Medical Device Link.

Originally Published IVD Technology July 2004

Molecular Diagnostics 

Figure 1. Workflow of the BD ProbeTec ET system for direct genotyping of SNPs from whole blood, dried blood, buccal swab, mouthwash, and urine samples (click to enlarge).

The analysis of genetic differences among individuals can potentially play a valuable role in the management of disease. It holds great promise for disease diagnosis, determination of predisposition to illness, and prediction of drug metabolism and therapeutic response. Single nucleotide polymorphisms (SNPs) are the most common form of genetic variation within the human genome, occurring with a frequency of once approximately every 1000 bases.^1,2 With some medical conditions, such as sickle-cell anemia, these subtle genetic differences are known to be directly associated with the disease phenotype, whereas with others, such as the Factor V Leiden mutation associated with hereditary thrombophilia, they are merely markers of possibly higher susceptibility to complex diseases.

In addition to being associated with disease states, SNPs also moderate the efficacy of therapeutic regimens to some extent, either through their effect on drug metabolism or by directly affecting drug targets or the pathways associated with their function. Patients often exhibit a great deal of heterogeneity in the way they respond to particular medications as a result of their individual SNP distributions. Genetic screening may be able to ameliorate such variation in therapeutic efficacy by identifying patients who will not respond, or who will respond idiosyncratically, thus allowing the drug regimen or dosage to be tailored specifically to suit an individual’s genetic profile. Genetic testing and pharmacogenomics are already contributing to the improvement of healthcare and the development of more-efficacious drugs. The genotyping of an individual in order to diagnose a disease, predict response to a drug, and determine an appropriate course of treatment is becoming an important, if not yet routine, diagnostic procedure.

Table I. Technologies used for the detection of SNPs (click to enlarge).

This article briefly reviews the state of the art in the detection of SNPs and other genetic variations, and then provides experimental data to illustrate the potential utility of one system as a platform for routine genetic analysis.³ Based on an alternative to polymerase chain reaction (PCR) technology, the novel system features the following:

• A homogeneous format for amplification and detection, with no manipulation of amplicons.
• A universal detection system and generic reaction buffer that allow discrimination at any given locus of two alternative alleles in a single well under standardized assay conditions.
• Direct genotyping of DNA from clinical samples without the need for extensive sample processing.
• Applicability to a wide range of specimen types.
• Streamlined work flow and rapid time to results.

Techniques of SNP Analysis

Figure 2. The sequence of events in the first stage of strand displacement amplification. S1 and S2 are SDA primers. B1 and B2 are bumper primers (click to enlarge).

The sequencing of the human genome and a rapidly growing understanding of the genetic basis of certain diseases and of how specific drugs are metabolized have led to an urgent need to develop fast, reliable, and economical methods of detecting SNPs and other genetic variations in the clinical laboratory. In fact, many high-throughput technologies for genetic analysis have been developed in recent years, which have been employed extensively in the discovery of SNPs and other genetic variations (see Table I).^{4, 5} 

Reliance on PCR Amplification. The techniques listed in the table have various pros and cons in terms of throughput, cost, and ease of use. However, a common feature among the large majority of them is reliance on PCR amplification of the target locus to provide a highly concentrated population of homogeneous DNA molecules for subsequent analysis. While it is possible to interrogate relatively small amounts of starting genetic material in this manner, many of these procedures are practically restricted to use in the research laboratory owing to the complexity and cost of the specialized instrumentation required. Some of them—for example, mass spectrometry—are amenable to automation and have utility for screening large numbers of samples in a search for novel markers. But others, for example, real-time nucleic acid amplification, can be employed only to detect known mutations.

Techniques based on real-time amplification and detection are unquestionably the ones most convenient and appropriate for use in a clinical setting. This is because they avoid the need to manipulate amplicons, thereby minimizing the pitfalls associated with contamination of the laboratory environment. Nevertheless, because almost all are based on PCR technology that is sensitive to the presence of inhibitors, these methods typically require the use of purified genomic DNA as the starting material.

In this regard, strand displacement amplification (SDA) may offer a particular advantage over PCR with TaqMan probes or molecular beacons. SDA is an isothermal nucleic acid amplification method based on the primer-directed nicking activity of a restriction enzyme and the strand displacement activity of an exonuclease-deficient DNA polymerase.^6-8 A specific target sequence may be amplified exponentially up to 10 billion–fold in less than 15 minutes, without the need for temperature cycling; typical doubling times are 20–30 seconds.⁸

Resource Requirements. A variety of clinical samples are used in genetic analysis. The most common types are fresh or frozen whole blood, buccal swabs, and mouthwashes. Alternative specimen materials that may be useful in particular circumstances include sputum, urine, stool, biopsy material, and dried blood spots.

Figure 3. The exponential phase of SDA (click to enlarge).

