Ancient DNA: Methods and Protocols (2 page)

We express our sincere thanks to all authors for their willingness to share their time and their trade secrets, and to Prof. John Walker at Humana Press for giving us the opportunity to assemble this collection of protocols.

Santa Cruz, CA, USA

Beth Shapiro

York, UK

Michael Hofreiter

Contents

Preface
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

v

Contributors
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xi

1 Setting Up an Ancient DNA Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

Tara L. Fulton

2 A Phenol–Chloroform Protocol for Extracting DNA

from Ancient Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13

Ross Barnett and Greger Larson

3 DNA Extraction of Ancient Animal Hard Tissue Samples

via Adsorption to Silica Particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

21

Nadin Rohland

4 Case Study: Recovery of Ancient Nuclear DNA from Toe Pads of the Extinct Passenger Pigeon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

29

Tara L. Fulton, Stephen M. Wagner, and Beth Shapiro

5 Extraction of DNA from Paleofeces. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

37

Melanie Kuch and Hendrik Poinar

6 DNA Extraction from Keratin and Chitin . . . . . . . . . . . . . . . . . . . . . . . . . . . .

43

Paula F. Campos and Thomas M.P. Gilbert

7 Case Study: Ancient Sloth DNA Recovered from Hairs

Preserved in Paleofeces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

51

Andrew A. Clack, Ross D.E. MacPhee, and Hendrik N. Poinar

8 Ancient DNA Extraction from Soils and Sediments . . . . . . . . . . . . . . . . . . . . .

57

James Haile

9 DNA Extraction from Fossil Eggshell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

65

Charlotte L. Oskam and Michael Bunce

10 Ancient DNA Extraction from Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

71

Logan Kistler

11 DNA Extraction from Formalin-Fixed Material . . . . . . . . . . . . . . . . . . . . . . . .

81

Paula F. Campos and Thomas M.P. Gilbert

12 Case Study: Ancient DNA Recovered from Pleistocene-Age Remains of a Florida Armadillo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

87

Brandon Letts and Beth Shapiro

13 Nondestructive DNA Extraction from Museum Specimens . . . . . . . . . . . . . . .

93

Michael Hofreiter

14 Case Study: Using a Nondestructive DNA Extraction

Method to Generate mtDNA Sequences from Historical

Chimpanzee Specimens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

Elmira Mohandesan, Stefan Prost, and Michael Hofreiter

ix

x

Contents

15 PCR Amplification, Cloning, and Sequencing of Ancient DNA . . . . . . . . . . . . 111

Tara L. Fulton and Mathias Stiller

16 Quantitative Real-Time PCR in aDNA Research . . . . . . . . . . . . . . . . . . . . . . . 121

Michael Bunce, Charlotte L. Oskam, and Morten E. Allentoft

17 Multiplex PCR Amplification of Ancient DNA . . . . . . . . . . . . . . . . . . . . . . . . 133

Mathias Stiller and Tara L. Fulton

18 Preparation of Next-Generation Sequencing Libraries

from Damaged DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

Adrian W. Briggs and Patricia Heyn

19 Generating Barcoded Libraries for Multiplex

High-Throughput Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

Michael Knapp, Mathias Stiller, and Matthias Meyer

20 Case Study: Targeted high-Throughput Sequencing of Mitochondrial Genomes from Extinct Cave Bears via Direct Multiplex

PCR Sequencing (DMPS). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171

Mathias Stiller

21 Target Enrichment via DNA Hybridization Capture . . . . . . . . . . . . . . . . . . . . 177

Susanne Horn

22 Case Study: Enrichment of Ancient Mitochondrial DNA

by Hybridization Capture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189

Susanne Horn

23 Analysis of High-Throughput Ancient DNA Sequencing Data. . . . . . . . . . . . . 197

Martin Kircher

24 Phylogenetic Analysis of Ancient DNA using
BEAST
. . . . . . . . . . . . . . . . . . . . 229

Simon Y.W. Ho

Index
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
243

Contributors

ADRIAN W. BRIGGS
• Department of Genetics , Harvard Medical School , 77 Avenue Louis Pasteur , Boston 02115 , MA , USA

