Diet and the evolution of human amylase gene copy number variation.
ABSTRACT Starch consumption is a prominent characteristic of agricultural societies and hunter-gatherers in arid environments. In contrast, rainforest and circum-arctic hunter-gatherers and some pastoralists consume much less starch. This behavioral variation raises the possibility that different selective pressures have acted on amylase, the enzyme responsible for starch hydrolysis. We found that copy number of the salivary amylase gene (AMY1) is correlated positively with salivary amylase protein level and that individuals from populations with high-starch diets have, on average, more AMY1 copies than those with traditionally low-starch diets. Comparisons with other loci in a subset of these populations suggest that the extent of AMY1 copy number differentiation is highly unusual. This example of positive selection on a copy number-variable gene is, to our knowledge, one of the first discovered in the human genome. Higher AMY1 copy numbers and protein levels probably improve the digestion of starchy foods and may buffer against the fitness-reducing effects of intestinal disease.
- SourceAvailable from: ncbi.nlm.nih.gov[show abstract] [hide abstract]
ABSTRACT: Copy number variation is surprisingly common among humans and can be involved in phenotypic diversity and variable susceptibility to complex diseases, but little is known of the extent of copy number variation in nonhuman primates. We have used two array-based comparative genomic hybridization platforms to identify a total of 355 copy number variants (CNVs) in the genomes of 20 wild-born chimpanzees (Pan troglodytes) and have compared the identified chimpanzee CNVs to known human CNVs from previous studies. Many CNVs were observed in the corresponding regions in both chimpanzees and humans; especially those CNVs of higher frequency. Strikingly, these loci are enriched 20-fold for ancestral segmental duplications, which may facilitate CNV formation through nonallelic homologous recombination mechanisms. Therefore, some of these regions may be unstable "hotspots" for the genesis of copy number variation, with recurrent duplications and deletions occurring across and within species.Proceedings of the National Academy of Sciences 06/2006; 103(21):8006-11. · 9.74 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Given that gene duplication is a major driving force of evolutionary change and the key mechanism underlying the emergence of new genes and biological processes, this study sought to use a novel genome-wide approach to identify genes that have undergone lineage-specific duplications or contractions among several hominoid lineages. Interspecies cDNA array-based comparative genomic hybridization was used to individually compare copy number variation for 39,711 cDNAs, representing 29,619 human genes, across five hominoid species, including human. We identified 1,005 genes, either as isolated genes or in clusters positionally biased toward rearrangement-prone genomic regions, that produced relative hybridization signals unique to one or more of the hominoid lineages. Measured as a function of the evolutionary age of each lineage, genes showing copy number expansions were most pronounced in human (134) and include a number of genes thought to be involved in the structure and function of the brain. This work represents, to our knowledge, the first genome-wide gene-based survey of gene duplication across hominoid species. The genes identified here likely represent a significant majority of the major gene copy number changes that have occurred over the past 15 million years of human and great ape evolution and are likely to underlie some of the key phenotypic characteristics that distinguish these species.PLoS Biology 08/2004; 2(7):E207. · 12.69 Impact Factor
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ABSTRACT: Polymorphic amylase protein patterns have suggested the presence in the human genome of various haplotypes encoding these allozymes. To investigate the genomic organization of the human α-amylase genes, we isolated the pertinent genes from a cosmid library constructed of DNA from an individual expressing three different salivary amylase allozymes. From the restriction maps of the overlapping cosmids and a comparison of these maps with the restriction enzyme patterns of DNA from the donor and family members, we were able to identify two haplotypes consisting of very different numbers of salivary amylase genes. The short haplotype contains two pancreatic genes (AMY2A and AMY2B) and one salivary amylase gene (AMY1C), arranged in the order 2B-2A-1C, encompassing a total length of approximately 100 kb. The long haplotype spans about 300 kb and contains six additional genes arranged in two repeats, each one consisting of two salivary amylase genes (AMY1A and AMY1B) and a pseudogene lacking the first three exons (AMYP1). The order of the amylase genes within the repeat is 1A-1B-P1. All genes are in a head-to-tail orientation except AMY1B, which has the reverse orientation with respect to the other genes. Analysis of somatic cell hybrids confirmed the presence of these short and long haplotypes. Furthermore, we present evidence for the existence of additional haplotypes in the human population and propose a general model for the evolution of the human α-amylase multigene family. A general designation 2B-2A-(1A-1B-P)n-1C can describe these haplotypes, n being 0 and 2 for the short and the long haplotypes presented in this paper, respectively.Genomics 08/1989; · 3.01 Impact Factor
Diet and the evolution of human amylase gene copy
George H Perry1,2, Nathaniel J Dominy3, Katrina G Claw1,4, Arthur S Lee2, Heike Fiegler5, Richard Redon5,
John Werner4, Fernando A Villanea3, Joanna L Mountain6, Rajeev Misra4, Nigel P Carter5, Charles Lee2,7,8&
Anne C Stone1,8
Starch consumption is a prominent characteristic of
agricultural societies and hunter-gatherers in arid
environments. In contrast, rainforest and circum-arctic hunter-
gatherers and some pastoralists consume much less starch1–3.
