SNPSTRs: Empirically Derived, Rapidly Typed,
Autosomal Haplotypes for Inference of Population
History and Mutational Processes
Joanna L. Mountain,1,2,3Alec Knight,1Matthew Jobin,1Christopher Gignoux,1
Adam Miller,1Alice A. Lin,2and Peter A. Underhill2
1Department of Anthropological Sciences, Stanford, California 94305, USA;2Department of Genetics,
Stanford, California 94305, USA
Each independently evolving segment of the genomes of a sexually reproducing organism has a separate history
reflecting part of the evolutionary history of that organism. Uniparentally or clonally inherited DNA segments
such as the mitochondrial and chloroplast genomes and the nonrecombining portion of the Y chromosome have
provided, to date, most of the known data regarding compound haplotypic variation within and among
populations. These comparatively small segments include numerous polymorphic sites and undergo little or no
recombination. Recombining autosomes, however, comprise the major repository of genetic variation. Technical
challenges and recombination have limited large-scale application of autosomal haplotypes. We have overcome
this barrier through development of a general approach to the assessment of short autosomal DNA segments.
Each such segment includes one or more single nucleotide polymorphisms (SNPs) and exactly one short tandem
repeat (STR) locus. With dramatically different mutation rates, these two types of genetic markers provide
complementary evolutionary information. We call the combination of a SNP and a STR polymorphism a
SNPSTR, and have developed a simple, rapid method for empirically determining gametic phase for double and
triple heterozygotes. Here, we illustrate the approach with two SNPSTR systems. Although even one system
provides insight into population history, the power of the approach lies in combining results from multiple
[Supplemental material is available online at http://www.genome.org. The following individual kindly provided
reagents, samples, or unpublished information as indicated in this paper: L. Luca Cavelli-Sforza.]
In 1996, researchers reported the global pattern of haplotype
frequency variation and linkage disequilibrium (LD) for a pair
of linked genetic markers on human chromosome 12 (Tish-
koff et al. 1996). The pattern, the authors concluded, provides
evidence in support of a common and recent African origin
for all non-African human populations. In reaching this con-
clusion, the authors took advantage of the different mutation
rates of the two linked markers. One of the markers is a rap-
idly evolving short tandem repeat (STR, or microsatellite) lo-
cus, whereas the other is a partial deletion of an ancient Alu
retroposon insertion, a unique event. That report served to
demonstrate the evolutionary information content of autoso-
mal haplotypes composed of different classes of markers.
Other research groups investigating human evolutionary
history have studied linked sets of genetic markers with very
different mutation rates on the nonrecombining portion of
the Y chromosome (NRY) (e.g., Ruiz-Linares et al. 1999; Gre-
sham et al. 2001; Nebel et al. 2001). Compound single nucleo-
tide polymorphism (SNP) and STR haplotypes on the NRY
provide powerful tools for inferring the histories of popula-
tions (de Knijff 2000). For example, estimates of the age of the
SNP have been inferred from the STR diversity of SNP-defined
monophyletic groups (clades or haplogroups) of lineages (e.g.,
Hurles et al. 1999). Conversely, each SNP provides insight
into STR diversity as the SNP defines a clade for which STR
diversity may then be interpreted free of independently
evolved, homoplastic diversity in other clades (Bosch et al.
1999; Makova et al. 2000).
Although the Y chromosome has proven highly infor-
mative, that segment of the human genome reflects only a
fraction of human history. The mitochondrial genome, al-
though also highly informative, similarly reflects only a frac-
tion of the history of a species. A more complete history re-
quires information from recombining chromosomes that
trace to many ancestors.
