Download Homogeneous Real-Time Detection of Single

Clinical Chemistry 49:10
1599 –1607 (2003)
Molecular Diagnostics
and Genetics
Homogeneous Real-Time Detection of
Single-Nucleotide Polymorphisms by Strand
Displacement Amplification on the
BD ProbeTec ET System
Sha-Sha Wang,* Keith Thornton,* Andrew M. Kuhn, James G. Nadeau, and
Tobin J. Hellyer†
Background: The BD ProbeTecTM ET System is based
on isothermal strand displacement amplification (SDA)
of target nucleic acid coupled with homogeneous realtime detection using fluorescent probes. We have developed a novel, rapid method using this platform that
incorporates a universal detection format for identification of single-nucleotide polymorphisms (SNPs) and
other genotypic variations.
Method: The system uses a common pair of fluorescent
Detector Probes in conjunction with unlabeled allelespecific Adapter Primers and a universal buffer chemistry to permit analysis of multiple SNP loci under
generic assay conditions. We used Detector Probes labeled with different dyes to facilitate differentiation of
two alternative alleles in a single reaction with no
postamplification manipulation. We analyzed six SNPs
within the human ␤2-adrenergic receptor (␤2AR) gene,
using whole blood, buccal swabs, and urine samples,
and compared results with those obtained by DNA
sequencing.
Results: Unprocessed whole blood was successfully
genotyped with as little as 0.1–1 ␮L of sample per
reaction. All six ␤2AR assays were able to accommodate
>20 ␮L of unprocessed whole blood. For the 14 individuals tested, genotypes determined with the six ␤2AR
assays agreed with DNA sequencing results.
BD Diagnostic Systems, 54 Loveton Circle, Sparks, MD 21152.
*Sha-Sha Wang and Keith Thornton contributed equally to the performance of this study.
†Author for correspondence. Fax 410-316-3690; e-mail Tobin_Hellyer@
bd.com.
Received April 2, 2003; accepted July 9, 2003.
Conclusion: SDA-based allelic differentiation on the
BD ProbeTec ET System can detect SNPs rapidly, using
whole blood, buccal swabs, or urine.
© 2003 American Association for Clinical Chemistry
Single-nucleotide polymorphisms (SNPs)1 are the most
common source of human genetic variation, occurring in
human populations with a mean frequency of ⬃1 per 1000
bp (1, 2 ). These small changes in nucleotide sequence
have great potential for identification of disease genes or
markers for genetic disorders, predicting drug metabolism and therapeutic response, and in the study of population genetics. Such analyses require fast, reliable, and
economical methods for detection of SNPs and other
genetic variations. Although several high-throughput
platforms exist for such applications in a research environment, there is a growing need for systems that are
sufficiently robust and easy to use for routine testing in
the clinical laboratory.
Apart from direct sequencing of PCR products, a
variety of molecular methods are currently used for
scoring or genotyping SNPs. Virtually all of these methods require PCR amplification of a target fragment bearing the SNP of interest before actual detection of the
variant nucleotide. Such methods include hybridization
of target to allele-specific oligonucleotides on DNA chip
arrays (3 ) or other solid-phase formats [e.g., dot blots (4 )],
oligonucleotide ligation assays (5 ), allele-specific PCR (6 ),
primer extension reactions or minisequencing (7 ), molecular beacons (8 ), 5⬘-nuclease assays (9 ), Invader technol-
1
Nonstandard abbreviations: SNP, single-nucleotide polymorphism; BD,
Becton Dickinson and Company; SDA, strand displacement amplification;
␤2AR, human ␤2-adrenergic receptor; ␳-Max, maximum density; ROX, rhodamine; FAM, fluorescein; and RFU, relative fluorescence unit(s).
1599
1600
Wang et al.: Real-Time Detection of SNPs
ogy (10 ), and mass spectrometry (11 ). Several homogeneous, fluorescence-based methods have been reported
that enable detection of SNPs during PCR (8, 9, 12, 13 ). By
combining the processes of amplification and detection
into a single step, these methods are amenable to higher
throughput and are less prone to contamination than
methods that involve manipulation of amplicons after the
completion of the amplification reaction.
