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METHODS IN MOLECULAR BIOLOGY™

Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK

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sdfsdf
 Molecular Methods
for Evolutionary Genetics
Edited by

Virginie Orgogozo
CNRS UMR7592, Université Paris VII, Paris, France

Matthew V. Rockman
Department of Biology and Center for Genomics and Systems Biology,
New York University, New York, NY, USA
Editors
Virginie Orgogozo Matthew V. Rockman
CNRS UMR7592 Department of Biology and
Université Paris VII Center for Genomics and Systems Biology
15 rue Hélène Brion New York University
75013 Paris New York, NY 10003-6688, USA
France [email protected]
[email protected]

ISSN 1064-3745 e-ISSN 1940-6029

ISBN 978-1-61779-227-4 e-ISBN 978-1-61779-228-1
DOI 10.1007/978-1-61779-228-1
Springer New York Dordrecht Heidelberg London

Library of Congress Control Number: 2011934052

© Springer Science+Business Media, LLC 2011

All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the
publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA),
except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information
storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or
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as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights.

Printed on acid-free paper

Humana Press is part of Springer Science+Business Media (www.springer.com)

Preface

Rapid technological progress is transforming the investigation of genetic variation within

and between species. We are entering a particularly fruitful period in evolutionary genetics,
as two of its main goals – reconstruction of population histories and identification of the
mutations responsible for evolutionary changes in phenotypes – have now turned into trac-
table problems.
This book is a collection of molecular biology protocols and general overviews intended
to represent the essential methods currently bringing evolutionary genetics to fruition. It
begins with methods for the basic characterization of an unknown genome and ends with
the functional dissection of individual allelic variants. We chose to focus on protocols that
are widely applicable to all species and that require, when possible, little money and equip-
ment. Part I includes two essential traditional methods for characterizing genomes, genome
size determination and chromosomal analysis. It also describes the construction, screening
and sequencing of genomic libraries, a classic tool for genome walking and for obtaining
the DNA sequence of a genomic region of interest. Part II describes various approaches to
enrich DNA for subsets of the genome prior to sequencing, including methods for isolating
and sequencing the transcriptome. Even as next-generation sequencing technologies are
rapidly improving in terms of efficiency and cost, sequencing targeted regions of the genome
is likely to remain a compelling strategy, particularly in non-model organisms and popula-
tion samples. Part III contains five powerful protocols for sampling genetic variation for
genetic mapping studies and for population genetic studies. Many evolutionary studies
aiming at identifying the mutations responsible for evolutionary change converge on
genomic regions containing one or several candidate genes that must be tested and vali-
dated. The last three parts of this book focus on such candidate gene studies. Part IV
describes three molecular biology methods that can be used to obtain the DNA sequence
of a region of interest: degenerate PCR, circular RACE and inverse PCR. Part V includes
protocols for measuring gene expression, including two allele-specific methods that can
identify cis-regulatory changes. In part VI, two general strategy chapters outline the main
steps for testing the functional import of candidate cis-regulatory and protein-coding muta-
tions. Testing a candidate mutation usually involves the construction of artificial DNA
sequences. Part VI includes several powerful methods for making these constructs, Gateway
cloning, PCR stitching, homologous recombination using yeast and fosmid recombineer-
ing in liquid culture. A functional assay applicable to many species, RNAi injection, is
described in the last chapter of our book.
It has been a real pleasure for us to work with the authors of this book. They have
shown an admirable willingness to share their knowledge and expertise. We deeply appreci-
ate their efforts to make their protocols widely accessible to our readers. We thank them all
for their work and their dedication.
We hope that this volume will provide a rich resource for biologists interested in evolu-
tion, whether they are specialists or beginners in molecular biology.

Paris, France Virginie Orgogozo

New York, NY, USA Matthew V. Rockman

v
sdfsdf
Contents

Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

PART I CHARACTERIZING THE GENOME

1 Genome Size Determination Using Flow Cytometry

of Propidium Iodide-Stained Nuclei . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Emily E. Hare and J. Spencer Johnston
2 Chromosome Analysis in Invertebrates and Vertebrates. . . . . . . . . . . . . . . . . . . . . 13
David M. Rowell, Shu Ly Lim, and Frank Grutzner
3 Genomic Libraries: I. Construction and Screening
of Fosmid Genomic Libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Mike A. Quail, Lucy Matthews, Sarah Sims, Christine Lloyd,
Helen Beasley, and Simon W. Baxter
4 Genomic Libraries: II. Subcloning, Sequencing,
and Assembling Large-Insert Genomic DNA Clones . . . . . . . . . . . . . . . . . . . . . . 59
Mike A. Quail, Lucy Matthews, Sarah Sims, Christine Lloyd,
Helen Beasley, and Simon W. Baxter

