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Clinical Chemistry 47: 852-857, 2001;
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(Clinical Chemistry. 2001;47:852-857.)
© 2001 American Association for Clinical Chemistry, Inc.

Articles

Fluorescence-based Detection of the CETP TaqIB Polymorphism: False Positives with the TaqMan-based Exonuclease Assay Attributable to a Previously Unknown Gene Variant

Daniel Teupser1a, Wolfgang Rupprecht2, Peter Lohse2 and Joachim Thiery1

1 Institute of Laboratory Medicine, Clinical Chemistry, and Molecular Diagnostics, University Hospital Leipzig, Liebigstrasse 27, 04103 Leipzig, Germany.

2 Institute of Clinical Chemistry, Grosshadern, University Hospital Munich, Marchioninistrasse 15, 81377 Munich, Germany.

aAuthor for correspondence. Fax 49-341-9722209; e-mail daniel{at}teupser.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Previous studies have shown an association between the TaqIB polymorphism of the cholesteryl ester transfer protein (CETP) gene with plasma CETP and HDL concentrations and the progression of coronary artery disease (CAD). The aim of the present study was to determine the performance of two new fluorescence-based detection systems in the analysis of the TaqIB genotype.

Methods: CAD patients (n = 150) with known TaqIB genotype, as determined by restriction fragment length polymorphism (RFLP) analysis, were selected, including three groups of 50 patients, carrying the B1/1, B1/2, and B2/2 genotypes, respectively. The genotypes were also analyzed by fluorescence-based allele-specific TaqMan PCR and melting curve analysis (LightCycler). In addition, DNA sequencing was applied.

Results: The TaqIB genotypes obtained by fluorescence analysis corresponded to those determined by RFLP analysis with the exception of three heterozygous patients (B1/2), who were misclassified as homozygous B2 carriers with the TaqMan system. Melting curve analysis of these samples demonstrated an additional melting point at 59.1 °C, which was also found in four patients homozygous for the B1 allele. DNA sequencing revealed a previously unknown C270T nucleotide exchange in intron 1 of the CETP gene, only nine base pairs from the TaqIB site.

Conclusions: Determination of the TaqIB polymorphism with the TaqMan system led to misclassifications because of a previously unknown C270T polymorphism of the CETP gene. The base substitution was detected with the LightCycler because of the occurrence of an additional melting point. Our data indicate the importance of thorough evaluation of new gene analysis systems before using them on a routine basis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cholesteryl ester transfer protein (CETP)1 is a key protein in reverse cholesterol transport. It is a 74 000-kDa plasma glycoprotein, facilitating the transfer of cholesteryl esters from HDL to apolipoprotein B-containing lipoproteins. Several molecular defects leading to CETP-deficiency have been identified [for a review, see Ref. (1)]. In addition, several polymorphisms of the CETP gene have been described (2). The significance of CETP polymorphisms in the development of atherosclerosis is still controversial (3)(4).

The TaqIB polymorphism is one of the polymorphisms of the CETP gene that has been studied in detail. It is located in intron 1 of the CETP gene (5). The presence of the TaqI restriction site was designated B1 and its absence as B2 (6). Individuals carrying the B2 allele showed lower CETP activity (7) and CETP mass concentrations (8) and higher HDL-cholesterol (5)(6)(8)(9)(10) and apolipoprotein AI plasma concentrations (6)(8)(10).

The role of the TaqIB polymorphism in the progression of coronary atherosclerosis has recently been studied in a subgroup of the Regression Growth Evaluation Statin Study (REGRESS) collective. It was confirmed that the B1 variant of the CETP gene was associated with both higher CETP and lower HDL-cholesterol concentrations. In addition, the B1 allele was dose-dependently associated with a faster progression of coronary atherosclerosis, as indicated by a decrease in mean luminal diameter, in the placebo group. This association was abolished by pravastatin. Pravastatin therapy slowed the progression of coronary atherosclerosis in patients homozygous for the B1 allele but not in those homozygous for the B2 allele (4).