The BD ProbeTecTM ET System (BD Diagnostic Systems) is a real-time amplification and detection platform
for the detection of Chlamydia trachomatis and Neisseria
gonorrhoeae in the clinical laboratory. The system is based
on isothermal strand displacement amplification (SDA)
(14, 15 ) of target nucleic acids in sealed microwells, coupled with homogeneous real-time detection by fluorescent probes (16, 17 ). Here we describe the use of the BD
ProbeTec ET System for the detection of multiple SNPs
with a single pair of universal fluorescent energy transfer
detector probes. The dual-dye capabilities of the instrument permit differentiation at any given locus of two
alternative alleles in a single reaction without postamplification manipulation.
Materials and Methods
principles of sda-based snp analysis
The principles of SDA for qualitative detection of nucleic
acids have been described previously (14 –17 ). Fig. 1
illustrates the homogeneous SDA-based universal fluorescent detection format for differentiation of multiple SNPs
with use of a single pair of labeled detector probes. The
system is based on allele-specific extension of an unlabeled Adapter Primer that hybridizes to the target sequence downstream of an SDA Primer. The Adapter
Primer comprises a 3⬘ target-specific region and 5⬘ generic/universal tail sequence. In the present study, each SDA
reaction contained a pair of Adapter Primers, each of
which was specific for one of two allelic variants (i.e.,
either allele A or allele B of a given nucleotide locus). The
5⬘ tail sequences of the two Adapter Primers are different,
permitting differentiation of alternative alleles through
comparison of the fluorescent signals obtained in each of
the two optical channels of the BD ProbeTec ET instrument. Importantly, the same pair of adapter tail sequences
and generic fluorescent Detector Probes can be used for
differentiation of multiple allelic pairs, thereby appreciably reducing the cost of assay development.
primers and probes
Six polymorphisms within the human ␤2-adrenergic receptor (␤2AR) gene were chosen as targets to investigate
the capabilities of SDA and the BD ProbeTec ET system
for analysis of SNPs. Reactions contained two each of the
following oligonucleotides: SDA Primers, Bumper Primers, Adapter Primers, and universal fluorescent Detector
Probes. The target-specific regions of the primers and
adapters were based on the published sequence of the
␤2AR gene [GenBank accession no. M15169 (18 )] and
were synthesized by Integrated DNA Technologies, Inc. A
complete listing of the primers and probes used in genotyping the six targeted SNPs of the ␤2AR locus is given in
Table 1 in the Data Supplement that accompanies the
online version of this article at http://www.clinchem.
org/content/vol49/issue10/.
cloning of control target sequences
Control sequences containing either allele A or allele B of
each of the six targeted ␤2AR SNPs [⫺654, ⫺367, ⫺47,
⫹46, ⫹491, and ⫹523) (18 )] were prepared by PCR
amplification of pooled human placental DNA (Sigma).
PCR fragments of 1.5 kb spanning the six SNP loci were
cloned into the Escherichia coli plasmid vector pUC19.
DNA sequencing of purified plasmid DNA was performed to verify the genotype of each cloned target.
specimens
Whole blood, buccal swabs, and urine samples were
collected with informed consent from healthy volunteers
at BD Diagnostic Systems (Sparks MD), according to
protocols approved by the company’s Institutional Review Board.
Blood Samples were collected in PPTTM plasma preparation tubes containing dipotassium EDTA as the anticoagulant (BD Vacutainer Systems). After collection, specimens were stored at ⫺20 °C before testing. For analysis of
unprocessed blood, samples were thawed, and the desired volume of specimen was mixed directly with SDA
buffer, heated for 5 min in a boiling water bath to lyse the
cells and denature the DNA, cooled, and centrifuged at
10 000g to pellet cellular debris. The blood– buffer supernatant was then added directly to priming microwells,
and the standard SDA assay protocol was followed. For
processed blood, 200 ␮L of thawed sample was extracted
with use of a QIAamp® DNA Blood Mini Kit (QIAGEN
Inc.) according to the manufacturer’s instructions. An
aliquot of purified DNA was mixed with SDA buffer,
boiled for 5 min to denature the DNA, cooled, and
assayed.
Buccal specimens were collected using BD BBL CultureSwabTM EZ polyurethane swabs (BD Diagnostic Systems). Three swabs were collected from each individual at
each collection time point. Swabs were stored frozen at
⫺20 °C until analysis, at which time two swabs were
thawed and expressed into 1 mL of SDA assay buffer.
