118.01.2016Markus Müller
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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
# 1318.01.2016Markus Müller
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.
# 1418.01.2016Markus Müller
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Relative Quantification
# 1518.01.2016© 2004 Bio-Rad Laboratories
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Probe Types
# 1618.01.2016Markus Müller
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
# 1718.01.2016
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
# 1818.01.2016Markus Müller
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
# 2218.01.2016Markus Müller
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Bridge Amplification
# 2318.01.2016Illumina Inc.
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Sequencing by Synthesis
# 2418.01.2016Markus Müller
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Emulsion PCR
# 2518.01.2016http://users.ugent.be/~avierstr/nextgen/nextgen.html
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SOLiD Sequencing by Ligation
# 2618.01.2016
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
# 2718.01.2016Markus Müller
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
# 2818.01.2016Markus Müller