PART II TARGETING REGIONS OF THE GENOME

5 Reduced Representation Methods for Subgenomic

Enrichment and Next-Generation Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Jeffrey M. Good
6 Accessing the Transcriptome: How to Normalize mRNA Pools . . . . . . . . . . . . . . 105
Heiko Vogel and Christopher W. Wheat
7 Transcriptome Sequencing Goals, Assembly, and Assessment . . . . . . . . . . . . . . . . 129
Christopher W. Wheat and Heiko Vogel
8 Rapid Retrieval of DNA Target Sequences by Primer Extension Capture. . . . . . . . 145
Adrian W. Briggs

PART III MEASURING GENETIC DIVERSITY

9 SNP Discovery and Genotyping for Evolutionary

Genetics Using RAD Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Paul D. Etter, Susan Bassham, Paul A. Hohenlohe, Eric A. Johnson,
and William A. Cresko

vii
viii Contents

10 DNA Microarray-Based Mutation Discovery and Genotyping. . . . . . . . . . . . . . . . 179

David Gresham
11 Genotyping with Sequenom . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
Martina Bradić , João Costa, and Ivo M. Chelo
12 Isolating Microsatellite Loci: Looking Back, Looking Ahead. . . . . . . . . . . . . . . . . 211
José A. Andrés and Steven M. Bogdanowicz
13 Design of Custom Oligonucleotide Microarrays for Single
Species or Interspecies Hybrids Using Array Oligo Selector . . . . . . . . . . . . . . . . . 233
Amy A. Caudy

PART IV OBTAINING CANDIDATE GENE SEQUENCES

14 Identification of Homologous Gene Sequences

by PCR with Degenerate Primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
Michael Lang and Virginie Orgogozo
15 Characterizing cDNA Ends by Circular RACE . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
Patrick T. McGrath
16 Identification of DNA Sequences that Flank a Known
Region by Inverse PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
Anastasios Pavlopoulos

PART V ANALYZING CANDIDATE GENE TRANSCRIPTS

17 Quantification of Transcript Levels with Quantitative RT-PCR . . . . . . . . . . . . . . . 279

Karen L. Carleton
18 Using Pyrosequencing to Measure Allele-Specific mRNA Abundance
and Infer the Effects of Cis- and Trans-regulatory Differences . . . . . . . . . . . . . . . 297
Patricia J. Wittkopp
19 Whole-Mount In Situ Hybridization of Sectioned Tissues of Species
Hybrids to Detect Cis-regulatory Changes in Gene Expression Pattern. . . . . . . . . 319
Ryo Futahashi
20 Identifying Fluorescently Labeled Single Molecules
in Image Stacks Using Machine Learning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329
Scott A. Rifkin

PART VI TESTING CANDIDATE GENES AND CANDIDATE MUTATIONS

21 Experimental Approaches to Evaluate the Contributions of

Candidate Cis-regulatory Mutations to Phenotypic Evolution. . . . . . . . . . . . . . . . 351
Mark Rebeiz and Thomas M. Williams
22 Experimental Approaches to Evaluate the Contributions of
Candidate Protein-Coding Mutations to Phenotypic Evolution . . . . . . . . . . . . . . 377
Jay F. Storz and Anthony J. Zera
23 Making Reporter Gene Constructs to Analyze Cis-regulatory Elements . . . . . . . . 397
José Bessa and José Luis Gómez-Skarmeta
 Contents ix

24 PCR-Directed In Vivo Plasmid Construction

Using Homologous Recombination in Baker’s Yeast. . . . . . . . . . . . . . . . . . . . . . . 409
Erik C. Andersen
25 Production of Fosmid Genomic Libraries Optimized for Liquid Culture
Recombineering and Cross-Species Transgenesis. . . . . . . . . . . . . . . . . . . . . . . . . . 423
Radoslaw Kamil Ejsmont, Maria Bogdanzaliewa,
Kamil Andrzej Lipinski, and Pavel Tomancak
26 Recombination-Mediated Genetic Engineering of Large
Genomic DNA Transgenes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 445
Radoslaw Kamil Ejsmont, Peter Ahlfeld, Andrei Pozniakovsky,
A. Francis Stewart, Pavel Tomancak, and Mihail Sarov
27 Overlap Extension PCR: An Efficient Method for Transgene Construction. . . . . . 459
Matthew D. Nelson and David H.A. Fitch
28 Gene Knockdown Analysis by Double-Stranded RNA Injection . . . . . . . . . . . . . . 471
Benjamin N. Philip and Yoshinori Tomoyasu
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499
sdfsdf
Contributors