Tubes containing expressed swab material were heated
for 5 min in a boiling water bath to denature the DNA,
cooled, and centrifuged 1 min at 10 000g to pellet cellular
debris. Samples were then added directly to the SDA
priming microwells and assayed.
Urine specimens were collected in sterile, plastic, preservative-free specimen collection cups that were stored
at 4 °C for up to 3 days or frozen at ⫺20 °C until analysis.
A 2-mL portion of each urine sample was centrifuged at
600g for 10 min. The supernatant was then decanted, and
the concentrated urine was resuspended in 0.5–1.0 mL
Clinical Chemistry 49, No. 10, 2003
1601
Fig. 1. Homogeneous SDA-based universal
fluorescent detection format for differentiation of multiple SNPs with use of a single pair
of labeled detector probes.
(A), universal detection. An Adapter Primer hybridizes to the amplified target downstream of an SDA
primer (1). The Adapter Primer comprises an allelespecific 3⬘ sequence (A) and 5⬘ generic tail (B).
Extension from the 3⬘ ends of both the Adapter
Primer and the upstream SDA Primer leads to
displacement of the Adapter Primer extension product into solution (2), which is in turn is captured by
a complementary SDA Primer (3). Simultaneous
extension from the 3⬘ ends of the SDA Primer and
Adapter Primer extension product generates the
complement of the 5⬘ Adapter Primer tail sequence
and a double-stranded restriction recognition site
(4). Nicking of the restriction site and extension
from the nick displaces a single-stranded copy of
the Adapter Primer complement into solution (5).
The 3⬘ end of this molecule is complementary to the
3⬘ end of the fluorescent Detector Probe. Hybridization of the Adapter Primer complement to the Detector Probe and extension from the 3⬘ ends of
these molecules (6) leads to opening of the Detector Probe hairpin, separation of the fluorophore and
quencher, and the generation of target-specific fluorescence (7). Additional fluorescent signal is produced by cleavage of the double-stranded Detector
Probe restriction site (8). (B), allele-specific signal
generation. Each SDA reaction contains two
Adapter Primers with different 5⬘ tail sequences and
two fluorescent Detector Probes labeled with different dyes. Each Adapter Primer is specific for one of
the allelic variants (A or B) at each SNP locus.
Hybridization and extension of a correctly matched
Adapter Primer (i and iii) generates allele-specific
fluorescent signal (panel A). The diagnostic nucleotide (underlined) is located one base (N-1) from the
3⬘ end of the Adapter Primer to maximize discriminatory power when it is mismatched with the target
sequence. A mismatch at the N-1 position reduces
the efficiency of extension of the Adapter Primer
relative to that of the perfectly matched Adapter (ii
and iv). A ratio of fluorescent signals obtained in
each optical channel is used to determine the
identity of the nucleotide at the targeted locus.
(depending on the assay) of universal SDA assay buffer.
Samples were then boiled for 5 min to denature the target
nucleic acid and assayed according to standard procedures.
genotyping with the BD ProbeTec ET system
The workflow used in the present study for genotyping
on the BD ProbeTec ET System is summarized in Fig. 2.
The assay reagents include a Priming Microwell containing SDA Primers, Bumper Primers, Adapter Primers,
universal Detector Probes, and nucleotides; and an Amplification Microwell containing the amplification enzymes. The same Amplification Microwell formulation
and Universal Buffer were used for each of the six ␤2AR
assays. For two of the assays that targeted particularly
GC-rich regions of the ␤2AR gene (⫺47 and ⫹46 loci;
1602
Wang et al.: Real-Time Detection of SNPs
Specimens were processed as described above. Denatured target nucleic acid in SDA buffer was dispensed
into the Priming Microwells and incubated at room temperature for 5 min. The Priming Microwells were then
placed on a special heating block set at 72 °C; at the same
time, the Amplification Microwells were prewarmed on
an adjacent block at 54 °C. The two heating blocks comprise part of the standard hardware for the BD ProbeTec
ET System. After a 10-min incubation, the samples were
transferred from the Priming to the Amplification Microwells, which were then permanently sealed and loaded in
the BD ProbeTec ET reader set at the standard operating
temperature of 52 °C. The final reagent concentrations in
each well were as follows (140-␮L volume): 24.5 mM
potassium phosphate (pH 7.6); 90.5 mM Bicine; 54.3 mM
KOH; 70 mL/L dimethyl sulfoxide; 70 mL/L glycerol; 5
mM magnesium acetate; 100 mg/L bovine serum albumin; 100 –500 nM SDA Primers; 50 nM Bumper Primers;
100 – 400 nM Adapter Primers; 150 –500 nM fluorescent
Detector Probes; 0.1 mM each of dATP, dGTP, and dTTP;
0.5 mM 2⬘-deoxycytidine 5⬘-O-(1-thiotriphosphate) S-isomer; ⬃0.8 U/␮L of Bst DNA polymerase; and ⬃2.6 U/␮L
of BsoBI restriction enzyme (NEB). For the ⫺47 and ⫹46
assays located in particularly GC-rich regions of the ␤2AR
gene, additional glycerol and dimethyl sulfoxide were
included in the Priming Microwell to bring the final
concentrations to 140 mL/L and 125 mL/L, respectively.