The results of the studies mentioned above make the CETP TaqIB polymorphism an interesting genetic variation to study in large atherosclerosis trials. In previous studies, the TaqIB polymorphism was analyzed by digestion of DNA with the restriction enzyme TaqI and subsequent detection of the fragments either by agarose gel electrophoresis or by hybridization. However, restriction fragment length polymorphism (RFLP) analysis is time- consuming and difficult to automate because it requires postamplification procedures, such as restriction enzyme digestion and electrophoresis. Currently, two fluorescence-based PCR detection systems, the LightCycler (Roche Diagnostics) and the TaqMan SDS 7700 (Applied Biosystems), have become available that can be applied to screen large study cohorts for polymorphisms within a short period of time. The short analysis time is mainly achieved by performing PCR and the subsequent fluorescence analysis of the PCR products in the same run.

Thus, the aim of our study was to compare the performance of the new detection systems (the TaqMan SDS 7700 and LightCycler) to determine the CETP TaqIB polymorphism in 150 patients with known TaqIB genotypes previously characterized by RFLP analysis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
patients and dna preparation
Patients who had been admitted for cardiac bypass grafting (n = 150), were included in the study. The CETP TaqIB polymorphism of the patients had been previously determined by RFLP analysis. Three groups of 50 patients each carrying the B1/1, B1/2, or B2/2 genotypes were selected. All patients had given informed consent to participate in the study. DNA was isolated from whole EDTA blood, using the QIAamp DNA Blood Mini Kit according to the recommendations of the supplier (QIAGEN).

genotyping by rflp
Genotyping of the TaqIB polymorphism by RFLP was essentially performed as described previously (5). Briefly, a 991-bp fragment of intron 1 of the CETP gene was amplified using the following primer pair: 5'-CAG GGG TCT TTT CAT GGA CAC-3' (forward) and 5'-CAC TTG TGC AAC CCA TAC TTG ACT-3' (reverse). The PCR product was subsequently digested at 65 °C with the restriction enzyme {alpha}-TaqI (New England BioLabs) and analyzed on a 2% agarose gel. The presence of the TaqI restriction site led to the generation of two fragments (249 and 742 bp) and was designated B1, the absence of the TaqI restriction site was designated B2 (5).

genotyping by allele-specific taqman pcr
This PCR method is based on the accumulation of fluorescence during the nucleolytic degradation of an internally quenched allele-specific probe. However, probes that differ from the template sequence by as little as a single nucleotide exhibit a lower melting point temperature and remain intact during PCR. They will be displaced from the template strand but not degraded by the exonuclease activity of Taq polymerase. The correct extension temperature is crucial for the performance of the assay.

Allelic discrimination is achieved by the use of two oligonucleotide probes, each complementary to one of the two alleles and labeled with a different fluorescent reporter dye. The design of suitable probes is dependent on the sequence of the two alleles. To our knowledge, the sequence of the B2 allele has not been published; therefore, we sequenced DNA from three patients carrying the B2/2 isoform as determined by RFLP analysis. In the B2 allele, we found a single G->A base exchange at nucleotide position 279 of intron 1 in all three patients (start of intron 1 according to GenBank Accession Nos. M32992 or J02898, corresponding to nucleotide position 784 of that sequence). With this information, we designed primers and probes for the allelic discrimination assay. The TaqMan probe for detection of the B1 allele (5'-TGA ACC CTA ACT CGA ACC CCA GTG AT-3') was 5'-labeled with the reporter dye 6-carboxyfluorescein (FAM), the TaqMan probe for the B2 allele (5'-TCT GAA CCC TAA CTT GAA CCC CAG TGA T-3') was 5'-labeled with VIC® (Fig. 1A ). The underlined nucleotides indicate the position of the base exchange of the TaqMan polymorphism. The primers 5'-TGT CTG CGA CCC AGA ATC ACT-3' (forward) and 5'-ACC CCC TAA CCT GGC TCA GA-3' (reverse) were used for amplification. The primers and TaqMan probes were synthesized by Applied Biosystems.



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  		Figure 1. Orientation of the fluorescence-labeled TaqMan probes (A) and representative experiment showing the automatic assignment of the genotypes as B1/1, B1/2, and B2/2 by the TaqMan system (B).

