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Articles |
32, CCR2-V64I, and SDF1-G801A Related to Pathogenesis of AIDS
1
Laboratory of Genomic Diversity, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702.
2
PE Applied Biosystems, 850 Lincoln Centre Dr., Foster
City, CA 94404.
a Author for correspondence. Fax 301-846-6468; e-mail waters{at}avpaxp1.ncifcrf.gov
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: TaqMan assays based on the 5' nuclease activity of Taq
polymerase and fluorescent resonance energy transfer were developed to
score alleles at the biallelic loci
CCR5-+/
32,
CCR2-V64I and SDF1-G801A. For each assay,
72 samples were analyzed. Data collection and analysis were performed
on the Prism 7700 Sequence Detection System. For comparison with gel
electrophoresis methods, each locus was also scored on a subset of 24
samples, using restriction enzymes or single-strand conformational
polymorphism (SSCP).
Results: Clear allelic discrimination was obtained on each of the 72 samples for all three TaqMan assays. The TaqMan scores for the subset of 24 samples were concordant with the restriction enzyme and SSCP scores.
Conclusions: Because of its simplicity, speed, and potential for automation and miniaturization, TaqMan is an excellent candidate for investigation of genetic variation in clinical, research, and forensic settings.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Three alleles that protect against AIDS and that can be used for
large-scale population studies are CCR5-32
(8), CCR2-64I (9), and
SDF1-801A (10). HIV-1, the virus that
causes AIDS, uses the chemokine receptors CCR5 and CCR2 as coreceptors
with CD4 for entry into lymphoid cells. The CCR5-32
allele is a 32-bp deletion that truncates the CCR5 protein
(8). The truncated protein fails to reach the cell surface,
leading to reduced density of CCR5 receptors. This receptor loss slows
HIV-1 cell entry and delays AIDS progression (8). For
CCR2-V64I, a G-to-A substitution in the coding region causes
valine to be replaced by isoleucine (9). People who carry
isoleucine, or a completely linked allele, have delayed progression of
AIDS (11). Stromal-derived factor (SDF1, also known as
pre-B-cell growth-stimulating factor) is the natural ligand for the
chemokine receptor CXCR4. A G-to-A substitution in the 3' untranslated
region has a recessive protective effect in HIV-1-infected patients for
extended periods of exposure (10).
Worldwide population surveys indicate that CCR5-32,
CCR2-64I, and SDF1-801A
attain frequencies of 
15% in some populations
(9)(10)(12). Recently, it has been
reported that CCR5-32 and SDF1-A protect
against AIDS-related non-Hodgkin lymphoma (13). These
worldwide frequencies and disease results suggest that
CCR5-32, CCR2-64I, and
SDF1-801A are good candidates for protection
against additional infectious agents, AIDS-related cancers, and
non-AIDS-related cancers.
Several methods are available for scoring SNPs (14). These include restriction endonucleases (15), single-strand conformational polymorphism (SSCP) (16), heteroduplex analysis (17), sequencing by hybridization chips (18), primer extension (19), and direct sequencing (20). All of these methods require molecular biology steps after PCR, and most require gel or capillary electrophoresis. Such steps can be complex, time-consuming, expensive, and labor-intensive. Two additional methods, molecular beacons (21)(22)(23) and the 5' nuclease assay (24)(25), have an advantage in that allelic discrimination occurs during PCR. This eliminates post-PCR molecular biology steps and the need for electrophoresis. Molecular beacons currently require fluorescent detection of assay plates during PCR, thus limiting throughput to one plate per unit of PCR thermal cycling time on a dedicated real-time PCR instrument. For this study, the 5' nuclease assay, or TaqMan, was chosen because assay plates can be processed in parallel on a battery of thermal cyclers and transferred to a fluorescent plate reader for analysis.
Each TaqMan assay requires two fluorogenic allele-specific probes
(24)(26). One probe matches the wild-type
sequence, and one probe matches the mutant sequence. Each probe is
labeled at the 5' end with a fluorescent reporter dye and at the 3' end
with the quencher dye 6-carboxytetramethylrhodamine (TAMRA). The
reporter dyes used in this study were 6-carboxyfluorescein (FAM) for
the wild-type sequence and VIC for the mutant sequence. In the PE
Biosystems TaqMan platform, the reporter dye VIC recently replaced the
reporter dyes JOE (2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein) and
HEX (hexachloro-6-carboxyfluorescein) because VIC has an intensity more
equal to that of FAM and because its narrower spectrum has less overlap
with FAM. Both qualities improve signal processing and thus allelic
discrimination (27). In solution, the proximity of the dyes
on each probe allows TAMRA to absorb energy from the reporter dye
through fluorescent resonance energy transfer (Fig. 1
) (26). During PCR amplification, reporter signal is
increased when hybridized probes are cleaved by the 5' nuclease
activity of the Taq polymerase. Mismatches between a probe and its
target sequence reduce probe hybridization efficiency and subsequent
cleavage. An allele is detected as significantly increased intensity of
its reporter dye over background. The fluorescent dye
6-carboxy-X-rhodamine (ROX) is included in each assay to normalize
variation in signal strength among wells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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primers and probes
Primers (Operon Technologies.) and probes (PE Biosystems; Table 1
) were designed using the Primer Express computer-aided
primer design software (PE Biosystems) following the manufacture’s
published guidelines. Briefly, the polymorphic site lies in the middle
one-third of the probes. Probe lengths were adjusted such that both
probes had approximately the same melting temperature (67.0 °C)
(24)(25). The probe melting temperature was
7.0–8.0 °C above the primer melting temperatures of
59.0–60.0 °C as determined by the nearest neighbor algorithm
implemented in Primer Express. For
CCR5-+/32, the wild-type probe is
the 32 bp that are deleted in the mutant allele. The mutant allele
probe spans the junction of the deleted sequence.