Evolutionary histories of individual species would be
most accurately elucidated from the combined histories of a
large number (>50) of DNA regions (Wall 2000). Within most
DNA regions short enough for recombination to be rare (on a
geographically global scale), however, few informative SNPs
exist. A second complication is that with conventional meth-
ods, empirical determination of gametic phase for double het-
erozygotes requires cloning of PCR products. The expense and
time involved limit the numbers of samples or genetic sys-
tems that may be studied to the point that population studies
based on empirical data are precluded. For these reasons, gene
histories have been inferred for a relatively small set of auto-
somal regions. One such region is MS205 (Rogers et al. 2000),
wherein 10 SNPs were identified within 2 kb of a minisatellite
array. The relatively large number of SNPs combined with
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inference of phase through typing of the linked microsatellite
made possible inference of the history of the MS205 region.
Given current estimates of the average frequency of polymor-
phism across the genome (∼1 per 1000 np), regions with these
characteristics are exceedingly rare. An alternative with wide
potential application is to consider a large number of short
DNA regions with at least two polymorphisms, one of which
is a rapidly evolving STR.
With this goal in mind, we recently undertook develop-
ment of a set of independent, compound haplotypic systems.
We chose to focus on SNPs linked tightly to STR polymor-
phisms. Such combinations of polymorphisms, which we re-
fer to as SNPSTRs (Fig. 1), satisfy the following three require-
ments: (1) close physical linkage of two or more polymor-
phisms; (2) significant difference in mutation rate between
polymorphisms; and, (3) potential for a large number of in-
dependent compound haplotypic systems.
Close Physical Linkage
The Alu and STR originally studied by Tishkoff et al. (1996) are
9.8 kb apart, and the PLAT locus (Tishkoff et al. 2000b) spans
22 kb. We have considered SNPs and STRs fewer than 400
bases apart in order to facilitate data generation. For such
tightly linked markers, we have developed a method to em-
pirically determine the homologous SNP and STR allelic states
of each individual, including gametic phase for double het-
erozygotes, using conventional fluorescent fragment analysis.
The result is an individual’s genohaplotype, in which a geno-
haplotype is one diploid individual’s pair of haplotypes at a
given SNPSTR system. The method described below enables us
to accomplish these goals rapidly and cost effectively with the
potential to be scaled up via automation. Closely linked mark-
ers also simplify interpretation of data by minimizing the
number of parameters.
Different Mutation Rates
Each independent SNPSTR system combines a slowly evolving
polymorphic locus and a more rapidly evolving polymorphic
locus. SNPs mutate at a rate on the order of 2.0–2.5 ? 10?8
mutations per nucleotide position per generation (Nachman
and Crowell 2000). Although estimates of autosomal STR mu-
tation rates vary widely on the basis of motif type
(Chakraborty et al. 1997), an effective mutation rate of
∼1.5 ? 10?3per STR per generation has been obtained for
dinucleotide repeat polymorphisms (Zhivotovsky 2001).
These different rates enable us to take advantage of the dif-
ferent time scales for which each type of marker is informa-
tive. Potentially, each SNP provides information into the his-
tory of the linked STR, and each STR provides insight into the
history of the linked SNP(s). In the context of molecular evo-
lutionary and population genetic models, we can draw infer-
ences regarding both molecular events and processes (e.g.,
mutation and recombination) and population history.
Large Number of Independent Systems
We consider a class of linked-marker systems that is large and
broadly distributed in the genomes of sexually reproducing
species. SNPs and STR polymorphisms are frequent in the ge-
nomes of many species. Numerous SNPSTR systems exist
whenever recombination decouples the histories of short
DNA regions that include SNPs and a STR polymorphism. The
independent histories of these short genetic regions can be
combined to infer population histories. For any given re-
search question, a particular number of these compound hap-
lotypic systems are required to draw a robust conclusion.
Here, we report our method for developing and screen-
ing SNPSTRs. Data obtained for two SNPSTR systems, one on
human chromosome 22 and one on human chromosome 5,
demonstrate the utility of the method and provide additional
evidence of the information content inherent in such com-
Development of New SNPSTR Systems
The Protocol for development of a SNPSTR system consists of
a sequence of three steps, summarized as follows.