(A), the polymorphic site of the TaqIB polymorphism is underlined. The boxed C indicates the position of the previously unknown C270T polymorphism on the B1 allele. (B), standardization of the assay was performed with eight control samples without DNA, eight control samples containing DNA from a patient homozygous for the B1 allele, and eight control samples with DNA from a patient homozygous for the B2 allele. The assignment of the genotypes in the samples was automatically performed by the software with a series of mathematical transformations according to the fluorescence signal in the tubes after PCR. The quantitative data on the axes of the graph show the correlation of the unknown samples with the controls. One sample from a patient with the B1/2 genotype was incorrectly identified as B2/2 homozygous. TAMRA, 6-carboxytetramethylrhodamine.

The PCR reaction was performed in a volume of 25 µL containing 12.5 µL of TaqMan 2x Universal PCR Master Mix, with ROX as passive reference (PE Applied Biosystems) and 4 µL of genomic DNA (80–240 ng). The final concentration of the oligonucleotides was 400 nM for each primer and 600 nM and 175 nM for the labeled B1 and B2 probes, respectively. The PCR program included one hold at 50 °C for 2 min, followed by one hold at 95 °C for 10 min, and 43 cycles at 95 °C for 15 s for denaturing and 62 °C for annealing/extension. PCR, endpoint reading of fluorescence, and analysis of the data were performed with the 7700 Sequence Detection System (Applied Biosystems).

genotyping by melting curve analysis (lightcycler)
In contrast to the TaqMan system, allelic discrimination with the LightCycler is based on the generation of a melting profile after PCR amplification of the target sequence. The polymorphic site of the sequence is spanned by a fluorescein-labeled "detection probe", complementary to the sequence of one of the alleles (the B1 allele in this case). Next to it, a second probe, designated the "anchor probe", is designed to bind more stably to the target DNA. It is labeled with LightCycler Red 640 at its 5' end, emitting fluorescence through fluorescence resonance energy transfer when the adjacent fluorescein of the detection probe is excited. A melting curve can be generated by continuous monitoring of fluorescence while increasing the temperature from annealing to denaturing conditions. The melting point of the probe can be detected by a rapid decrease of fluorescence, allowing the discrimination of different alleles by their specific melting points.

The primers 5'-TCT TTT CAT GGA CAC CCA CTA TG-3' (forward) and 5'-CCC CAA CAC CAA ATA TAC ACC A-3' (reverse) were used to amplify a 328-bp fragment of intron 1 of the CETP gene. The sequence of the detection probe 5'-AAC CCT AAC TCG AAC CCT AGT GAT TCT-3' was complementary to the CETP B1 allele with the exception of an additional mismatch at nucleotide 272 to increase the discrimination of the alleles (11). The detection probe was labeled with fluorescein at the 3' end. The anchor probe 5'-TCG CAG ACA AAC ACA AAT CCC TAT ACC TGG-3' was labeled with LightCycler Red 640 at its 5' end and 3'-phosphorylated to prevent extension. The anchor probe of the LightCycler assay was constructed on the basis of the information obtained from GenBank. The sequencing of intron 1 of the CETP gene later showed an adenine at position 257 instead of a cytosine (GenBank Accession Nos. M32992 or J02898) in all patients. The mismatch did not interfere with the LightCycler assay.

PCR was performed in a reaction volume of 10 µL containing 500 nM of each primer, 188 nM of the detection probe, and 113 nM of the anchor probe. DNA (1 µL), 0.9 µL of 25 mM MgCl2, and 1 µL of a LightCycler-DNA Master Hybridization Probes reaction mixture (containing Taq DNA Polymerase, reaction buffer, dNTP mixture with dUTP instead of dTTP, and 10 mM MgCl2; Roche Diagnostics) were added. The cycling conditions included one initial denaturation step (2 min at 95 °C) and 43 cycles of 95 °C for 0 s, 59 °C for 5 s, and 72 °C for 18 s with a temperature transition rate of 20 °C/s. After amplification was complete, melting curves were generated by denaturing the reaction at 95 °C for 0 s, followed by 10 s at 45 °C, and then by slowly heating the samples to 85 °C at 0.1 °C/s with continuous monitoring of fluorescence (F2/F1).

genotyping by sequencing
To determine the DNA sequence in the vicinity of the TaqIB site, we first amplified DNA samples by PCR with the primers and conditions as described above (genotyping by RFLP). The PCR products (40 µL) were subsequently purified using the QIAquick PCR purification method (QIAGEN), and the DNA concentration was determined. DNA (400 ng) was sequenced in each direction by dye-terminator chemistry using a 377 DNA sequencer (Applied Biosystems).