Data were collected over a period of 60 min and downloaded from the instrument for analysis.
data analysis
Fig. 2. Workflow for SDA-based genotyping of clinical specimens,
without sample processing, in the BD ProbeTec ET System.
Sample lysis and target denaturation take place in Universal SDA Buffer, which
is then mixed with the contents of the Priming Microwells. After a brief
incubation, the contents of the Priming Microwells are transferred to the
Amplification Microwells, which are sealed and loaded into the BD ProbeTec ET
reader.
Table 1 in the online Data Supplement), additional glycerol and dimethyl sulfoxide were included in the Priming
Microwell formulation to increase the stringency of reaction conditions, alleviate primer-primer interactions, and
reduce nonspecific amplification. The Universal Buffer
conditions (below) were derived through a series of
strategically designed multifactorial experiments that
were conducted with different SNP assay systems. Analysis of the results identified a set of compromise conditions for amplification and detection of all six ␤2AR SNP
targets.
We have developed SNP analysis software to analyze BD
ProbeTec ET data and generate genotyping results. The
instrument measures output from both optical channels at
discrete intervals (passes) throughout the course of the
reaction. Raw optical data are processed by dark correction and dynamic normalization using internal fluorescence calibrators, followed by step detection/repair and
smoothing with running mean and median filters to
remove periodic noise. The background fluorescence is
then calculated over a predetermined number of readings
from early in the course of amplification and subtracted
from all subsequent data points. The maximum density
(␳-Max) algorithm is applied to the background-corrected
data to determine the ratio (r) of fluorescent signals from
the two Detector Probes at each pass during the reaction
according to the equation: r ⫽ ln(ROX/FAM), where ROX
and FAM correspond to the signals in relative fluorescence units (RFU) obtained from the rhodamine- and
fluorescein-labeled probes, respectively. A maximum
density function is then used to determine the most likely
value of r for the sample, and this number is output as the
␳-Max score. For the ␤2AR assays, ␳-Max values greater
than ⫹1 corresponded to high ROX signals and were
considered indicative of a homozygous allele A genotype.
Similarly, ␳-Max values less than ⫺1 corresponded to
high FAM fluorescence and a homozygous allele B geno-
Clinical Chemistry 49, No. 10, 2003
type. ␳-Max values between ⫺1 and ⫹1 indicated the
detection of similar fluorescence intensities in the two
optical channels and a heterozygous allele A/B genotype.
sequence analysis
The results of SDA-based genotyping of processed and
unprocessed blood, urine, and buccal swabs were compared with those obtained by DNA sequencing of PCR
products obtained from amplification of purified DNA
from blood. DNA sequencing was performed in a blinded
fashion without reference to the SDA genotyping results
(Commonwealth Biotechnologies, Inc.). Heterozygous genotypes were confirmed by visual analysis of the sequencing electropherograms.
Results
differentiation of homozygous and
heterozygous alleles
Fig. 3 shows representative amplification curves generated with the SDA assay for the ⫺47 ␤2AR SNP locus, the
amplicon for which has a G⫹C content of 86%. The assays
were performed with 3 ␮L of whole, unprocessed blood
added directly to the reaction mixture. SDA results were
in complete agreement with DNA sequencing of PCR
products. The two optical channels of the BD ProbeTec ET
1603
instrument were used to detect the two alternative alleles
that are possible at the ⫺47 locus. The ROX-labeled
Detector Probe corresponded to allele A (C nucleotide at
position ⫺47), whereas the FAM-labeled probe corresponded to allele B (T nucleotide at position ⫺47). Data
were analyzed using the ␳-Max algorithm as described in
the Materials and Methods.