Locate a STR locus and its flanking regions via GenLink
(http://mapper.wustl.edu/), GenBank (http://www.ncbi.
nlm.nih.gov/), or another database. STRs with a larger num-
ber of repeat units are more likely to be informative of evo-
lutionary history; STRs with fewer than 10 repeat units often
reveal little variation. STRs with perfect repeats (relatively
common in the human genome) are optimal as the evolution
(specifically, mutational mechanisms and rates) of imperfect
STRs is less well understood (Macaubas et al. 1997).
Determine whether at least one SNP is present within ∼400 bp
of the STR on either or both upstream and downstream flank-
ing regions. We design PCR primers from the STR-flanking
regions available in GenBank clones. These primers are de-
signed to encompass a target from as close as possible to the
STR (within constraints of primer design) to ∼400 bp away
from the STR, for both upstream and downstream flanking
regions. The amplified flanking regions are then examined
for SNPs using a rapid, inexpensive screening method such
as denaturing high-performance liquid chromatography
(DHPLC, Oefner and Underhill 1998) or SSCP analysis (Orita
et al. 1989). The appropriate set of samples used for SNP dis-
covery (the screening set) depends on the nature of the sci-
entific questions to be addressed. To minimize ascertainment
gote autosomal genohaplotype for a diploid organism. In this ex-
ample, one homolog has a C allele at the SNP and an STR of 21 repeat
units. The other homolog has a T allele at the SNP and an STR of 23
repeat units. The C allele is amplified via PCR with an allele-specific
primer, with C at the 3? terminus, and is labeled with the fluorescent
dye 6-FAM. The T allele is amplified with an allele-specific primer, with
T at the 3? terminus, and is labeled with the fluorescent dye HEX. Both
labeled PCR products are produced with the same reverse primer “r.”
As the length of the PCR product varies depending solely upon the
repeat number of the STR, and length is determined by fluorescent
detection of electrophoretic mobility, the allelic states of the SNP and
STR, and gametic phase, are all determined simultaneously by fluo-
rescent electrophoretic fragment analysis.
Schematic of SNPSTR system depicting double heterozy-
Autosomal Haplotyping Systems
bias (Mountain et al. 1994; Wakely et al. 2001) and to maxi-
mize the applicability of SNPSTR systems, we use a global
screening set composed of individual samples representing
geographically and linguistically diverse human groups. Pu-
tative polymorphic samples are sequenced to determine the
basis of apparent polymorphism.
After discovering a SNP, design allele-specific primers, each
labeled with a different fluorescent dye (Fig. 1). Design an
unlabeled reverse primer across the STR region to produce
labeled PCR product with either of the fluorescent allele-
specific primers. The fluorescently labeled PCR product then
encompasses the SNP at one end and the STR at the other. The
length of the PCR product varies among chromosomes, de-
pending solely on the copy number of repeats at the STR.
Once these primers and corresponding PCR parameters are
optimized, determine the genohaplotypes of each individual
by electrophoresis on a genetic analysis instrument using
fluorescent detection (Fig. 2). As with any electrophoretic
fragment analysis, for each SNPSTR locus at least one indi-
vidual PCR product, homozygous at the STR, must also be
sequenced for comparison with the fragment analysis to en-
sure accurate determination of repeat number of STR alleles
by fragment mobility.
Generation of Labeled Fragments
We developed two procedures for generation of allele-specific
fluorescently labeled fragments for electrophoretic determi-
nation of SNPSTR genohaplotypes. The first approach in-
volves exponential PCR amplification of labeled fragments
starting with genomic sample DNA. Using this exponential
approach, for each SNPSTR locus, each individual sample
DNA is amplified separately with each allele-specific, fluores-
cently labeled primer and the same reverse primer (two sepa-
rate PCR reactions per individual are carried out). Incorpora-
tion of a deliberate mismatch one base from the 3? terminus
of allele-specific primers increases specificity. Optimal anneal-
ing temperature must be determined experimentally. For
some loci, complete specificity is difficult to achieve. If low-
frequency amplification of the alternative allele occurs during
early cycles, these nonspecific amplicons increase exponen-
tially, so that detectable levels are produced by later cycles. A
build-up of nonspecific, labeled fragments then prevents de-
termination of the genohaplotype.