PETER AHLFELD s Max Planck Institute of Molecular Cell Biology and Genetics,
Dresden, Germany
ERIK C. ANDERSEN s Lewis-Sigler Institute for Integrative Genomics,
Princeton University, Princeton, NJ, USA
JOSÉ A. ANDRÉS s Department of Biology, University of Saskatchewan, Saskatoon,
SK, Canada
SUSAN BASSHAM s Center for Ecology and Evolutionary Biology, University of Oregon,
Eugene, OR, USA
SIMON W. BAXTER s Department of Zoology, University of Cambridge, Cambridge, UK
HELEN BEASLEY s Sequencing, DNA Pipelines, Wellcome Trust Sanger Institute,
Cambridge, UK
JOSÉ BESSA s Centro Andaluz de Biología del Desarrollo (CABD),
CSIC-Universidad Pablo de Olavide, Seville, Spain
STEVEN M. BOGDANOWICZ s Department of Ecology and Evolutionary Biology,
Cornell University, Ithaca, NY, USA
MARIA BOGDANZALIEWA s Max Planck Institute of Molecular Cell Biology and Genetics,
Dresden, Germany
MARTINA BRADIÃ s Department of Biology, New York University, New York, NY, USA
ADRIAN W. BRIGGS s Department of Genetics, Harvard Medical School, Boston,
MA, USA
KAREN L. CARLETON s University of Maryland, College Park, MD, USA
AMY A. CAUDY s Lewis-Sigler Institute for Integrative Genomics, Princeton University,
Princeton, NJ, USA
IVO M. CHELO s Instituto Gulbenkian de Ciência, Oeiras, Portugal
JOÃO COSTA s Instituto Gulbenkian de Ciência, Oeiras, Portugal
WILLIAM A. CRESKO s Center for Ecology and Evolutionary Biology,
University of Oregon, Eugene, OR, USA
RADOSLAW KAMIL EJSMONT s Max Planck Institute of Molecular Cell Biology
and Genetics, Dresden, Germany
PAUL D. ETTER s Institute of Molecular Biology, University of Oregon, Eugene,
OR, USA
DAVID H. A. FITCH s Department of Biology, New York University, New York,
NY, USA
RYO FUTAHASHI s Bioproduction Research Institute, National Institute of Advanced
Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan
JOSÉ LUIS GÓMEZ-SKARMETA s Centro Andaluz de Biología del Desarrollo (CABD),
CSIC-Universidad Pablo de Olavide, Seville, Spain
JEFFREY M. GOOD s Division of Biological Sciences, University of Montana, Missoula,
MT, USA