This behavioral variation raises the possibility that different
selective pressures have acted on amylase, the enzyme
responsible for starch hydrolysis4. We found that copy number
of the salivary amylase gene (AMY1) is correlated positively
with salivary amylase protein level and that individuals from
populations with high-starch diets have, on average, more
AMY1 copies than those with traditionally low-starch diets.
Comparisons with other loci in a subset of these populations
suggest that the extent of AMY1 copy number differentiation is
highly unusual. This example of positive selection on a copy
number–variable gene is, to our knowledge, one of the first
discovered in the human genome. Higher AMY1 copy numbers
and protein levels probably improve the digestion of starchy
foods and may buffer against the fitness-reducing effects
of intestinal disease.
Hominin evolution is characterized by significant dietary shifts,
facilitated in part by the development of stone tool technology,
the control of fire and, most recently, the domestication of plants
and animals5–7. Starch, for instance, has become an increasingly
prominent component of the human diet, particularly among
agricultural societies8. It stands to reason, therefore, that studies of
the evolution of amylase in humans and our close primate relatives
may provide insight into our ecological history. Because the human
salivary amylase gene (AMY1) shows extensive variation in copy
number9,10, we first assessed whether a functional relationship exists
between AMY1 copy number and the amount of amylase protein
in saliva. We then determined if AMY1 copy number differs
among modern human populations with contrasting amounts of
We estimated diploid AMY1 gene copy number for 50 European
Americans using an AMY1-specific real-time quantitative PCR
(qPCR) assay. We observed extensive variation in AMY1 copy number
in this population sample (Fig. 1a and Supplementary Table 1
online), consistent with previous studies10,11. Next, we performed
protein blot experiments with saliva samples from the same indivi-
duals in order to estimate salivary amylase protein levels (Fig. 1b).
These experiments showed a significant positive correlation between
salivary amylase gene copy number and protein expression (P o
0.001; Fig. 1c).
2468 10 1214 16
AMY1 diploid gene copy number
mg AMY1 protein / ml saliva
AMY1 diploid copy number
Figure 1 AMY1 copy number variation and salivary amylase protein
expression. (a,b) For the same European American individuals,
we estimated diploid AMY1 gene copy number with qPCR (a) and
estimated amylase protein levels in saliva by protein blot (b). Error bars
indicate s.d. (c) Relationship between AMY1 diploid copy number and
salivary amylase protein level (n ¼ 50 European Americans). A considerable
amount of variation in AMY1 protein expression is not explained by copy
number (R2¼ 0.351), which may reflect other genetic influences on
AMY1 expression, such as regulatory region SNPs or nongenetic factors
that may include individual hydration status, stress level and short-term
Received 9 May; accepted 3 August; published online 9 September 2007; doi:10.1038/ng2123
1School of Human Evolution and Social Change, Arizona State University, Tempe, Arizona 85287, USA.2Department of Pathology, Brigham and Women’s Hospital,
Boston, Massachusetts 02115, USA.3Department of Anthropology, University of California, Santa Cruz, California 95064, USA.4School of Life Sciences, Arizona
State University, Tempe, Arizona 85287, USA.5The Wellcome Trust Sanger Institute, The Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.
6Department of Anthropological Sciences, Stanford University, Stanford, California 94305, USA.7Harvard Medical School, Boston, Massachusetts 02115, USA.