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pcr conditions
TaqMan Universal Master Mix (PE Biosystems) was used at a final
concentration of 1x and volume of 25 µL along with 50–100 ng of
genomic DNA. Probe concentrations were initially optimized for each
assay to yield equal relative fluorescence. However, it was found that
excellent allelic discrimination was obtained for all assays when 200
nmol/L final probe concentration and 900 nmol/L final primer
concentration were used. The thermal cycling profile was that
recommended by the manufacturer: 50 °C for 2 min and 95 °C for 10
min, followed by 40 cycles of 95 °C for 15 s and 62 ° C for
1 min. Optical plates (PE Biosystems) were thermal cycled in the Prism
7700 Sequence Detection System (SDS) for real-time detection for Fig. 2
, A–D. Otherwise, plates were thermal cycled on GeneAmp PCR System
9700 (PE Biosystems) and analyzed on the 7700 using end-point analysis.
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other tests
Restriction enzyme digests, SSCP, and subsequent electrophoresis
were performed following standard procedures.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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dye contributions
The composite spectra are then processed to estimate the
contribution of each dye. Estimation is accomplished by the SDS
software, using a multicomponent linear model based on the spectrum of
each pure dye. For the CCR2-V64I assay, the estimated
contributions of individual dyes to the four possible emission patterns
are illustrated in Fig. 2
, A–D. Readings were taken during the course of a real-time PCR
experiment. Fig. 2A
depicts the no-template control. As can be seen,
there was little change in fluorescent intensity of either reporter dye
after thermal cycling. In contrast, Fig. 2B
illustrates a 2.5-fold
increase in FAM and a 1.1-fold increase in VIC for a G/G homozygote.
The slight increase in VIC is attributable to nonspecific
hybridization. Similarly, Fig. 2C
depicts a 2.1-fold increase in VIC
and a 1.4-fold increase in FAM for an A/A homozygote. Lastly, Fig. 2D
shows a 2.0-fold increase in FAM and a 1.8-fold increase in VIC for a
G/A heterozygote. In Fig. 2
, B–D, the quencher TAMRA decreases because
of probe cleavage and disassociation of reporter and quencher dyes. In
Fig. 2
, A–D, the intensity of ROX remains unchanged.
allelic contributions
To infer genotype, the allelic contribution to each sample is
estimated in the SDS software by substituting homozygote fluorescent
spectra for the pure dye spectra in the multicomponent linear model.
For this reason, eight replicate samples inferred to be homozygous for
each allele must be included on each assay plate and designated as
Allele 1 and Allele 2 controls in the SDS software. In this study, FAM
was designated as Allele 1 and VIC was designated as Allele 2. The SDS
software makes an additional normalization for background to an
approximate 0.0–1.0 scale by inclusion of eight replicate samples for
which reagents but no template DNA is added. These replicates are
designated as No Template Controls. In total, control samples occupy 24
wells of the 96-well plate. Samples for which genotype were to be
determined are designated in the SDS software as Unknowns.
For the CCR2-V64I assay, Fig. 2E
contrasts the normalized
allele G (FAM) and allele A (VIC) contributions for 72 samples and 24
controls. This plot clearly reveals four distinct clusters. These
clusters are further defined visually by genotype-specific colors.
Similar results were obtained for the SDF1-G801A and
CCR5-+/32 assays, using a single
set of reagent concentrations and thermal cycling conditions. The
scatter plot patterns of individual samples were highly reproducible
across repeated experiments performed on different days by different
people using PCR reagents freshly aliquoted from individual stock
vials.
Allelic contributions normalized to background for 22 samples for the
CCR2-V64I assay are listed in Table 2
. The first seven samples were estimated to have no contribution
from either Allele 1 or Allele 2 and thus were inferred by the software
to have failed PCR amplification (No Amp). The next seven samples were
estimated to have intermediate contributions of both alleles and were
assigned heterozygous status. The next sample had a low estimated
Allele 1 contribution and a high Allele 2 contribution. Because of
this, it was inferred by the software to have only an Allele 2
contribution and thus to be an A/A homozygote. The remaining seven
samples listed in Table 1
had a high estimated Allele 1 contribution
and a low estimated Allele 2 contribution. These samples were inferred
by the software as to match the Allele 1 control and thus to be G/G
homozygotes.
