analysis of nucleic acids - carell group€¦ · detection and analysis of nucleic acids nucleic...
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Markus Müller
T1OM-M: Chemical Biology“Basics of Cloning , Genomics and Proteomics“
Analysis of Nucleic Acids
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
# 404.12.2017TriLink Biotechnologies website: http://www.trilinkbiotech.com/tech/extinction_intro.asp
E = ε ∙ c ∙ d
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
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
Ethidium and Propidium
# 712/4/2017
• 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
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
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
Minor groove binder
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DAPI
Hoechst 33258
• Minor groove binder• Prefer AT-rich regions• UV-excitation • Not or badly cell permeant
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
# 1204.12.2017http://www.thermofisher.com/de/de/home/references/molecular-probes-the-handbook/reagents-for-modifying-groups-other-than-thiols-or-amines/click-chemistry.html
Quantitative PCR
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0
500000
1000000
1500000
2000000
2500000
3000000
0 5 10 15 20 25
Sig
nal i
nten
sity
Cycle
exponential amplification
one copy
two copies
five copies
ten copies
Calibration
Absolute Quantification
• Dilution series of reference material
Relative Quantification
• Use of „housekeeping“ genes.
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Relative Quantification
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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
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
Genome Size Quantification
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Jochen Wilhelm et al. Nucl. Acids Res. 2003;31:e56 ©2003 by Oxford University Press
Digital PCR
# 1904.12.2017http://strategy.gene-quantification.info/
# 2004.12.2017http://strategy.gene-quantification.info/
Pyrosequencing
# 2104.12.2017https://www.qiagen.com/de/resources/technologies/pyrosequencing-resource-center/technology-overview/
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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Library Preparation – First Strand
# 2304.12.2017NEB & RNA-seqlopedia (https://rnaseq.uoregon.edu)
- High quality RNA is required- RNA is fragmented to 100 – 300 bases to allow
complete sequencing- Random priming for reverse transcriptase ensures
unbiased cDNA synthesis
cDNA can also be synthesized from adapters:
Library Preparation – Second Strand
# 2404.12.2017NEB & RNA-seqlopedia (https://rnaseq.uoregon.edu)
- RNase H nicks the RNA andcreate priming-sites
- DNA polymerase fills-up the gaps
- Ligase closes the nicks
- A-tailing allows sticky-end ligation of the adapters
Different technology can ensure that asymmetric molecules are produced for specific amplification. Left: cleavable hairpins (NEB), right: Y-shaped adapters (Illumina)
Bridge Amplification
# 2504.12.2017Illumina Inc.
Sequencing by Synthesis
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Illumina Chemistry
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Emulsion PCR
# 2804.12.2017http://users.ugent.be/~avierstr/nextgen/nextgen.html
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
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
Single-Molecule-Real-Time sequencing
Phosphor-labelled Nucleotides
Nanopore Sequencing
# 3304.12.2017http://www2.technologyreview.com/news/427677/nanopore-sequencing/
Nanopore Sequencing
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