xi
xii Contributors

DAVID GRESHAM s Department of Biology and Center for Genomics and Systems Biology,
New York University, New York, NY, USA
FRANK GRUTZNER s School of Molecular & Biomedical Science,
The University of Adelaide, SA, Australia
EMILY E. HARE s Locus of Development, Inc., San Francisco, CA, USA
PAUL A. HOHENLOHE s Center for Ecology and Evolutionary Biology,
University of Oregon, Eugene, OR, USA
ERIC A. JOHNSON s Institute of Molecular Biology, University of Oregon, Eugene,
OR, USA
J. SPENCER JOHNSTON s Department of Entomology, Texas A&M University, College
Station, Austin, TX, USA
MICHAEL LANG s CNRS, Institut Jacques Monod, Paris, France
SHU LY LIM s School of Molecular & Biomedical Science,
The University of Adelaide, SA, Australia
KAMIL ANDRZEJ LIPINSKI s Max Planck Institute of Molecular Cell Biology and Genetics,
Dresden, Germany
CHRISTINE LLYOD s Sequencing, DNA Pipelines, Wellcome Trust Sanger Institute,
Cambridge, UK
LUCY MATTHEWS s Sequencing, DNA Pipelines, Wellcome Trust Sanger Institute,
Cambridge, UK
PATRICK T. MCGRATH s The Rockefeller University, New York, NY, USA
MATTHEW D. NELSON s Department of Biology, New York University, New York,
NY, USA
VIRGINIE ORGOGOZO s CNRS UMR7592, Université Paris VII, Paris, France
ANASTASIOS PAVLOPOULOS s Department of Zoology, University Museum
of Zoology, Laboratory for Development and Evolution, University
of Cambridge, Cambridge, UK
BENJAMIN N. PHILIP s Department of Biology, Rivier College, Nashua, NH, USA
ANDREI POZNIAKOVSKY s Max Planck Institute of Molecular Cell Biology and Genetics,
Dresden, Germany
MIKE A. QUAIL s Sequencing Research and Development, Wellcome Trust
Sanger Institute, Cambridge, UK
MARK REBEIZ s Department of Biological Sciences, University of Pittsburgh,
Pittsburgh, PA, USA
SCOTT A. RIFKIN s Division of Biological Sciences, Section of Ecology, Behavior
and Evolution, University of California, San Diego, CA, USA
MATTHEW V. ROCKMAN s Department of Biology and Center for Genomics and Systems
Biology, New York University, New York, USA
DAVID M. ROWELL s Department of Evolution, Ecology and Genetics, Research
School of Biology, Australian National University, Canberra, ACT, Australia
MIHAIL SAROV s Max Planck Institute of Molecular Cell Biology and Genetics,
Dresden, Germany
SARAH SIMS s Sequencing, DNA Pipelines, Wellcome Trust Sanger Institute,
Cambridge, UK
A. FRANCIS STEWART s Max Planck Institute of Molecular Cell Biology and Genetics,
Dresden, Germany
 Contributors xiii

JAY F. STORZ s School of Biological Sciences, University of Nebraska, Lincoln, NE, USA
PAVEL TOMANCAK s Max Planck Institute of Molecular Cell Biology and Genetics,
Dresden, Germany
YOSHINORI TOMOYASU s Department of Zoology, Miami University, Oxford, OH, USA
HEIKO VOGEL s Department of Entomology, Max Planck Institute for Chemical Ecology,
Jena, Germany
CHRISTOPHER W. WHEAT s Department of Biological and Environmental Sciences,
University of Helsinki, Helsinki, Finland;Centre for Ecology and Conservation, School
of Biosciences, University of Exeter, Cornwall Campus, UK
THOMAS M. WILLIAMS s Department of Biology, University of Dayton,
Dayton, OH, USA
PATRICIA J. WITTKOPP s University of Michigan, Ann Arbor, MI, USA
SHOZO YOKOYAMA s Emory University, Atlanta, GA, USA
ANTHONY J. ZERA s School of Biological Sciences, University of Nebraska,
Lincoln, NE, USA
sdfsdf
Abbreviations

aDNA Ancient DNA

Ara Arabinose
BAC Bacterial artificial chromosome
BSA Bovine serum albumin
CHEF Clamped homogeneous electric field
Cm Chloramphenicol
CRE Cis-regulatory element
ddH2O Double-distilled water
DMSO Dimethylsulfoxide
DSN Duplex-specific nuclease
EDTA Ethylene diamine tetraacetic acid
EGFP Enhanced green fluorescent protein
FCS Foetal calf serum
FRT Flippase recombination target
HBSS Hank’s balanced saline solution
HCS Head capsule slippage
HMP High melting point
iPCR Inverse PCR
LB Luria-Bertani broth
LMP Low melting point
OTE Off-target effect
PBS Phosphate-buffered saline
PCR Polymerase chain reaction
PFGE Pulse-field gel electrophoresis
PI Propidium iodide
RISC RNA-induced silencing complex
RT-PCR Reverse transcription-polymerase chain reaction
SDS Sodium dodecyl sulfate
SEM Scanning electron microscopy
SOC Super optimal broth with catabolite repression
SRD Sequencing reaction diluent
ss single stranded
SSC Saline-sodium citrate
SSH Sonic hedgehog

xv
xvi Abbreviations

TBE Tris/borate/EDTA
TE Tris/EDTA
UV UltraViolet
qRT-PCR Quantitative reverse transcription-polymerase chain reaction
siRNA Small interfering RNA
YENB Yeast extract, nutrient broth
v Volume
vol Volume
w Weight
 Part I