A second approach that circumvents the problem of
nonspecific amplification involves linear production of la-
beled fragments and requires two steps. Consider the case of a
SNP upstream of a STR. First, genomic DNA is amplified via
PCR with primers that target a region from a forward primer
upstream of the SNP to a reverse primer downstream of the
STR. Both primers are unlabeled. Successful amplification is
confirmed by visualization of an aliquot on an agarose gel.
Next, an aliquot (typically 10 µL) of the completed PCR reac-
tion is added as template to an equal volume of a new, sec-
ond-step reaction mixture. The second-step mixture contains
the same PCR reagents as the first reaction, except that only a
single, labeled, allele-specific primer is included. No reverse
primer is included. Residual reverse primer will not interfere
with this step, so no clean-up is needed. With this linear pro-
cedure, we have found no need to
introduce a deliberate mismatch to
enhance specificity; each allele-
specific primer is a perfect match to
its respective SNP allele. The second
step reaction mixture is then sub-
jected to a single cycle of denatur-
ing, annealing, and extension. This
linear procedure produces ample la-
beled allele-specific fragments with-
out exponential build-up of non-
specific fragments. A second advan-
tage is that the method is not
sensitive to annealing temperature.
A single, stringent annealing tem-
perature will work for most primers.
As exemplified below, we have suc-
cessfully used both the exponential
and linear methods to determine
SNPSTR System 22SR1
The SNPSTR system 22SR1 includes
the CA STR spanning np 112278 to
112323 in Homo sapiens clone CTA-
390C10 on chromosome 22q11.21–
22q12.1, GenBank Accession Num-
ber AL008721 (see supplemental
data for further details). We ampli-
fied the flanking regions of the STR
and screened for SNPs via DHPLC.
No heteroduplexes were apparent
in the downstream flanking region.
system with a C/T SNP and a CA STR. Detection was performed using a 310 Genetic Analyzer. As in
Fig. 1, the C SNP was labeled with 6-FAM (blue). The T SNP was labeled with HEX (green). Size
standard HD400 ROX is red. (A) Genohaplotype that is homozygous C at the SNP locus and hetero-
zygous at the STR locus, with repeat numbers of 21 and 23. (B) Doubly homozygous T genohaplotype
with a CA repeat number of 22. (C) Double heterozygous genohaplotype depicted schematically in
Figure 1. The chromosome with the C SNP has an STR with 21 repeat units. The chromosome with the
T allele has an STR with 23 repeat units.
GeneScan (Applied Biosystems) analysis of chromosome 22 SNPSTR system 22SR1, a
Mountain et al.
1768 Genome Research
In the upstream flanking region, we observed a heteroduplex
and verified a C/T SNP at 112227 by sequencing (update sub-
mitted to GenBank). We synthesized allele-specific primers
spanning np 112207 to the SNP. One allele-specific primer
terminated in C and was labeled with 6-FAM. The other allele-
specific primer terminated in T and was labeled with HEX. We
incorporated a deliberate mismatch one base from the 3? ter-
minus to increase specificity (C and T, respectively). We ob-
tained consistent allele specificity at 58°C annealing for 15
sec, using Taq DNA polymerase (Promega) in the presence of
2.5 mM MgCl2, by use of the exponential procedure described
above. Using this system, we obtained 22SR1 SNPSTR geno-
haplotypes for 52 individuals representing globally diverse
populations, about half from across the African continent (see
supplemental data). Outgroup comparison using chimpanzee
(Pan troglodytes) provided evidence that the ancestral SNP al-
lele is T, whereas C is derived. Table 1 provides absolute fre-
quencies of the 19 different SNPSTR haplotypes observed in
the sample. A total of 21 of 52 individuals (40%) are doubly
heterozygous. Highly significant levels of LD were detected
for the non-African (P <0.001), but not for the African
(P = 0.838) segment of the sample.
