analysis of nucleic acids - carell group€¦ · detection and analysis of nucleic acids nucleic...

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

# 204.12.2017Markus Müller

Detection and Analysis of Nucleic Acids


Oligomer Quantification

# 404.12.2017TriLink Biotechnologies website: http://www.trilinkbiotech.com/tech/extinction_intro.asp

E = ε ∙ c ∙ d

Polymer Quantification

# 504.12.2017

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


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


SYBR Green

# 804.12.2017

• 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

# 904.12.2017

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


Minor groove binder


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


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


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.


Relative Quantification

# 1504.12.2017© 2004 Bio-Rad Laboratories

Probe Types


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

# 1704.12.2017

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


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


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


Illumina Chemistry


Emulsion PCR

# 2804.12.2017http://users.ugent.be/~avierstr/nextgen/nextgen.html

SOLiD Sequencing by Ligation

# 2904.12.2017

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

SOLiD data assembly


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


analysis of nucleic acids - carell group€¦ · detection and analysis of nucleic acids nucleic...

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