For specimen B (Fig. 3), there was a strong ROX signal
and corresponding weak FAM fluorescence with a ␳-Max
score of ⫹2.70, indicating the presence of the homozygous
C/C allelic pair. In contrast, for specimen E, there was a
strong FAM signal with little increase in ROX fluorescence over the course of the reaction. The ␳-Max score for
this sample was ⫺2.33, indicating the presence of the
homozygous T/T allelic pair. With specimen G, there
were approximately equivalent increases in both FAM
and ROX fluorescence during the course of the reaction,
yielding a ␳-Max score of ⫹0.05. This value lies between
⫺1 and ⫹1, thereby indicating that the sample was
heterozygous at the ⫺47 position (C/T allelic pair). The
control reaction containing no target DNA yielded only
background signal in both optical channels. In this case,
because neither optic yielded a detectable signal, no
␳-Max score was calculated and the sample was therefore
recorded as indeterminate.
Fig. 3. Genotyping of human blood specimens by the BD ProbeTec ET System.
Genotyping of the ⫺47 ␤2AR SNP locus was performed on three blood samples, using 3 ␮L of whole blood per reaction. Graphs show the relative increase in
fluorescence over time in each of the two optical channels corresponding to fluorescein (FAM) and rhodamine (ROX).
1604
Wang et al.: Real-Time Detection of SNPs
accuracy of allelic differentiation
The accuracy of SDA-based SNP analysis relative to DNA
sequencing was demonstrated by genotyping the six
targeted ␤2AR loci (⫺654, ⫺367, ⫺47, ⫹46, ⫹491, and
⫹523) in 19 different blood specimens from 14 healthy
volunteers and by analysis of cloned target nucleic acid.
Blood specimens were genotyped either by directly adding whole blood to SDA reactions or by analysis of
purified genomic DNA prepared as described in the
Materials and Methods. We used 3 ␮L of whole blood or 100
ng of purified genomic DNA in each SDA reaction. In
parallel with analysis of processed and unprocessed
blood, two plasmid clones of the ␤2AR gene were genotyped with use of 0.45 pg of purified plasmid DNA (1 ⫻
105 copies) per reaction. A single genotyping reaction for
each SNP locus was performed with each sample. A total
of 7 different genotypes [haplotype pairs (18 )] was identified among the 14 individuals tested. For all six SNP loci
and for all samples, the SDA-based genotyping results
were in complete concordance with those obtained by
DNA sequencing (data not shown).
reproducibility of allelic differentiation
The reproducibility of SDA for differentiation of allelic
variants was demonstrated with the ⫺654 ␤2AR SNP
assay. Various amounts of processed and unprocessed
blood were analyzed from individuals who had previously been genotyped by DNA sequence analysis. Between 0.1 and 3 ␮L of whole blood or 2–105 ng of purified
genomic DNA was used for genotyping in each SDA
Fig. 4. Cluster analysis of data obtained with the ⫺654 ␤2AR assay.
A and C, relative fluorescent signals emitted by the FAM- and ROX-labeled detector probes. Ellipses correspond to 95% probability limits based on the bivariate gaussian
distribution. Genotyping reactions contained 0.1–3 ␮L of whole blood (A) or 2–105 ng of purified genomic DNA (C) as target. B and D, ␳-Max analysis of fluorescent
signals shown in panels A and C, respectively.
Clinical Chemistry 49, No. 10, 2003
reaction. Three replicates were run at each target concentration for a total of 15 data points with each sample type
plus negative controls. Fig. 4 shows the clustering of
signals within the three possible genotypes at the ⫺654
locus. All SDA-based genotyping results agreed with the
corresponding DNA sequence analysis. For both unprocessed blood and purified DNA, there was a clear distinction in terms of the relative FAM and ROX signals
between the homozygous samples B and E and the
heterozygous sample G. Regardless of the quantity of
sample analyzed or the purity of the target DNA, results
clustered into three distinct groups, representing the
homozygous and heterozygous genotypes and in agreement with DNA sequence analysis. This clustering of data
was particularly evident when the ␳-Max algorithm was
been applied (Fig. 4, B and D). All homozygous samples
yielded ␳-Max scores that were either greater than ⫹1 or
less than ⫺1 depending on the allele. In contrast, all
heterozygous samples yielded ␳-Max scores between ⫺1
and ⫹1.