SNPSTR System 5SR1
The SNPSTR system 5SR1 includes the GT (CA) STR spanning
np 147561 to 147594 in Homo sapiens chromosome 5 clone
RP11–121L11, GenBank Accession Number AC026743 (Table
1). We amplified the flanking regions of the STR and screened
for SNPs via DHPLC. No heteroduplexes were apparent in the
downstream flanking region. In the upstream flanking region
we observed a heteroduplex and verified a G/T SNP at np
147511 by sequencing (update submitted to GenBank). We
synthesized allele-specific primers spanning np 147488 to the
SNP. One allele-specific primer terminated in G and was la-
beled with 6-FAM. The other allele-specific primer terminated
in T and was labeled with HEX. Each primer was a perfect
match to its respective allele. We obtained consistent allele
specificity at 58°C annealing for 15 sec, using Taq DNA poly-
merase (Promega) in the presence of 2.5 mM MgCl2, using the
two-step linear procedure (above). We obtained 5SR1 SNPSTR
genohaplotypes for 52 individuals (see Supplemental Data).
Table 1 provides haplotype frequencies for the global, African,
and non-African segments of the overall sample. A total of 17
of 52 individuals (33%) are doubly heterozygous. Highly sig-
nificant levels of LD were detected for both the African
(P <0.001) and non-African (P <0.001) segments of the
The 22SR1 and 5SR1 SNPSTR systems described above meet
the three goals stated in the introduction. (1) Within each
system the SNP and STR are physically linked; (2) mutation
rates of the SNP and STR differ significantly; and (3) the two
systems, located on different chromosomes, evolve indepen-
dently. In addition, typing is rapid and cost effective now that
the systems have been developed and optimized. With ge-
netic marker systems that meet these goals, we have flexible,
powerful tools suitable for a variety of applications. SNPSTR
systems may be used wherever genetic markers such as restric-
tion fragment-length polymorphisms (RFLPs), SNP, and STR
markers have been applied. As demonstrated by the example
of Tishkoff et al. (1996), pairs of such markers provide more
information than any single polymorphism system, or than a
pair of polymorphisms considered without empirical determi-
nation of gametic phase. For instance, by comparing the STR
alleles on the multiple SNP backgrounds, we gain information
regarding the extent of homoplasy at the STR locus. The ad-
ditional information derives from the resetting of the STR at
the time of the SNP-generating mutation. The derived SNP
allele arises on a single chromosome with, by necessity, zero
One informative component of SNPSTR data is empiri-
cally determined haplotypic phase. One-third or more of the
individuals tested for the 22SR1 and 5SR1 systems were dou-
bly heterozygous and, therefore, required phase determina-
Globally Diverse Populations
22SR1 and 5SR1 SNPSTR Haplotype Frequencies Observed in a Sample of 52 Individuals (104 Haplotypes) Representing
Linked STR CA repeat number
Total15 1617 1819 20 21222324 25
The sample included 25 individuals from across the continent of Africa, and 27 from the Middle East, Europe, Central Asia, East Asia, Oceania,
Australia, and the Americas. Global, African, and non-African distributions are given. Individual genohaplotype information (two specific
haplotypes per individual) from SNPSTR analysis is provided as Supplemental Table 2.