pcr mutagenesis
A fragment of the CETP gene with the C->T base exchange at position 270 on the B2 allele was generated by PCR mutagenesis using two complementary mismatch primers (5'-AGA ATC ATT GGG GTT CAA GTT AGG-3' and 5'-CCT AAC TTG AAC CCC AAT GAT TCT-3') and the forward and reverse primers used for LightCycler PCR. The presence of the nucleotide exchanges at positions 270 (C->T) and 279 (G->A) of the resulting fragment was confirmed by DNA sequencing.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Of the 150 patients with known CETP TaqIB genotype, as determined by RFLP analysis, 50 patients were homozygous for the B1 allele, 50 were homozygous for the B2 allele, and 50 patients were heterozygous (B1/2).

The samples were analyzed by allele-specific TaqMan PCR. Eight samples from a patient with the B1/1 genotype and eight samples from a patient carrying the B2/2 genotype, previously determined by DNA sequencing, were included in each assay for calibration. After PCR amplification, the fluorescence was read and the genotypes of the samples were automatically assigned with an algorithm according to the fluorescence signal of the calibrators (Fig. 1BUp ). All patients homozygous for the B1 or B2 alleles were correctly genotyped, in agreement with the results of the RFLP analysis. However, unexpectedly, 3 of 50 patients carrying the B1/2 genotype were misclassified as homozygous for the B2 allele by this method.

Samples from all patients were also analyzed by melting curve analysis in the LightCycler. With the increase of temperature, samples from patients carrying the B2 isoform showed a rapid drop of fluorescence at 55.4 °C corresponding to the dissociation of the fluorescence-labeled probe from the template. In most samples from patients carrying the B1 isoform, the decrease occurred at 63.3 °C because of the higher homology of sample and probe. In the first negative derivative (-dF/dT), the melting temperatures appeared as maxima, as shown in Fig. 2A . Heterozygotes (B1/2) showed composite melting peaks, representing the signals obtained from the B1 and B2 alleles.



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  		Figure 2. Melting profiles of representative samples of the three possible TaqIB genotypes (A) and melting profiles of one patient with the B1 270T/B1 genotype and from one patient with the B1 270T/B2 genotype (B).

(A), melting profiles of representative samples of the three possible TaqIB genotypes obtained by the LightCycler analysis. The two characteristic melting points were at 55.4 °C and 63.3 °C. (B), melting profiles of representative samples from one patient with the B1/1 genotype and from one patient with the B1/2 genotype, both carrying the C270T polymorphism on the B1 allele with the characteristic melting point at 59.1 °C. The melting curve of a B1/2 heterozygous patient without the polymorphism is given as the control.

The melting patterns in samples from four patients with the B1/1 genotype and from three patients with the B1/2 genotype were clearly divergent from the characteristic melting curves of the corresponding genotypes. The four B1/1 patients showed a melting point at 59.1 °C in addition to that at 63.3 °C. Samples from the three B1/2 patients also showed a shift in melting temperatures with one melting point at 59.1 °C and one at 55.4 °C (Fig. 2BUp ). Careful analysis of the melting curves of the samples from patients homozygous for the B2 genotype revealed no shift in the melting points. Interestingly, the three B1/2 patients showing the shift of the melting point from 63.3 °C to 59.1 °C were the same who had been previously misclassified as B2/2 by TaqMan allelic discrimination.

This observation prompted us to analyze the DNA sequences of all samples showing a melting point at 59.1 °C. We could confirm that the four B1/1 patients were homozygous for the B1 genotype (G at nucleotide position 279). In addition, we found that these B1/1 patients were heterozygous for a previously unknown C->T base exchange at position 270. The same single nucleotide polymorphism was also found in the samples from the three B1/2 patients with a melting point at 59.1 °C.

A comparison of the specificities of the three methods to analyze the TaqIB genotype in the study population is given in Table 1 . The melting-point data indicated that the C->T polymorphism occurred only on the B1 allele and led to a reduction of the melting temperature of the detection probe. To investigate the effect of the polymorphism on the reduction of the melting temperature if it occurred on the B2 allele, we generated a fragment of the B2 allele bearing the 270T variant. As expected, melting curve analysis of this fragment in the LightCycler led to a further destabilization of the detection probe with a melting point at 50.2 °C. We have not observed the occurrence of this melting point in any of our patient samples.