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comparison with restriction enzyme and sscp assays
SDF1-G801A is a MspI restriction site
polymorphism, which allows PCR-restriction enzyme analysis. Fig. 3
aligns the SDS genotype call (Fig. 3A
) with the 3% agarose gel
electrophoretic pattern (Fig. 3B
) for 24 samples that were subjected to
both TaqMan and MspI restriction enzyme analysis. In all
cases, the SDS call was concordant with the genotype inferred from the
electrophoretic band pattern. The electrophoretic band patterns of the
genotype controls match those predicted from electronic digestion of
allele-specific 302-bp reference amplicons. Genotype calls were also
concordant for CCR5-+/32, for which
the 32-bp deletion was resolved simply by length differences of PCR
products on a 6% polyacrylamide gel, and for the SSCP pattern of
CCR2-V64I.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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32,
CCR2-64I, and SDF1-801A.
Whereas the CCR5-+/32 locus
comprised alleles of different lengths, CCR2-V64I and
SDF1-G801A were examples of SNPs. Thus, the
CCR2-V64I and SDF1-G801A assays increase the
number of SNPs for which a successful TaqMan assay has been reported.
A major consideration for large-scale SNP scoring is the frequency with
which a designed assay actually yields acceptable discrimination. This
is particularly relevant to TaqMan assays because, whereas the probe
cost per sample is low when a large number of samples are scored, the
synthesis of dually labeled probes remains expensive. During
development of the three assays presented here, the first set of
CCR5-+/32 and CCR2-V64I
primers and probes designed with the aid of Primer Express yielded
excellent discrimination. Although the first combination of
SDF1-G801A primers and probes gave poor discrimination, a
second primer set lying completely outside the original pair yielded
excellent discrimination. These results suggest that probe
hybridization technology is advancing to the point of enabling routine
design of TaqMan assays.
Compared with restriction enzymes, SSCP, and gel electrophoresis, TaqMan allelic discrimination is much faster and easier. Primer Express simplifies complicated assay design, and the elimination of post-PCR molecular biology steps reduces labor and supply costs as well as the risk of contamination by PCR product. Use of a single set of reagent concentrations and thermal cycling conditions eases logistics at the bench. Genotype calls are electronically captured, thereby eliminating human error associated with manual recording of gel electrophoretic data. Because several thermal cyclers can be used in parallel and plates can be read on the Prism 7700 using SDS software in end-point mode, hundreds of samples can be processed daily by a single technician.
As indicated earlier in the text, several methods are available for SNP scoring (14). Each method has advantages and disadvantages. Some, like sequencing by hybridization chips, are currently more amenable to scoring many SNPs on a few people (18). Others, like TaqMan, currently are amenable to scoring a few SNPs on many people. Because of this, no one SNP scoring method is likely to have universal appeal. However, only a few methods are likely to have widespread use. The methods that dominate will do so based on cost and ease of use.
Interestingly, there are two organizational strategies for performing TaqMan assays on a microtiter plate. Here, we processed many DNA samples for a single SNP. The reverse strategy is to process a single DNA sample for many SNPs. This latter strategy is commercially available only for TaqMan gene expression studies. As density increases from 96 to 384 and on to 1536 wells, this strategy converges on the chip strategy (18).
One application of TaqMan technology is forensics. A recent report indicated that only 50 SNPs, each with a minor allele frequency of 0.20, are needed to equal the statistical power of the current forensic standard of 13 short-tandem repeat loci (6). This number of SNPs can be accommodated in the current 96-well format.
With respect to cost, the nature of the TaqMan assay eliminates much of the labor involved with SNP scoring. Because TaqMan requires only PCR and fluorescent detection, it has great potential for automation (26). Reagent costs can be reduced by reducing reaction volume, and a miniature TaqMan system has been reported (28). Although the practical limit of commercially available technology is currently in the 5-µL range, microfluidic devices may soon reduce practical volumes to 1 µL or less. Together, these advantages suggest that TaqMan SNP assays may be a generally useful SNP scoring strategy for clinical diagnostics, research, and forensics.
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Acknowledgments |
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Footnotes |
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1 Current address: Silicon Genomics, Greenbelt, MD 20770. 
2 Nonstandard abbreviations: SNP, single nucleotide polymorphism; SDF, stroma-derived factor; SSCP, single-strand conformational polymorphism; TAMRA, 6-carboxytetramethylrhodamine; FAM, 6-carboxyfluorescein; ROX, 6-carboxy-X-rhodamine; and SDS, Sequence Detection System.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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32 AIDS-resistance allele by the coalescence of haplotypes. Am J Hum Genet 1998;62:1507-1515.
[ISI][Medline]
[Order article via Infotrieve]
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, G/G (1/1) homozygote; 
, G/A (1/2) heterozygote.