Autosomal Haplotyping Systems
tion. In the absence of family data, the physical association of
an allele at one locus with an allele at a second locus is typi-
cally inferred via estimation methods (Hawley and Kidd
1995). Such estimation methods may underestimate the fre-
quency of rare haplotypes (Tishkoff et al. 2000a). As is true for
rare alleles, however, rare haplotypes are likely to be particu-
larly informative regarding gene flow between populations
(Barton and Slatkin 1986) and mutation rates (Chakraborty
Given the extensive number of STRs discovered in the
genomes of a wide array of species during recent years, nu-
merous potential SNPSTR systems exist. Because SNPs and
STRs are frequent in the nuclear genomes of most species,
closely linked pairs are quite common (e.g., Makova et al.
2000). According to the dbSNP summary (http://www.ncbi.
nlm.nih.gov/SNP/snp_summary.cgi), >2.5 million SNPs have
already been identified using human samples. This total cor-
responds to an average of ∼1 SNP every 1–2 kb. Consistent
with this estimate, in our experience, at least one SNP is found
within 400 bp of roughly 50% of STRs.
Recent studies (Daly et al. 2001; Jeffreys et al. 2001; Reich
et al. 2001) of the patterns of LD and distribution of recom-
bination hotspots within the human genome have implica-
tions for SNPSTR application. If the two polymorphisms (SNP
and STR) are located at a recombination hotspot, haplotype
frequencies may reflect reciprocal recombination as well as
other evolutionary forces. If current estimates of the distribu-
tion of such hotspots (Daly et al. 2001) are roughly accurate,
such cases are expected to be relatively rare. Consider a sim-
plified model of one hotspot of length Z every X nucleotides.
If a SNPSTR system (from the SNP through the STR) spans Y
nucleotides, the chance of the SNPSTR system overlapping
the hotspot is (Y + Z)/X. For instance, for SNPSTR systems of
length Y = 400 nucleotides, hotspots of length Z = 1000
nucleotides, and inter-hotspot (LD block) distances of
X = 50,000 nucleotides, overlap is expected <3% of the time.
Within LD blocks, we may be able to develop compound-
linked SNPSTR systems that extend beyond the typical 500 bp
and still assume that recombination is essentially absent.
Gene conversion has probably had greater influence on
haplotype frequencies at most SNPSTR systems than has re-
ciprocal recombination. Recent comparisons of LD and levels
of polymorphism indicate that gene conversion has played a
significant role in reducing LD at closely linked sites (Ardlie et
al. 2001; Frisse et al. 2001). Whereas interpretation of SNPSTR
haplotype frequencies must include the possibility of gene
conversion, STR mutation (on the order of 10?3per gen-
eration) plays a much larger role than gene conversion in
SNPSTR evolution. A large set of SNPSTR systems, however,
may provide insights into the rate of gene conversion across
the genome. The 5SR1 system, for instance, lacks evidence of
gene conversion, whereas the 22SR1 system is less informative
in this regard.
The set of applications for which a given SNPSTR system
is particularly suited depends in part on the date of the mu-
tation that gave rise to the SNP. When information regarding
the age of an allele (at a SNP or a STR polymorphism) is valu-
able, a sufficiently polymorphic SNPSTR system will be infor-
mative. Two obvious areas of application are in evolutionary
genetics and medical genetics. Other areas of potential appli-
cation include agriculture, entomology, wildlife manage-
ment, and forensics. Within evolutionary genetics, we expect
SNPSTRs to be informative regarding the evolutionary histo-
ries of humans and many other species. SNPSTRs may also be
informative regarding molecular evolutionary processes such
as gene conversion. Information resides in the extent of varia-
tion and of LD within populations and in differences in hap-
lotype frequencies and LD among populations (Tishkoff et al.
1996; Ardlie et al. 2002). SNPSTR haplotype frequency differ-
ences arise largely through the molecular process of mutation
and the population genetic processes of genetic drift, migra-
tion, and natural selection.