8These authors contributed equally to this work. Correspondence should be addressed to N.J.D. (email@example.com).
1256 VOLUME 39 [ NUMBER 10 [ OCTOBER 2007 NATURE GENETICS
© 2007 Nature Publishing Group http://www.nature.com/naturegenetics
Although there is a considerable range of variation in dietary starch
intake among human populations, a distinction can be made between
‘high-starch’ populations for which starchy food resources comprise a
substantial portion of the diet and the small fraction of ‘low-starch’
populations with traditional diets that incorporate relatively few
starchy foods. Such diets instead emphasize proteinaceous resources
(for example, meats and blood) and simple saccharides (for example,
from fruit, honey and milk). To determine if AMY1 copy number
differs among populations with high- and low-starch diets, we
estimated AMY1 copy number in three high-starch and four low-
starch population samples. Our high-starch sample included two
agricultural populations, European Americans (n ¼ 50) and Japanese
(n ¼ 45), and Hadza hunter-gatherers who rely extensively on starch-
rich roots and tubers (n ¼ 38)12. Low-starch populations included
Biaka (n ¼ 36) and Mbuti (n ¼ 15) rainforest hunter-gatherers, Datog
pastoralists (n ¼ 17) and the Yakut, a pastoralist, fishing society (n ¼
25). Additional details on the diets of these populations are provided
in Supplementary Table 2 online. We found that mean diploid AMY1
copy number was greater in high-starch populations (Fig. 2 and
Supplementary Fig. 1 online). Notably, the proportion of individuals
from the combined high-starch sample with at least six AMY1 copies
(70%) was nearly two times greater than that for low-starch popula-
tions (37%). To visualize the allele-specific number and orientation of
AMY1 gene copies, we performed high-resolution FISH on stretched
DNA fibers (fiber FISH); these results were consistent with diploid
AMY1 copy number estimates from our qPCR experiments (Fig. 3).
The among-population patterns of AMY1 copy number variation
do not fit expectations under a simple geographical region–based
model of genetic drift: our high- and low-starch samples include both
African and Asian populations, suggesting that diet more strongly
predicts AMY1 copy number than geographic proximity. Based on this
observation, we hypothesized that natural selection may have influ-
enced AMY1 copy number in certain human populations. However,
we cannot rigorously test such a hypothesis on the basis of our qPCR
results alone, in part because we lack comparative data from other
loci. Therefore, we next performed array-based comparative genomic
hybridization (aCGH) on the Yakut population sample with a whole-
genome tile path (WGTP) array platform that was previously used11
to describe genome-wide patterns of copy number variation in 270
individuals (the HapMap collection), including the same Japanese
population sample as in our study. For the Yakut aCGH experiments,
we used the same reference DNA sample (NA10851) as in the previous
study11, facilitating comparisons of Japanese and Yakut relative
intensity log2ratios for the 26,574 BAC clones on the array, including
two clones mapped to the AMY1 locus.
AMY1 diploid gene copy number
68 1012 14 15
Proportion of individuals
AMY1 diploid gene copy number
Cumulative proportion of individuals
Figure 2 Diet and AMY1 copy number variation. (a) Comparison of qPCR-
estimated AMY1 diploid copy number frequency distributions for populations
with traditional diets that incorporate many starch-rich foods (high-starch)
and populations with traditional diets that include little or no starch (low-
starch). (b) Cumulative distribution plot of diploid AMY1 copy number for
each of the seven populations in the study.
Figure 3 High-resolution fiber FISH validation of AMY1 copy number
estimates. Red (B10 kb) and green (B8 kb) probes encompass the entire
AMY1 gene and a retrotransposon directly upstream of (and unique to)
AMY1, respectively. (a) Japanese individual GM18972 was estimated by
qPCR to have 14 (13.73 ± 0.93) diploid AMY1 gene copies, consistent with
fiber FISH results showing one allele with ten copies and the other with four
copies. (b) Biaka individual GM10472 was estimated by qPCR to have six
(6.11 ± 0.17) diploid AMY1 gene copies, consistent with fiber FISH results.
(c) The reference chimpanzee (Clint; S006006) was confirmed to have two
diploid AMY1 gene copies.