ANDREW A. CLACK
• McMaster Ancient DNA Center, McMaster University , 1280 Main Street West Hamilton , ON , Canada L8S 4L9 ; Department of Biology , The Pennsylvania State University , 326 Mueller Laboratory, University Park , PA 16802 , USA

BETH SHAPIRO
• Department of Ecology and Evolutionary Biology , University of California Santa Cruz , A414 Earth & Marine Sciences, Santa Cruz , CA 95064 , USA

BRANDON LETTS
• Department of Biology , The Pennsylvania State University , 320 Mueller Laboratory, University Park , PA 16802 , USA CHARLOTTE L. OSKAM
• Ancient DNA Laboratory , School of Biological Sciences and Biotechnology, Murdoch University , South Street , Perth 6150 , WA , Australia ELMIRA MOHANDESAN
• Allan Wilson Centre for Molecular Ecology and Evolution, Institute of Natural Sciences, Massey University , Private Bag 102904 NSMC , Auckland , New Zealand

GREGER LARSON
• Department of Archaeology , Durham University , South Road, Durham DH1 3LE , UK

HENDRIK POINAR
• McMaster Ancient DNA Centre, McMaster University , Hamilton , ON , Canada

JAMES HAILE
• Ancient DNA Laboratory , School of Biological Sciences and Biotechnology, Murdoch University , South Street , Perth 6150 , WA , Australia LOGAN KISTLER
• Department of Anthropology , The Pennsylvania State University , 409 Carpenter Building, University Park , PA 16802 , USA MARTIN KIRCHER
• Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology , Deutscher Platz 6 , D-04103 Leipzig , Germany MATTHIAS MEYER
• Max Planck Institute for Evolutionary Anthropology , Deutscher Platz 6, 04103 Leipzig , Germany

MATHIAS STILLER
• Department of Biology , The Pennsylvania State University , 320 Mueller Laboratory , University Park , PA 16802 , USA MELANIE KUCH
• McMaster Ancient DNA Centre, McMaster University , Hamilton , ON , Canada

MICHAEL BUNCE
• Ancient DNA Laboratory , School of Biological Sciences and Biotechnology, Murdoch University , South Street, Perth 6150 , WA , Australia MICHAEL HOFREITER
• Department of Biology , The University of York , Wentworth Way, Heslington , York YO10 5DD , UK

MICHAEL KNAPP
• Allan Wilson Centre for Molecular Ecology and Evolution, Department of Anatomy and Structural Biology , University of Otago , Dunedin 9016 , New Zealand

xi

xii

Contributors

MORTEN E. ALLENTOFT
• Ancient DNA Laboratory , School of Biological Sciences and Biotechnology, Murdoch University , South Street , Perth 6150 , WA , Australia NADIN ROHLAND
• Department of Genetics , Harvard Medical School , 77 Avenue Louis Pasteur, Boston , MA 02115 , USA PATRICIA HEYN
• Max Planck Institute of Molecular Cell Biology and Genetics , Pfotenhauerstrasse 108 , 01307 Dresden , Germany

PAULA F. CAMPOS
• Natural History Museum of Denmark, University of Copenhagen , Østervoldgade 5-7 1350 , Copenhagen , Denmark

ROSS BARNETT
• Department of Archaeology , Durham University , South Road , Durham DH1 3LE , UK

ROSS D. E. MACPHEE
• American Museum of Natural History , New York , NY 10024 , USA

SIMON Y. W. HO
• School of Biological Sciences, University of Sydney , Sydney, 2006 NSW , Australia

STEFAN PROST
• Allan Wilson Centre for Molecular Ecology and Evolution, Department of Anatomy and Structural Biology , University of Otago , Dunedin , New Zealand ; Department of Integrative Biology , University of California , Berkeley , CA , USA