In the case of humans, our data demonstrate that even
single systems provide insights into history. Whereas only a
limited number of individuals have been screened for SNPSTR
systems 5SR1 and 22SR1, the resulting haplotype frequencies
reveal patterns consistent with patterns of previously gener-
ated genetic data. Both systems reveal greater genetic diversity
within Africa than outside of Africa, particularly in terms of
overall numbers of STR alleles. Considering the number of
STR alleles on each SNP background, however, reveals a more
complex pattern. Wheres for 22SR1, the set of African samples
reveals 11 STR alleles, 10 are found on the C background and
7 are found on the T background. The set of non-African
samples, with 8 STR alleles total, reveals only 5 on the C
background, but 8 on the T background. The set of non-
African samples, therefore, exhibits greater (in absolute terms)
diversity on the T background than does the set of African
samples. Further typing of this SNPSTR system in both African
and non-African populations may clarify whether the T diver-
sity in non-Africans reflects population expansion outside of
Africa or the diverse nature of the groups that have migrated
out of Africa at various points in human history.
The 5SR1 system is striking with respect to the level of LD
both within and outside of Africa. There is no overlap in the
STR alleles found on each of the SNP backgrounds. This sys-
tem is unusual for a SNPSTR in that we detect no evidence of
homoplasy at the STR locus. This pattern suggests that the
SNP arose much earlier than the initial spread of modern hu-
mans from Africa. In the context of a model of recent African
origin of anatomically modern humans, the overall STR fre-
quencies on the SNP backgrounds of the 5SR1 and 22SR1 sys-
tems indicate that when modern humans migrated out of
Africa, they carried with them only a subset of the total Afri-
can diversity. Furthermore, the diversity on each of the SNP
backgrounds in the Americas and Oceania is lower than the
diversity in Eurasia, which, in turn, is lower than that in Af-
rica (see supplemental data).
Within medical genetics, we expect SNPSTRs to be infor-
mative regarding the influence of genetic regions on disease
susceptibility. If the precise location of a gene is unknown,
association or linkage studies might take advantage of the
information contained within SNPSTR systems distributed
throughout the genome. As noted by Akey et al. (2001) and
Ardlie et al. (2002), among others, haplotype information sig-
nificantly improves the power to map disease genes. Where a
predisposing or candidate gene has been identified, nearby
SNPSTRs may be informative regarding the ages of mutations
or haplotypes associated with disease. Whereas SNPSTRs not
known to be influenced by natural selection are ideal for in-
ference of population history, SNPSTRs located in or near
genes may be developed to recover the evolutionary history of
The number and characteristics of a set of SNPSTR sys-
tems required to address a given research question depend on
the nature of that question. As with SNP and STR discovery,
many laboratories might develop SNPSTR systems and com-
pile a shared resource suitable for a range of applications.
Mountain et al.
Although the contributions of several laboratories would lead
to a valuable resource, even research groups focusing on less-
frequently studied species might take advantage of SNPSTR
technology. A single research group can develop a small num-
ber of systems and obtain insights into the evolutionary his-
tory of that species. Once developed, these systems can
be typed in a large sample very rapidly. Because typing of
SNPSTR systems is carried out using fluorescently labeled
primers, only very small quantities of DNA are needed (∼50
We are in the process of developing a number of human
SNPSTR systems using a global screening set. Some of these
will be informative at the global level, as are 22SR1 and 5SR1,
with geographically broad distributions of the SNP alleles.
Such systems make possible more precise inferences regarding
the initial spread of our human ancestors throughout the
world. Other SNPSTR systems, however, will be regionally in-
formative, say, for the peopling of the Americas. Regionally
informative systems are characterized by restricted distribu-
tion of one of the two SNP alleles, or low STR diversity within
one region relative to other regions. Screening sets may be
tailored (by including many individuals from a subset of re-
gions) to generate SNPSTR systems relevant to particular re-
gions of the world. Each SNPSTR system, when unlinked to
others on recombining chromosomes, provides independent
information regarding the evolutionary history of a species.