Characterizing the Genome

 Chapter 1

Genome Size Determination Using Flow Cytometry

of Propidium Iodide-Stained Nuclei
Emily E. Hare and J. Spencer Johnston

Abstract
With the rapid expansion of whole-genome sequencing and other genomic studies in nonmodel ­organisms,
there is a growing demand for robust and user-friendly methods for estimating eukaryotic genome sizes
across a broad range of taxa. Propidium iodide (PI) staining with flow cytometry is a powerful method for
genome sizing because it is relatively fast, works with a wide variety of materials, and provides information
on a very large number of nuclei. In this method, nuclei are stained with PI, which intercalates into the
major groove of DNA. Unknown samples are typically costained with standard nuclei of a known genome
size, and the relative fluorescence is used to calculate the genome size of the unknown.

Key words: Genome size, Propidium iodide, Flow cytometry, C-value

1. Introduction

With the availability of genome sizes for thousands of eukaryotic

organisms determined during the last 50 years, a fascinating pic-
ture is emerging regarding the function of and constraints on
genome size. Genome size has been shown to correlate with
numerous organismal characteristics, including egg size and devel-
opmental patterning mechanisms (1), cell size, mitotic and meiotic
cell division rates, deletion rate, developmental rate, and body size
(reviewed in (2)). However, the full spectrum of functional con-
straints on genome size is not yet understood and closely related
taxa can have significantly different genome sizes, as in the
Drosophila melanogaster subgroup species, D. simulans and
D. orena, with 150 and 275 Mb genomes, respectively (3, 4).
Lynch (5) has suggested that correlations may be due, not to direct
genome size effects, but rather to differential responses to the

Virginie Orgogozo and Matthew V. Rockman (eds.), Molecular Methods for Evolutionary Genetics,
Methods in Molecular Biology, vol. 772, DOI 10.1007/978-1-61779-228-1_1, © Springer Science+Business Media, LLC 2011

3
4 E.E. Hare and J.S. Johnston

mutational hazard of mobile elements that is associated with

species level effective population size differences. Ongoing research
is addressing these and other questions in the field of genome-size
evolution: What is the origin of noncoding DNA in eukaryotes,
and what are the mechanisms for its expansion and reduction
during evolution? What are the functions of this noncoding DNA,
and why do some taxa have vast amounts of it while others have
relatively little?
The substantial variation in genome sizes across even closely
related taxa has significant implications for the rapidly expanding
field of whole-genome sequencing and other genomic techniques,
including the screening of whole-genome libraries. An accurate
measurement of genome size is a necessary prerequisite for esti-
mating clone coverage in shotgun sequencing and library screen-
ing and is essential for estimating the necessary read coverage for
next-generation sequencing technologies. Thus, there is a high
demand for robust and user-friendly methods for estimating
genome sizes across a broad range of eukaryotic taxa.
The most commonly used methods for current genome sizing
in eukaryotes are densitometry and flow cytometry. The first
method utilizes Feulgen staining, in which nuclei are stained with
Schiff’s reagent, which stains DNA magenta. The intensity of
magenta stain is measured with a densitometer, thus quantifying
the amount of DNA in the nuclei. In the second method, nuclei
are stained with propidium iodide (PI), which intercalates into the
major groove of DNA and RNA without sequence preference,
with one dye molecule per 4–5 bp (6). This binding enhances the
fluorescence of PI by 20- to 30-fold, and the excitation and emis-
sion maxima are shifted (6). Unknown samples are typically
costained with standard nuclei of a known genome size, RNA is
removed by RNase, and the relative fluorescence of the DNA in
the unknown and standard is used to calculate the genome size of
the unknown. Relative fluorescence is quantified in a flow cytom-
eter with a laser tuned to 488 or 514 nm (see Fig. 1).
PI staining with flow cytometry has the advantage that it is
relatively fast, works with a wide variety of materials and provides
information on a very large number of nuclei. A potential limita-
tion of PI staining is that inhibitors and possibly chromatin con-
densation can bias the result (7). This limitation can be minimized
by suitable choice of run conditions, the most important of which
is the use of an internal standard that is coprepared with the ­sample.
Attempts to use an external standard have led to estimates as low
as 30% of the true genome size value (7). Feulgen densitometry,
because it uses acid hydrolysis, is less sensitive to inhibitors and to
chromatin condensation, but is not entirely free from inhibitor
bias. Good practice, as described in (7, 8), remains the best protec-
tion against bias.
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