NATURE GENETICS VOLUME 39 [ NUMBER 10 [ OCTOBER 2007 1257
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Results from the two AMY1-mapped clones on the WGTP array
supported our original observations: the log2ratios were strongly
correlated with the qPCR estimates of AMY1 diploid copy number
(Supplementary Fig. 1), and the population mean log2ratios for both
clones were greater for the Japanese sample (Fig. 4a and Supplemen-
tary Fig. 1). More importantly, with the WGTP data, we were able to
compare the extent of population differentiation at the AMY1 locus to
other loci in the genome for the two Asian population samples in our
study. We would expect the magnitude and direction of the Japanese-
Yakut mean log2ratio difference for the AMY1-mapped clones to be
similar to those for other copy number–variable clones, if these CNVs
have experienced similar evolutionary pressures. However, the two
AMY1-mapped clones are significant outliers in this distribution
(Fig. 4b and Supplementary Fig. 2 online), leading us to reject this
null hypothesis. In addition, we considered a database of genotypes for
783 genome-wide microsatellites for the same Yakut individuals and a
different Japanese population sample13, because microsatellite loci are
usually multiallelic (as is the AMY1 locus). We found that the extent of
Japanese-Yakut differentiation at the AMY1 locus exceeded that for
497% of the microsatellite loci (Supplementary Fig. 3 online).
Although this result should be interpreted with caution because we
do not know whether AMY1 copy number and microsatellite muta-
tion rates and patterns are similar, this finding is consistent with our
results from the WGTP comparison.
These observations suggest that natural selection has shaped AMY1
copy number variation in either the Japanese or the Yakut or in both
populations. We cannot fully test the null hypothesis for the other
high- and low-starch populations in our study, but the patterns of
copy number variation we observed in these populations are similar to
those for the Japanese and Yakut and thus may also reflect non-neutral
evolution. We favor a model in which AMY1 copy number has been
subject to positive or directional selection in at least some high-starch
populations but has evolved neutrally (that is, through genetic drift) in
low-starch populations. Although it is possible that lower AMY1 gene
copy numbers have been favored by selection in low-starch popula-
tions, such an interpretation is less plausible for the simple reason that
excessive amylase production is unlikely to have a significant negative
effect on fitness. Furthermore, several lines of evidence offer mechan-
isms by which higher salivary amylase protein expression may confer a
fitness advantage for individuals with a high-starch diet. First, a
significant amount of starch digestion occurs in the mouth during
mastication14. For example, blood glucose has been shown to be
significantly higher when high-starch foods such as corn, rice and
potatoes (but not low-starch foods such as apples) are first chewed
and then swallowed, rather than swallowed directly15. In addition, it
has been suggested that oral digestion of starch is critically important
for energy absorption during episodes of diarrhea4. Diarrheal diseases
can have a significant effect on fitness; for example, such diseases
caused 15% of worldwide deaths among children younger than 5 years
as recently as 2001 (ref. 16). Last, salivary amylase persists in the
stomach and intestines after swallowing17, thereby augmenting the
enzymatic activity of pancreatic amylase in the small intestine. Higher
AMY1 copy number and a concomitant increase in salivary amylase
protein thus are likely to improve the efficiency with which high-
starch foods are digested in the mouth, stomach and intestines and
may also buffer against the potential fitness-reducing effects of
To understand better the evolutionary context of human
AMY1 copy number variation, we analyzed patterns of AMY1 copy
number variation in chimpanzees (Pan troglodytes) and bonobos
(Pan paniscus). In contrast to the extensive copy number variation
we observed in humans, each of 15 wild-born western chimpanzees
(P. t. verus) showed evidence of only two diploid AMY1 copies (Fig. 3c
and Supplementary Fig. 4 online), consistent with previous find-
ings18–21. Although we observed evidence of a gain in AMY1 copy
number in bonobos relative to chimpanzees (Supplementary Fig. 4),
our sequence-based analyses suggest that each of these AMY1 copies
has a disrupted coding sequence and may be nonfunctional (Supple-
mentary Fig. 5 online). Therefore, the average human has roughly
three times more AMY1 copies than chimpanzees, and bonobos may
not have salivary amylase at all. Outgroup comparisons with other
great apes suggest that AMY1 copy number was probably gained in the
human lineage, rather than lost in chimpanzees21,22. Given that AMY1
copy number is positively correlated with salivary amylase protein
expression in humans, it stands to reason that the human-specific
increase in copy number may explain, at least in part, why salivary
amylase protein levels are approximately six to eight times higher in
humans than in chimpanzees23. These patterns are consistent with the
general dietary characteristics of Pan and Homo; chimpanzees and
bonobos are predominantly frugivorous and ingest little starch relative
to most human populations24.