As with other sets of polymorphisms (Pamilo and Nei 1988),
the combination of information from several SNPSTR systems
dramatically increases the power to distinguish between al-
ternative hypotheses regarding molecular processes and
PCR primers were designed on the basis of sequences sur-
rounding SNPSTR loci retrieved from GenBank. Within the
constraints of the region, design considerations included
melting temperature, thermal profile, secondary structure,
dimer formation, and 3? terminal base. For allele-specific
primers, as they must terminate at the SNP, there is little one
may do to influence some of these factors, other than adjust-
ing primer length. Certain SNPs are not amenable to allele-
specific PCR (and therefore SNPSTR analysis) due to the na-
ture of the sequences adjacent to the SNP.
All PCR was performed using Taq DNA polymerase (Promega)
and supplied 10? buffer, at 2.5 mM MgCl2, 0.8 µM each
primer, for 35 cycles. Reaction reagents and concentrations
were standard (Sambrook et al. 1989). Reactions were pre-
pared on ice and transferred to a preheated thermal cycler
block at 94°C for 15 sec, followed by 35 cycles of 94°C for 15
sec, 58°C for 15 sec, and 72°C for 40 sec.
For exponential amplification of SNPSTR system 22SR1, a
range of annealing temperatures was tested. Almost perfect
specificity was found at 58°C. This was improved by addition
of Perfect Match PCR Enhancer (Stratagene) used according to
the manufacturer’s instructions. For linear amplification of
labeled 5SR1, both initial and second step (fluorescent) PCR
was performed at 58°C. Addition of Perfect Match was unnec-
essary, as sufficient allele specificity was obtained with the
linear, two-step procedure. Primer concentration was 0.8 µM.
Fluorescent Fragment Analysis
For fragment-length determination, fluorescently labeled PCR
products were separated by capillary electrophoresis on a 310
Genetic Analyzer in POP-4 (Applied Biosystems). Individual
samples were prepared as follows: 2.0 µL of labeled PCR prod-
uct and 0.7 µL of HD400 ROX size standard (Applied Biosys-
tems) were added to 15.0 µL of formamide, heated at 94°C for
2 min, and snap cooled in an ice bath.
Significance of Linkage Disequilibrium
For each population, for each pair of alleles at the two linked
polymorphisms, we test the significance of linkage disequi-
librium. LD for each SNPSTR system is measured by a Monte
Carlo implementation of a ?2test of the D statistic. D is cal-
culated (Weir 1996) for each allele in a SNPSTR as follows:
Duv= puv− pupv
The ?2statistic to test the hypothesis that none of the
Duv’s is significantly different from zero is:
The ?2statistic is calculated given the haplotype frequen-
cies observed for a SNPSTR system. To test significance of
the statistic, we generate 1000 random SNPSTR haplotype
frequency distributions with the same row and column
sums. The fraction of occurrences in which the randomized
SNPSTR’s ?2statistic exceeds the observed SNPSTR’s statistic is
taken to be the probability that the observed SNPSTR’s LD
occurred by chance. The chance of falsely rejecting the null
hypothesis is dependent on the number of randomizations in
the test (Roff and Bentzen 1989).
We thank NIH (GM 28428) and NSF (BCS-9905574) for par-
tially funding this research. We thank Prescott L. Deininger
for suggesting the linear method and for technical advice,
Todd Ward for technical advice, and L. Luca Cavalli-Sforza for
providing DNA samples for the screening set as well as labo-
ratory facilities during the initial phase of the project.
The publication costs of this article were defrayed in part
by payment of page charges. This article must therefore be
hereby marked “advertisement” in accordance with 18 USC
section 1734 solely to indicate this fact.
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WEB SITE REFERENCES
http://mapper.wustl.edu/; GenLink Home Page
http://www.ncbi.nlm.nih.gov/; National Center for Biotechnology
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Received March 4, 2002; accepted in revised form September 10, 2002.
Mountain et al.