sample volume
All six assays yielded the correct genotyping results as
determined by comparison with DNA sequence analysis
with 1–20 ␮L of blood per reaction. Four assays (⫺654,
⫺367, ⫹491, and ⫹523) gave positive results with 0.3 ␮L
of blood, and two (⫺654 and ⫹491) also yielded results
with as little as 0.1 ␮L of blood per reaction (Table 2 in the
online Data Supplement). With purified genomic DNA
extracted from blood, correct results were obtained from
1605
the six assays when we used 3.5–35 ng (980 –9800 genomic-equivalents) per reaction.
analysis of matched blood, buccal swabs, and
urine
Because sample types such as hair, buccal swabs, or
fingernails may be appropriate for widespread genotyping applications (19 ), we performed a series of experiments to demonstrate the ability to genotype specimens
other than blood. Ten matched blood, buccal, and urine
specimens from different donors were analyzed in the
⫺654, ⫺367, ⫹491, and ⫹523 ␤2AR SNP assays (Fig. 5).
With the exception of specimens from donor G, for each
SNP locus, homozygous samples yielded ␳-Max scores
either greater than ⫹1 or less than ⫺1 depending on the
allele, whereas heterozygous samples yielded values between ⫺1 and ⫹1. Matched samples from the same
individual yielded identical ␤2AR genotypes that were in
complete concordance with DNA sequence analysis.
Initial testing of buccal swab specimens from donor G
incorrectly identified the individual as heterozygous at
the ⫺367 locus (␳-Max score ⫽ ⫹1.1). This was related to
an abnormally low yield of DNA from the swab specimens from this donor. Repeat testing of additional buccal
swabs from the same donor yielded the correct genotyping result. Similarly, the indeterminate results obtained
with urine from donors E (⫹491 and ⫹523 assays) and L
(⫹523 assay) and from buccal swabs from donor I (⫹523
assay) were associated with low target loads present in
these samples. These data emphasize that control over
Fig. 5. Use of alternative specimen types from the same individual for genotyping with the BD ProbeTec ET System.
SDA-based SNP analysis was conducted on four loci within the human ␤2AR gene using matched blood, buccal swabs, and urine specimens from 10 individuals (A–L).
Dashed lines represent the ⫺1 and ⫹1 ␳-Max cutoff values segregating homozygous and heterozygous genotypes. ␳-Max greater than ⫹1 indicates homozygous allele
A; ␳-Max less than ⫺1 indicates homozygous allele B; ␳-Max between ⫺1 and ⫹1 indicates heterozygous alleles A/B. Solid lines represent the mean ␳-Max score
obtained for each individual across the different sample types. Specimens that yielded indeterminate results are not shown. Indeterminate results were obtained with
the following samples: locus ⫹491, donor L, urine; locus ⫹523, donors E and L, urine; donor I, buccal swabs.
1606
Wang et al.: Real-Time Detection of SNPs
input target DNA concentration and the establishment of
appropriate ␳-Max cutoffs are required to ensure accurate
reporting of correct genotypes and to identify inadequate
specimens.
Discussion
In the present study we developed a novel SDA-based
method for the detection of SNPs with the BD ProbeTec
ET System. The method permits the use of a single pair of
universal fluorescently labeled probes for the detection of
multiple polymorphisms, thereby reducing the cost and
complexity of assay development. The utility of this
method was demonstrated by the development of six
individual assays for polymorphisms within the human
␤2AR gene (18 ). Through the use of two fluorescent
probes labeled with different fluorophores, each assay
enables the differentiation of two alternative alleles at the
targeted SNP locus. Importantly, all six assays described
here were optimized to work with the same universal
buffer, further streamlining assay set-up and workflow.
The adapter-mediated universal detection system we
have described therefore provides a simple, rapid, sensitive, and specific method for SNP analysis and haplotyping. Furthermore, through the appropriate location and
design of Adapter and SDA Primer sequences, the principles of SNP analysis described here are also amenable to
the differentiation of other nucleotide acid sequence variations, such as insertions, deletions, and splice variations.