Among other primates, New World monkeys do not produce
salivary amylase and tend to consume little starch, but cercopithecines
(a subfamily of Old World monkeys including macaques and
Number of individuals
Chr1tp-6D2 log2 ratio
–0.30.00.3 0.60.9 1.21.5
Japanese (n = 45; mean = 0.433; s.d. = 0.335)
Yakut (n = 25; mean = 0.071; s.d. = 0.451)
Japanese mean log2 ratio
Yakut mean log2 ratio
Figure 4 Japanese-Yakut copy number differentiation at AMY1 versus
other genome-wide loci. (a) Frequency distributions of WGTP aCGH relative
intensity log2ratios from AMY1-mapped clone Chr1tp-6D2 for Japanese and
Yakut individuals. (b) Relationship between Japanese and Yakut mean log2
ratios for all autosomal WGTP clones that were copy number variable in both
populations. AMY1-mapped clones Chr1tp-6D2 and Chr1tp-30C7 are
depicted as filled red and blue circles, respectively.
1258 VOLUME 39 [ NUMBER 10 [ OCTOBER 2007 NATURE GENETICS
© 2007 Nature Publishing Group http://www.nature.com/naturegenetics
mangabeys) have relatively high salivary amylase expression, even
compared to humans23. Although the genetic mechanisms are
unknown, this expression pattern may have evolved to facilitate
the digestion of starchy foods (such as the seeds of unripe fruits)
stowed in the cheek pouch, a trait that, among primates, is unique
The initial human-specific increase in AMY1 copy number may
have been coincident with a dietary shift early in hominin evolu-
tionary history. For example, it is hypothesized that starch-rich plant
underground storage organs (USOs) such as bulbs, corms and tubers
were a critical food resource for early hominins26,27. Changes in USO
consumption may even have facilitated the initial emergence and
spread of Homo erectus out of Africa5,28. Yet such arguments are
difficult to test, mainly because direct evidence for the use of USOs is
difficult to obtain, particularly for more remote time periods. USOs
themselves are perishable, as are many of the tools used to collect and
process them. Therefore, understanding the timing and nature of the
initial human-lineage AMY1 duplications may provide insight into
our ecological and evolutionary history. The low amount of nucleotide
sequence divergence among the three AMY1 gene copies found in the
human genome reference sequence (hg18; d ¼ 0.00011 to 0.00056)
implies a relatively recent origin that may be within the time frame of
modern human origins (that is, within the last B200,000 years; based
on human-chimpanzee AMY1 d ¼ 0.027 and using an estimate of
6 million years ago for divergence of the human and chimpanzee
lineages). However, given the possibility for gene conversion, we do
not necessarily consider this estimate to be reliable. The generation of
AMY1 sequences from multiple humans may ultimately help to shed
light on this issue.
In summary, we have shown that the pattern of variation in copy
number of the human AMY1 gene is consistent with a history of diet-
related selection pressures, demonstrating the importance of starchy
foods in human evolution. Although the amylase locus is one of
the most variable in the human genome with regard to copy
number10, it is by no means unique; a recent genome-wide survey
identified 1,447 copy number–variable regions among 270 pheno-
typically normal human individuals11, and many more such regions
are likely to be discovered with advances in copy number variation
detection technology. It is reasonable to speculate that copy number
variants other than AMY1 are or have been subject to strong pressures
of natural selection, particularly given their potential influence
on transcriptional and translational levels (for example, see ref. 29).
The characterization of copy number variation among humans and
between humans and other primates promises considerable insight
into our evolutionary history.
