Read Ancient DNA: Methods and Protocols Online
Authors: Beth Shapiro
14 Case Study: Using a Nondestructive DNA Extraction Method…
107
Fig. 1. Geographical distribution of chimpanzee subspecies.
extractions and amplifi cations as well as obtaining multiple clonal sequences are an absolute requirement in such studies.
Chimpanzee subspecies are divided into two geographically
and genetically defi ned groups: a central/eastern African group (
P. t. schweinfurthii
and
P. t. troglodytes
) and a western African group (
P. t. verus
and
P. t. vellerosus
) with a signifi cant phylogeographic break at the Sanaga River in central Camer
oon (Fig. 1 ).
( 18
) reconstructed from our historical sequences and modern chimpanzee sequences obtained from
GenBank shows that all historical haplotypes are closely related to moder
n ones (Fig. 2
), although some of them have not (yet) been found in the extant gene pool.
With 51%, the DNA extraction success rate in this study is lower than in previous studies reporting the method
( 3, 19,
20 )
, but still suffi ciently high to obtain DNA from about half of the investigated specimens. Similarly, the length of the obtained PCR
products is large enough to obtain, by using several overlapping fragments, DNA sequences suffi ciently long for phylogeographic and phylogenetic analyses. However, the high incidence of contaminating sequences found also indicates that a substantial failure rate has to be taken into account when planning a study, although there seem to be large differences among collections and species, probably depending on storage and handling.
108
E. Mohandesan
et al.
2
1
1 1
1
1
1
1
1
1
1
1
1
2
1
1
1
1
1
1
1
1
1 1 1
1
1
2
1
3
1
1
1 1 1 1
2
1 1
1 1 1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
1
2
2
1
1
1
2
1
2
2
1
1
1
1
1
1
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
111
1
1
1
1
1
1
1
2
1
1
2
2
1
1
Fig. 2. Temporal statistical parsimony network of modern and ancient chimp sequences. The upper layer comprises modern-day sequences obtained from GenBank, whereas the lower layer consists of ancient DNA samples generated in this study.
Haplotypes sampled in a given time layer are represented as
gray ellipses
. Those present in the overall network, but not in the individual time layer are shown as
small white ellipses
. Haplotypes shared between the two layers are connected by vertical lines . Haplotypes present in a time-horizon are connected by
solid lines
, whereas lines connecting at least one unsampled haplotype for this time-horizon are
dotted
. Those separated by more than one mutation are indicated by one small black circle for each additional mutation. Please note that for graphical reasons, not all modern sequences available were used in the network. Therefore, a larger proportion of museum sequences than shown in this fi gure are actually still present in the modern population.
Both success rate and total length of the DNA sequences that can be obtained should increase considerably when using DNA hybridization capture methods
( 22– 25
) rather than PCR for targeting specifi c DNA regions. These methods have recently been used successfully for targeting both mitochondrial (up to complete mtDNA genomes
( 25, 26
) ) and nuclear DNA
( 27
)
.
Due their ability to target very short DNA fragments, they are ideally suited for the analysis of fragmented DNA such as that recovered from museum specimens. It needs to be noted, though, that measures used to distinguish endogenous ancient DNA obtained from
Pleistocene specimens from contaminating modern DNA such as fragmentation or nucleotide substitution patterns may not be applicable to museum specimen DNA for several reasons. First, due to their younger age, museum specimens may not have accumulated DNA damage to the extent that fossil DNA dating to the Pleistocene has. Second, and perhaps even more importantly, the sequences contaminating museum specimens probably originate 14 Case Study: Using a Nondestructive DNA Extraction Method…
109
quite frequently from cross-contamination with DNA from other museum specimens, which is likely to display highly similar damage patterns. However, as our results show, this problem can be addressed at least partially by performing two consecutive extractions and by preferential use of the second extract.
Acknowledgments
We thank the lab members of the Research Group Molecular
Ecology at the Max Planck Institute for Evolutionary Anthropology, and especially Tim Heupink, for their assistance in laboratory work, museum curators for providing us with chimpanzee samples, and the Max Planck Society for fi nancial support.