SUSANNE HORN
• Max Planck Institute for Evolutionary Anthropology, Germany and German Cancer Research Center (DKFZ) , Heidelberg , Germany STEPHEN M. WAGNER
• Department of Biology , The Pennsylvania State University , 320 Mueller Laboratory , University Park , PA 16802 , USA TARA L. FULTON
• Department of Biology , The Pennsylvania State University , 320 Mueller Laboratory, University Park , PA 16802 , USA THOMAS M.P. GILBERT
• Natural History Museum of Denmark, University of Copenhagen , Østervoldgade 5-7, DK 1350 , Copenhagen , Denmark

Setting Up an Ancient DNA Laboratory

Tara L. Fulton

Abstract

Entering into the world of ancient DNA research is nontrivial. Because the DNA in most ancient specimens is degraded to some extent, the potential for contamination of ancient samples and DNA extracts with modern DNA is considerable. To minimize the risk associated with working with ancient DNA, experimental protocols specifi c to handling ancient specimens have been introduced. Here, I outline the challenges associated with working with ancient DNA and describe guidelines for setting up a new ancient DNA laboratory. I also discuss steps that can be taken at the sample collection and preparation stage to minimize the potential for contamination with exogenous sources of DNA.

Key words:
Ancient DNA , aDNA , DNA damage , Laboratory setup , Contamination , Subsampling , Sample preparation , Guidelines

1. Introduction

The fi eld of ancient DNA (aDNA) was born in 1984, when DNA sequences were successfully recovered from the extinct quagga, a relative of the zebra
( 1 )
. With the advent of the polymerase chain reaction (PCR)
( 2 )
, the fi eld began to take shape
( 3
) and has taken off during the last two decades. The power of aDNA is that it offers a window into the past that modern DNA or paleontological studies alone cannot provide. It has been widely adopted to address questions relating to, for example, the history and relationships of hominids
( 4
)
, plant and animal domestication ( 5– 8 )
, population dynamics and diversity through time
( 9– 13
) , and phylogenetics of extinct species
( 14– 16 )
. While aDNA can be a powerful tool, it is one that should be handled with caution.

Beth Shapiro and Michael Hofreiter (eds.),
Ancient DNA: Methods and Protocols
, Methods in Molecular Biology, vol. 840, DOI 10.1007/978-1-61779-516-9_1, © Springer Science+Business Media, LLC 2012

1

2

T.L. Fulton

1.1. Diffi culties

DNA is frequently damaged while the organism is alive, but this
of aDNA Work

damage is repaired via a suite of host repair mechanisms. DNA damage continues after death, but the repair pathways no longer 1.1.1. Postmortem

function. As a result, few intact copies of aDNA tend to survive in Degradation

old specimens, and those that remain are often highly fragmented and damaged (T
able 1
). Preservation in cold environments may slow or inhibit nuclease activity, reducing some of the damage that occurs immediately after death. However, environmental factors still work to cause DNA damage. Hydrolytic damage leads to single-strand breaks through direct cleavage or following depuri-nation, fragmenting the DNA. Hydrolysis can also induce miscoding lesions, most commonly the deamination of cytosine to uracil,
causing C–T transitions ( 17
)
. Oxidation can induce lesions ( 17,

18 )
that block polymerases and either stop amplifi cation or lead to
“jumping PCR” ( 3
) and the production of chimeric sequences.

Table 1

Types of ancient DNA damage

Damage type

Mechanisms

Effects

Solutions

Strand breaks Nuclease activity

Low quantity of

Amplify short

Microorganism degradation

surviving DNA;

(<100–300 bp)

Desiccation, heat, chemicals,
etc.

short fragment

overlapping fragments

Direct cleavage (hydrolysis)

length

Depurination causes a basic

site (hydrolysis)

Miscoding

Deamination causes