Samples. Buccal swabs and saliva were collected under informed consent from
50 European Americans ages 18–30 (Arizona State University institutional
review board (IRB) protocol number 0503002355). Saliva was collected for
3 min from under the tongue. Buccal swabs were collected from the Hadza
(n ¼ 38) and Datog (n ¼ 17) from Tanzania (Stanford University IRB protocol
number 9798-414). Genomic DNA samples from the Biaka (Central African
Republic; n ¼ 32), Mbuti (Democratic Republic of Congo; n ¼ 15) and Yakut
(Siberia; n ¼ 25) are from the HGDP-CEPH Human Genome Diversity Cell
Line Panel. Lymphoblastoid cell lines from 45 Japanese, 4 additional Biaka and
the donor for the chimpanzee genome sequence (Clint) were obtained from the
Coriell Institute for Medical Research. Whole blood samples were collected
during routine veterinary examinations from chimpanzees and bonobos
housed at various zoological and research facilities. Two additional bonobo
samples were obtained from the Integrated Primate Biomaterials and Informa-
tion Resource. DNA was isolated using standard methods.
Copy number estimation. Primers for qPCR (Supplementary Table 3 online)
were designed to be specific to AMY1 (that is, to have sequence mismatches
with AMY2A and AMY2B) based on the human and chimpanzee reference
genome sequences. A previous study reported a single (haploid) copy of AMY1
for one chimpanzee18, and a recent analysis19did not find any evidence of
recent AMY1 duplication for Clint. We used fiber FISH to confirm that Clint
has two diploid copies of AMY1 (Fig. 3c). Therefore, we were able to estimate
diploid copy number based on relative AMY1 quantity for human DNA
compared to a standard curve constructed from the DNA of Clint. A fragment
from the TP53 gene was also amplified to adjust for DNA dilution quantity
variation. Samples were run in triplicate and standards in duplicate. Experi-
ments were performed and analyzed as described previously20.
Protein blot analysis. Protein samples were prepared by solubilizing saliva
samples in 2% SDS and heating at 100 1C for 5 min. These samples were
analyzed on mini SDS-polyacrylamide gels and transferred to polyvinylidene
difluoride (PVDF) membranes (Immobilon-Millipore). For quantification
purposes, a human salivary amylase protein sample of known quantity (Sigma)
was run on each gel, with 5 mL of saliva for each sample. After transfer,
the membranes were incubated for 1.5 h with primary antibodies raised
against human salivary amylase (Sigma). The membranes were washed and
goat anti-rabbit alkaline phosphatase–conjugated IgG secondary antibodies
(Pierce) were added for 1 h. The membranes were exposed to ECF substrate
(Amersham Biosciences) for 5 min and then analyzed using a phosphorimager.
Quantification of protein bands was performed using ImageQuant software
Fiber FISH. DNA fibers were prepared by gently lysing cultured lymphoblast
cells with 300 ml Cell Lysis Buffer (Gentra Systems) per 5 million cells. 10 ml of
lysate was placed on a poly-L-lysine–coated slide (LabScientific) and mechani-
cally stretched with the edge of a coverslip. After 30 s, 300 ml of 100% methanol
was applied to fix the fibers. Slides were dried at 37 1C for 5 min and then
stored at room temperature (22–25 1C).
PCR product probes were made from (i) the entire AMY1 gene itself
(B10 kb; red in images) and (ii) the retrotransposon found directly upstream
of all AMY1 copies but not of pancreatic amylase genes or amylase pseudogenes
(B8 kb; green in images). The gene probe may not be specific to AMY1 under
all hybridization conditions (AMY1 sequence divergence with AMY2A and
AMY2B ¼ 7.5% and 7.1%, respectively), but the upstream probe is. We used
long-range PCR followed by nested PCR for each region (primers and
conditions are provided in Supplementary Table 3). PCR products were
purified with DNA Clean and Concentrator columns (Zymo).
For each nested PCR product, 750 ng was combined with 20 ml 2.5?
random primer (BioPrime aCGH Labeling Module, Invitrogen) in a total
volume of 39 ml. Samples were incubated at 100 1C for 5 min and were then
placed on ice for 5 min. Next, 5 ml 10? dUTP and 1 ml Exo-Klenow Fragment
(BioPrime Module) and either 5 ml (5 nmol) Biotin-16-dUTP (Roche; gene
probe) or 5 ml (5 nmol) digoxigenin-11-dUTP (Roche; upstream probe) were
added, and samples were incubated at 37 1C for 5 h. Labeled products were
purified with Microcon Centrifugal Filter Devices (Millipore) using three
washes of 300 ml 0.1? SSC, eluted with 50 ml H2O. For each 1 mg of labeled
DNA, we added 10 mg human Cot-1 DNA (Invitrogen).