References
1. Krings M, Stone A, Schmitz RW et al (1997)
Nigeria and Cameroon: a multilocus analysis.
Neandertal DNA sequences and the origin of
In: Lehman S, Fleagle J (eds) Primate biogeog—
modern humans. Cell 90:19–30
raphy. Springer, New York, pp 129–161
2. Hadly EA, Kohn MH, Leonard JA et al (1998)
11. Gonder MK, Disotell T, Oates JF (2006) New
A genetic record of population isolation in pocket
genetic evidence on the evolution of chimpan—
gophers during Holocene climatic change. Proc
zee populations and implications for taxonomy.
Natl Acad Sci USA 95:6893–6896
Int J Primatol 27:1103–1127
3. Rohland N, Siedel H, Hofreiter M (2004) 12. Becquet C, Patterson N, Stone AC et al (2007) Nondestructive DNA extraction method for
Genetic structure of chimpanzee populations.
mitochondrial DNA analyses of museum speci—
PLoS Genet 3:e66
mens. Biotechniques 36(814–816):818–821
13. Campbell G, Kuehl H, N’Goran KP et al
4. Schwarz E (1934) On the local races of chim-
(2008) Alarming decline of West African chimpanzees. Ann Mag Nat Hist Lond 13:
panzees in Côte d’Ivoire. Curr Biol
576–583
18:903–904
5. Hill WCO (1969) In: Bourne GH (ed) The 14. Walsh PD, Abernethy KA, Bermejo M et al chimpanzee; a series of volumes on the chim-
(2003) Catastrophic ape decline in western
panzee, vol. 1. S. Karger AG, Basel, NY, pp.
equatorial Africa. Nature 422:611–614
22–49
15. Greengrass E (2009) Chimpanzees are close to
6. Groves CP (2001) Primate taxonomy.
extinction in Southwest Nigeria. Prim Cons
Smithsonian Institution Press, Washington,
24:77–83
DC, 350p
16. Hall TA (1999) BioEdit: a user-friendly bio—
7. Gonder MK, Oates JF, Disotell TR et al (1997)
logical sequence alignment editor and analysis
A new West African chimpanzee subspecies?
program for Windows 95/98/NT. Nucl Acids
Nature 388:337
Symp Ser 41:95–98
8. Gonder MK, Disotell TR, Oates JF (2006) 17. Altschul SF, Madden TL, Schäffer AA et al New genetic evidence on the evolution of
(1997) Gapped BLAST and PSI-BLAST: a
chimpanzee populations and implications for
new generation of protein database search
taxonomy. Int J Primatol 27:1103–1127
programs (Review). Nucleic Acids Res 25:
9. Gonder MK (2000) Evolutionary genetics of
3389–3402
chimpanzees (
Pan troglodytes
) in Nigeria and 18. Prost S, Anderson CNK (2011) TempNet: a Cameroon. Ph.D. Dissertation, City University
method to display statistical parsimony net—
of New York, New York, 338pp
works for heterochronous DNA sequence data.
10. Gonder MK, Disotell TR (2006) Contrasting
Methods Ecol Evol 2:663–667. doi: 10.1111/
phylogeographic histories of chimpanzees in
j.2041-210X.2011.00129.x
110
E. Mohandesan
et al.
19. Asher RJ, Hofreiter M (2006) Tenrec phylog—
on custom-designed microarrays for massively
eny and the noninvasive extraction of nuclear
parallel sequencing. Nat Protoc 4:960–974
DNA. Syst Biol 55(2):181–194
24. Gnirke A, Melnikov A, Maguire J et al (2009)
20. Asher RJ, Maree S, Bronner G, Bennett NC,
Solution hybrid selection with ultra-long oligo—
Bloomer P, Czechowski P, Meyer M, Hofreiter
nucleotides for massively parallel targeted
M (2010) A phylogenetic estimate for golden
sequencing. Nat Biotechnol 27:182–189
moles (Mammalia, Afrotheria, Chryso—
25. Briggs AW, Good JM, Green RE et al (2009)
chloridae). BMC Evol Biol 10:69
Targeted retrieval and analysis of fi ve
21. Hofreiter M, Jaenicke V, Serre D, Haeseler Av
Neandertal mtDNA genomes. Science 325:
A, Pääbo S (2001) DNA sequences from
318–321
multiple amplifi cations reveal artifacts induced
26. Krause J, Briggs AW, Kircher M et al (2010) A
by cytosine deamination in ancient DNA.