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  		Table 1. Specificity of mutation detection assays applied in the study to analyze TaqIB genotypes in 150 coronary artery disease patients.

To study the underlying causes of the limitations of the TaqMan assay to classify B1 270T/B2 samples correctly, we analyzed endpoint fluorescence measurements and continual monitoring of fluorescence in the quantification mode of the SDS 7700. Because VIC fluorescence is derived from nucleolytic degradation of a VIC-labeled probe complementary to the B2 allele, one could assume that in a heterozygous B1/2 situation, the increase of VIC fluorescence would be one-half that of a homozygous B2 sample. However, VIC fluorescence exceeded this value in our experimental situation, and there was overlap with B2 homozygotes depending on PCR efficiency. Thus, the assignment of the genotype had to be made taking the FAM data also into account. With this information, B2 homozygotes and B1/2 heterozygotes (without 270T variation) could be clearly discriminated because the FAM signal showed no overlap between both genotypes. In contrast, FAM fluorescence in B1 270T/B2 heterozygotes was within the range of B2 homozygotes and among those B2/2 samples with highest FAM signal. This was true for endpoint reading, as well as continual monitoring of fluorescence during PCR. Thus, based on the signals of FAM and VIC fluorescence, B1 270T/B2 samples resembled B2 homozygotes.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results indicate the importance of thoroughly evaluating new fluorescence-based PCR procedures before using them on a routine basis. The initial aim of this study was to determine the performance of two new fluorescence-based PCR methods in the analysis of the TaqIB polymorphism located in intron 1 of the CETP gene in 150 coronary heart disease patients.

In our study, the determination of the TaqIB polymorphism by allelic discrimination using TaqMan PCR led to misclassifications because of a previously unknown C270T variation in intron 1 of the CETP gene located only 9 base pairs upstream of the TaqIB site. The misclassification only appeared in B1/2 heterozygotes with the 270T variant on the B1 allele, which were classified as B2 homozygous. B1 homozygotes with the 270T variant were correctly classified. It appears that the vicinity of this single nucleotide polymorphism led to a destabilization and decreased melting temperature of the FAM-labeled TaqMan probe, which was unable to bind firmly to the variant B1 strand. The probe was thus only displaced from the strand and not cleaved by the exonuclease activity of the Taq polymerase, leading to a low FAM fluorescence signal. B1 homozygous samples were correctly assigned in the presence of the 270T variant because the FAM fluorescence was high (presence of one B1 270C allele) and VIC fluorescence was low (absence of a B2 allele), resembling the fluorescence pattern of a B1 homozygous sample. However, in case of B1 270T/B2 heterozygosity, the FAM signal was in the range of B2 homozygous samples because of the low binding affinity of the FAM-labeled B1 probe to the variant B1 strand. Because there was overlap of the VIC signal between B1/2 heterozygotes and B2 homozygotes, this situation was misclassified as B2 homozygous by the allele discrimination software.

Our data show that a TaqMan assay, which has 100% specificity and sensitivity for detecting the B1 or B2 alleles of the TaqIB polymorphism in the "wild-type" situation, failed when a previously unknown sequence variation occurred at the probe binding site that was not taken into account when the assay was designed. Theoretically, this situation can happen in any gene sequence at any position, and our data suggest that because of its biochemical principle, the TaqMan system is less flexible to circumvent this problem.

In contrast to the TaqMan data, the previously unknown C270T polymorphism did not interfere with the LightCycler assay and could be detected by the occurrence of an additional melting point at 59.1 °C. These results show that the determination of single nucleotide polymorphisms by the melting pattern of fluorescently labeled hybridization probes added safety to the analysis that was not provided by the TaqMan procedure. Different mismatches destabilize oligonucleotides to different extents and can be often distinguished (12)(13)(14). However, it must be pointed out that melting curve analysis can also lead to misclassifications when unexpected additional nucleotide exchanges in one allele led to a melting point comparable to that of the other allele (12). The LightCycler detection probe used in this assay had a mismatch at position 272 that was initially added to increase discrimination. We must point out that probes with artificial mismatches theoretically do not detect wild type with 100% specificity. Misclassifications may be encountered in the unlikely event that a Watson–Crick base pair forms at the mismatch site. However, the results of our study show that the LightCycler assay presented here had a specificity of 100% in the collective of patients tested.