For each experiment, 500 ng of labeled DNA from each of the nested PCR
reactions was combined, lyophilized, reconstituted in 10 ml hybridization buffer
(50% formamide, 20% dextran sulfate, 2? SSC) and added to the slide (18 ?
18 mm cover glass; Fisher). Fibers and probes were denatured together
(95 1C for 3 min) and hybridized in a humidified chamber (37 1C for 40 h).
The slide was washed in 0.5? SSC at 75 1C for 5 min followed by three washes
in 1? PBS at room temperature (22–25 1C) for 2 min each. Next, fibers were
incubated with 200 ml CAS Block (Zymed) and 10% (vol/vol) normal goat
serum (Zymed) for 20 min at room temperature (22–25 1C) under a HybriSlip
(Invitrogen). We used a three-step detection and amplification (with reagents
in 200 ml CAS Block with 10% (vol/vol) normal goat serum). Each step was
conducted for 30 min at room temperature under a HybriSlip followed by
three washes in 1? PBS for 2 min each at room temperature (22–25 1C).
Reagents were as follows for each step: step (i): 1:500 anti-digoxigenin-
fluorescein, Fab fragments (Roche) and 1:500 Strepavidin, Alexa Fluor 594
NATURE GENETICS VOLUME 39 [ NUMBER 10 [ OCTOBER 2007 1259
© 2007 Nature Publishing Group http://www.nature.com/naturegenetics
conjugate (Invitrogen); step (ii): 1:250 rabbit anti-FITC (Zymed) and 1:500
biotinylated anti-streptavidin (Vector Laboratories); step (iii): 1:100 goat anti-
rabbit IgG-FITC (Zymed) and 1:500 streptavidin, Alexa Fluor 594 conjugate.
Images were captured on an Olympus BX51 fluorescent microscope with
an Applied Imaging camera and were analyzed with Applied Imaging’s
aCGH analysis. For aCGH experiments, we used a large-insert clone DNA
microarray covering the human genome in tiling path resolution30. Test
genomic DNA samples (from Yakut individuals) and reference genomic
DNA samples (from NA10851) were labeled with Cy3-dCTP and Cy5-dCTP,
respectively (NEN Life Science Products) and were cohybridized to the array.
For each sample, a duplicate experiment was performed in dye-swap to reduce
false-positive error rates. Labeling, hybridization, washes and analyses were
performed as described11,30.
Note: Supplementary information is available on the Nature Genetics website.
We are grateful to all our study participants. We thank H. Cann and C. de Toma
of the Fondation Jean Dausset (CEPH), the Cincinnati Zoo, the Lincoln Park
Zoo, the New Iberia Research Center, the Primate Foundation of Arizona, the
Southwest Foundation for Biomedical Research, the Coriell Institute for Medical
Research and the Integrated Primate Biomaterials and Information Resource for
samples. C. Tyler-Smith and Y. Gilad provided comments on a previous version
of the manuscript. We would also like to thank the Wellcome Trust Sanger
Institute Microarray Facility for printing the arrays and T. Fitzgerald and D. Rajan
for technical support. This study was funded by grants from the L.S.B. Leakey
Foundation and Wenner-Gren Foundation (to N.J.D.), the Department of
Pathology, Brigham & Women’s Hospital (to C.L.), the National Institutes of
Health (to the University of Louisiana at Lafayette New Iberia Research Center;
numbers RR015087, RR014491 and RR016483) and the Wellcome Trust (H.F.,
R.R. and N.P.C.).
G.H.P. and N.J.D. contributed equally to this work. G.H.P., N.J.D., C.L. and
A.C.S. designed the study; G.H.P., F.A.V., J.L.M. and A.C.S. collected the samples;
G.H.P. and A.S.L. performed qPCR experiments; J.W. performed protein blot
experiments; G.H.P. performed fiber FISH experiments; H.F. and R.R. performed
and analyzed aCGH experiments; K.G.C. performed nucleotide sequencing
experiments; G.H.P. performed data analyses; R.M., N.P.C., C.L. and A.C.S.
supervised the experiments and analyses and G.H.P. and N.J.D. wrote the paper.
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