complete mtDNA genome of an early modern
Nucleic Acids Res 29(23):4793–4799
human from Kostenki, Russia. Curr Biol
22. Hodges E, Xuan Z, Balija V et al (2007)
20:231–236
Genome-wide in situ exon capture for selective
27. Burbano HA, Hodges E, Green RE et al (2010)
resequencing. Nat Genet 39:1522–1527
Targeted investigation of the Neandertal
23. Hodges E, Rooks M, Xuan Z et al (2009)
genome by array-based sequence capture.
Hybrid selection of discrete genomic intervals
Science 328:723–725
PCR Amplifi cation, Cloning, and Sequencing of Ancient DNA Tara L. Fulton and Mathias Stiller Abstract
PCR amplifi cation of DNA is routine in modern molecular biology. However, the application of PCR to ancient DNA (aDNA) experiments often requires signifi cant modifi cation to standard protocols. The degraded nature of most aDNA fragments requires targeting shorter fragments, performing replicate amplifi cations, incorporating multiple negative controls, combating PCR inhibition, using specifi c DNA polymerases to deal with damaged bases, working in a separate aDNA facility, and modifying the PCR
recipe to deal with damaged and low copy-number target DNA. In this chapter, we describe how and why these procedures are implemented, discuss aDNA-specifi c troubleshooting methodology, and suggest modifi cations to commercial cloning and sequencing procedures to reduce the expense of PCR
product cloning.
Key words:
Polymerase chain reaction , PCR optimization , BSA, inhibition , Ancient DNA , DNA polymerase
1. Introduction
The invention of the polymerase chain reaction (PCR)
( 1
) revolutionized the fi eld of ancient DNA (aDNA) research. In theory, only a single copy of the targeted DNA region is required for PCR, making it a powerful tool for amplifying aDNA from samples where only a handful of intact copies of the target region may remain.
PCR is not, by any means, a technique exclusive to aDNA research.
However, its use with aDNA requires modifi cations to the experimental design, the experiment itself, and post-experimental troubleshooting.
Ancient DNA is often highly degraded, and even exceptionally preserved permafrost specimens may contain only 5% of surviving DNA fragments longer than 300 base pairs (bp)
( 2
) . Thus, when fragments longer than 100–300 bp are targeted using PCR, it is 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_15, © Springer Science+Business Media, LLC 2012
111
112
T.L. Fulton and M. Stiller
possible that long fragments of undamaged, modern DNA may be preferentially amplifi ed. To overcome this, a series of overlapping primer sets can be used to obtain a long stretch of continuous DNA sequence in small, stepwise fragments. This has the added advantage of identifying any nontarget amplifi cations such as numts (nuclear insertions of the mitochondrial DNA), other pseudogenes, or nonhomologous copies of the target gene, if mismatches are observed between overlapping regions of the amplifi ed fragments. It is also routine to clone at least some of the amplifi cation products of aDNA experiments, as this can identify potential contaminants or PCR artifacts and allow evaluation of the extent of postmortem damage.
The high-performing Platinum Taq High Fidelity and
AmpliTaq Gold (both from Life Technologies) are among the
most common polymerases used in aDNA experiments. The choice of polymerase is important as commercial polymerases vary widely in their effi ciency in synthesizing aDNA
( 3 )
and in the particular way they interact with damaged bases
( 4 )
. Even with high-fi delity polymerases, it is important to consider the possibility of strand jumping which can produce chimeric products. An additional benefi t of both Platinum Taq and AmpliTaq Gold is that they are hot-start polymerases, a desirable attribute as PCR amplifi cation from ancient extracts is generally set up in a facility that is spatially distant from the thermocycler.