One of the objectives of this study was to develop a set
of generic amplification conditions that would permit the
differentiation of alternative alleles at multiple SNP loci
without the need for reoptimization of the assay buffer.
This was a particular challenge for the six ␤2AR assays,
the target regions for which ranged in G⫹C content from
53% to 86%. Reports of PCR-based SNP analysis indicate
difficulty in genotyping sequences with ⬎60% G⫹C content (20 ). Although the SDA assays we developed were
able to differentiate polymorphisms within highly G⫹Crich regions of DNA, we did observe differences in
analytical sensitivity among the six assays that correlated
broadly with G⫹C content. Nevertheless, we were successful in obtaining the correct genotypes with all six
assays, using as little as 1 ␮L of unprocessed blood per
reaction. We also observed that all of the ␤2AR assays
were tolerant to ⱖ20 ␮L of unprocessed blood (equivalent
to ⬎14% of total reaction volume). The system provides
an efficient method for direct determination of unphased
genotypes from a variety of sample types, including
blood, buccal swabs, and urine. However, we observed
that care is necessary in validating genotyping protocols
for use with different specimen types that may yield
widely varying quantities of nucleic acid. Such fluctuations in target load can impact amplification efficiency,
and the establishment of appropriate algorithm parameters to safeguard against incorrect assignment of genotypes and identify inadequate specimens is an important
aspect of assay development.
The assay format we have described here uses a final
reaction volume of 140 ␮L, although this may readily be
adjusted for specific applications. Using the standard BD
ProbeTec ET microwell format and assay workflow, we
have demonstrated the ability to genotype specimens in a
50-␮L reaction volume containing 10% whole blood by
volume. The ability to circumvent time-consuming and
costly sample processing represents a major advance in
the field of genetic analysis. The blood samples tested in
the present study were collected with dipotassium EDTA
as the anticoagulant, although we have also demonstrated
the compatibility of the ␤2AR assays with unprocessed
blood containing heparin or citrate as the anticoagulant
(data not shown).
The method we describe here provides differentiation
between single nucleotide variants through the positioning of the diagnostic nucleotide one base from the 3⬘ end
of the Adapter Primer. This differs from typical allelespecific PCR systems, in which high selectivity is accomplished only when the diagnostic nucleotide is positioned
at the very 3⬘end of the SNP-specific primer (21, 22 ). For
highly G⫹C-rich sequences, in which primer-primer interactions are common, artificially created mismatches in
the SDA primers or toward the 3⬘ terminus of the adapter
primer may be introduced to improve amplification efficiency and/or differentiation. For example, the ⫺654
␤2AR Adapter Primers include intentional mismatches 3
bases from the 3⬘ terminus to improve allelic differentiation (Table 1 in the online Data Supplement).
The novel genotyping procedure we have described
here involves isothermal amplification without the need
for temperature cycling. For the purposes of this study,
we used an amplification time of 60 min, although,
depending on the target concentration, there is no significant change in ␳-Max score after the first 20 –30 min of
incubation (data not shown). With suitable control over
the input of target DNA, the amplification and detection
time could therefore be reduced substantially to enhance
throughput. With a 30-min amplification reaction as many
as 960 specimens may be processed and genotyped in one
8-h shift on a single BD ProbeTec ET instrument.
In conclusion, the BD ProbeTec ET System permits rapid
genotype analysis using common fluorescent detector
probes and a streamlined workflow with a wide range of
specimen types and sample volumes.
We thank Gordon Franklin, Mark Hall, and Mike Little for
technical assistance and helpful discussions.
References
1. Kruglyak L, Nickerson DA. Variation is the spice of life. Nat Genet
2001;27:234 – 6.
2. The International SNP Map Working Group. A map of human
genome sequence variation containing 1.42 million single nucleotide polymorphisms. Nature 2001;409:928 –33.
Clinical Chemistry 49, No. 10, 2003
3. Hacia JG, Fan JB, Ryder O, Jin L, Edgemon K, Ghandour G, et al.
Determination of ancestral alleles for human single-nucleotide
polymorphisms using high-density oligonucleotide arrays. Nat
Genet 1999;22:164 –7.