Our results and findings from previous studies (12)(13)(14)(15) indicate that melting profiles generated by hybridization procedures should be interpreted with great care and sequencing should be applied when unusual patterns occur. With knowledge of the newly discovered polymorphism, an appropriate assay to determine the TaqIB polymorphism for the TaqMan system could be developed (e.g., by constructing a probe that does not overlap the polymorphic site at nucleotide 270). However, this was not the aim of the present study.

The interference of a previously unknown nucleotide exchange has been reported for the PCR-based restriction endonuclease assay of the factor V Leiden mutation (16). In one patient, a silent A->C transition at nucleotide 1692 (no amino acid exchange) led to the loss of the MnlI restriction site. Although the correlation of functional and restriction digestion assays suggests a low frequency of the nucleotide exchange (17)(18), it may nevertheless lead to an overestimation of the factor V Leiden mutation. This problem has been discussed by others describing fluorescence-based assays for factor V Leiden genotyping (14)(19). The effect of gene variants in the vicinity of mutations of interest on the melting pattern of hybridization probes has been shown for factor V, apolipoprotein B, cystic fibrosis, and hemochromatosis mutations (12)(13)(14)(15)(20). Recently, the discovery of previously unknown mutations with a probe-based design has been described (15)(21).

In the collective of coronary heart disease patients analyzed in our study, the newly discovered C270T polymorphism in intron 1 of the CETP gene was found only on the B1 allele with a frequency of ~5%. To confirm that we did not miss the polymorphism on the B2 allele, we generated a fragment with both polymorphisms (T at position 270 and A at position 279). As expected, the fragment could be distinguished clearly by its low melting point at 50.2 °C. These data indicate that, in our study population, the polymorphism was associated only with the B1 allele. We also did not observe patients homozygous for T at position 270 of intron 1, possibly because of the low frequency of the polymorphism. Determination of plasma HDL-cholesterol concentrations did not show an association with the C270T genotype, but the number of carriers of the T allele (n = 7) may have been too small (data not shown). We are currently studying the effect of the C270T variation on plasma lipids and lipoprotein concentrations and progression of coronary artery disease in a larger population.

In conclusion, we have shown that determination of the TaqIB polymorphism of the CETP gene with fluorescence-based PCR techniques is possible. However, thorough evaluation of the test systems is important to avoid unexpected misclassifications because of previously unknown sequence variations.


   Acknowledgments
 
We thank Wolfgang Wilfert for expert technical assistance and Olfert Landt of TIB MOLBIOL (Berlin, Germany) for design and synthesis of primers and probes for the LightCycler assay.


   Footnotes
 
1 Nonstandard abbreviations: CETP, cholesteryl ester transfer protein; RFLP, restriction fragment length polymorphism; and FAM, 6-carboxyfluorescein.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Single-Nucleotide Polymorphism Genotyping by Melting Analysis of Dual-Labeled Probes: Examples Using Factor V Leiden and Prothrombin 20210A Mutations
Clin. Chem., October 1, 2003; 49(10): 1669 - 1672.
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 J. Lipid Res.Home page
S. M. Boekholdt and J. F. Thompson
Natural genetic variation as a tool in understanding the role of CETP in lipid levels and disease
J. Lipid Res., July 1, 2003; 44(6): 1080 - 1093.
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 Clin. Chem.Home page
M. Erali, B. Schmidt, E. Lyon, and C. Wittwer
Evaluation of Electronic Microarrays for Genotyping Factor V, Factor II, and MTHFR
Clin. Chem., May 1, 2003; 49(5): 732 - 739.
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 Clin. Chem.Home page
K. Xu, R. H. Lipsky, W. Mangal, E. Ferro, and D. Goldman
Single-Nucleotide Polymorphism Allele Frequencies Determined by Quantitative Kinetic Assay of Pooled DNA
Clin. Chem., September 1, 2002; 48(9): 1605 - 1608.
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