4. Nollau P, Wagener C. Methods for detection of point mutations:
performance and quality assessment. Clin Chem 1997;43:1114 –
28.
5. Delahunty C, Ankener W, Deng Q, Eng J, Nickerson DA. Testing the
feasibility of DNA typing for human identification by PCR and an
oligonucleotide ligation assay. Am J Hum Genet 1996;58:1239 –
46.
6. Myakishev MV, Khripin Y, Hu S, Hamer DH. High-throughput SNP
genotyping by allele-specific PCR with universal energy-transferlabeled primers. Genome Res 2001;11:163–9.
7. Syvanen AC. From gels to chips: “minisequencing” primer extension for analysis of point mutations and single nucleotide polymorphisms. Hum Mutat 1999;13:1–10.
8. Tyagi S, Bratu DP, Kramer FR. Multicolor molecular beacons for
allele discrimination. Nat Biotechnol 1998;16:49 –53.
9. Livak KJ. Allelic discrimination using fluorogenic probes and the 5⬘
nuclease assay. Genet Anal 1999;14:143–9.
10. Olivier M, Chuang LM, Chang MS, Chen YT, Pei D, Ranade K, et al.
High-throughput genotyping of single nucleotide polymorphisms
using new biplex invader technology. Nucl Acids Res 2002;30:
e53.
11. Ross P, Hall L, Smirnov I, Haff L. High level multiplex genotyping
by MALDI-TOF mass spectrometry. Nat Biotechnol 1998;16:
1347–51.
12. Bengra C, Mifflin TE, Khripin Y, Manunta P, Williams SM, Jose PA,
Felder RA. Genotyping of essential hypertension single-nucleotide
polymorphisms by a homogeneous PCR method with universal
energy transfer primers. Clin Chem 2002;48:2131– 40.
13. Lee LG, Livak KJ, Mullah B, Graham RJ, Vinayak RS, Woudenberg
TM. Seven-color, homogeneous detection of six PCR products.
Biotechniques 1999;27:342–9.
14. Walker GT, Fraiser, MS, Schram JL, Little MC, Nadeau JG,
15.
16.
17.
18.
19.
20.
21.
22.
1607
Malinowski DP. Strand displacement amplification–an isothermal,
in vitro DNA amplification technique. Nucleic Acids Res 1992;20:
1691– 6.
Spargo CA, Fraiser MS, Van Cleve M, Wright DJ, Nycz CM, Spears
PA, et al. Detection of M. tuberculosis DNA using thermophilic
strand displacement amplification. Mol Cell Probes 1996;10:
247–56.
Nadeau JG, Pitner JB, Linn CP, Schram JL, Dean CH, Nycz CM.
Real-time, sequence-specific detection of nucleic acids during
strand displacement amplification. Anal Biochem 1999;276:177–
87.
Little MC, Andrews J, Moore R, Bustos S, Jones L, Embres C, et al.
Strand displacement amplification and homogeneous real-time
detection incorporated in a second-generation DNA probe system,
BD ProbeTec ET. Clin Chem 1999;45:777– 84.
Drysdale CM, McGraw DW, Stack CB, Stephens JC, Judson RS,
Nandabalan K, et al. Complex promoter and coding region
␤2-adrenergic receptor haplotypes alter receptor expression and
predict in vivo responsiveness. Proc Natl Acad Sci U S A 2000;
97:10483– 8.
Tanigawara Y, Kita T, Hirono M, Sakaeda T, Komada F, Okumura
K. Identification of N-acetyltransferase 2 and CYP2C19 genotypes
for hair, buccal cell swabs, or fingernails compared with blood.
Ther Drug Monit 2001;23:341– 6.
Vieux EF, Kwok PY, Miller RD. Primer design for PCR and sequencing in high-throughput analysis of SNPs. Biotechniques 2002;June
Suppl:28 –32.
Newton CR, Graham A, Heptinstall LE, Powell SJ, Summers C,
Kalsheker N, et al. Analysis of any point mutation in DNA. The
amplification refractory mutation system (ARMS). Nucleic Acids
Res 1989;17:2503–16.
Kwok S, Kellogg DE, McKinney N, Spasic D, Goda L, Levenson C,
et al. Effects of primer-template mismatches on the polymerase
chain reaction: human immunodeficiency virus type 1 model
studies. Nucleic Acids Res 1990;18:999 –1005.