Often, the labor-intensive and time-consuming chore of specimen processing can become a significant bottleneck in a busy laboratory, particularly if a large number of specimens is involved. All of the samples may require repeated centrifugation and hands-on manipulation in preparation for analysis. Another important resource factor is the cost of reagents for nucleic acid extraction. And while automated and semiautomated nucleic acid extraction platforms do exist, they are not necessarily able to handle all types of specimen. These platforms may also be prohibitively expensive for the small or medium-size laboratory.

The choice of a technology to use for a particular genetic test is driven, therefore, not only by the type of mutation or variation under investigation—as well as the perennial intellectual property and licensing issues—but also by considerations of test volume, reagent cost, and the available technical resources for both specimen processing and the assay system itself. Perhaps the key clinical laboratory attributes of concern are the degree of technical expertise required of the individuals running the assays and the ability of the analytical system to meet the anticipated test volume.

A System for SDA-Based SNP Analysis

Table II. Genotyping of b2AR alleles on the BD ProbeTec ET System using matched specimens from 10 donors. The r-Max scores above +1.5 indicate homozygous allele A, those below –1.5 homozygous allele B, and those between –1.5 and +1.5 heterozygous allele A/B (click to enlarge).

The BD ProbeTec ET system, developed by BD Diagnostics (Sparks, MD), is a temperature-controlled kinetic fluorescence reader specifically designed for use in a clinical setting. It has been widely adopted for screening urogenital specimens for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC), and is used to detect the presence of Mycobacterium tuberculosis (TB) in respiratory specimens.^9-15 The system is based on the isothermal SDA method with real-time homogeneous detection of the amplified products.^{6-8, 16} The CT, GC, and TB amplified DNA assays for this reader employ a 96-microwell format with dried reagents, including enzymes, that are rehydrated with the processed specimen. Adding another advantage to the speed of isothermal amplification, this reagent configuration provides a simple work flow and rapid time to results, making this probe system particularly amenable to high-throughput clinical laboratories.

With the BD ProbeTec ET system, real-time SDA technology was adapted for the detection of SNPs directly from clinical specimens without any need for extensive nucleic acid purification (see Figure 1). Experiments are described here in which whole blood or dried blood on FTA Micro cards (Whatman Inc.; Clifton, NJ) was added directly to SDA buffer, heated for 5 minutes to lyse the cells and denature the target DNA, and then centrifuged at 10,000 times gravity for 1 minute. The centrifugation removed debris from the sample prior to transfer to microwells containing a priming mix consisting of primers, detector probes, and nucleotides. In the case of mouthwashes and urine specimens, cells were first concentrated by centrifugation at 10,000 times gravity for 1 minute and then resuspended in SDA buffer prior to lysis, denaturation, and recentrifugation as just described.

At the same time that denatured target DNA was added to the priming microwells, an amplification mix of SDA enzymes was dispensed into a second set of microwells. The priming microwells were then heated to 72°C and the amplification microwells warmed at 54°C. After incubation for 10 minutes, liquid was transferred from the priming to the amplification microwells. The latter were then sealed and loaded into the instrument.

Figure 4. The universal fluorescence detection format for homogeneous SDA-based SNP analysis (click to enlarge).

Fluorescence readings were collected over 30–60 minutes, depending on the experiment, and downloaded for analysis. Although the results presented in this article were generated using liquid reagents, it is important to note that the procedure for genotypic analysis can readily be adapted to employ the same dried-reagent format that the commercially available CT, GC, and TB assays use.

Principles of Analysis

The concepts behind SDA-based detection of SNPs can be clearly illustrated (see Figures 2 and 3).

Primer Extension. The probe system allows two alternative alleles to be discriminated at any given locus. Its method is based upon the relative efficiencies of DNA polymerase extensions of unlabeled, allele-specific adapter primers. In each reaction, a pair of adapter primers is present. The target-specific regions of those differ by a single nucleotide corresponding to the SNP locus, which is at the penultimate base (N – 1) from the 3' end. Each adapter primer is specific for one of two allelic variants—either allele A or allele B at the targeted locus—and the relative signal intensity generated through extension of each primer is used to determine the genotype of the sample.

In Figure 2, step 1 depicts bumper primer B1 hybridizing to single-stranded DNA target upstream of SDA primer S1. DNA polymerase extension from the 3' ends of B1 and S1, shown in step 2, results in displacing the S1 extension product (S1-ext) into solution. In step 3, S1-ext hybridizes to SDA primer S2 and upstream bumper primer B2. Extension from the 3' end of B2 (step 4) displaces the downstream S2 extension product (S2-ext) into solution. The fifth event in the sequence is hybridization of an S1 primer to S2-ext. Next, extension from the 3' end of the hybridized S1 primer results in formation of a double-stranded molecule with nickable restriction sites at either end (nicking is indicated by small red arrows). Finally, in step 7, nicking and DNA polymerase extension from the nick sites displaces single-stranded molecules into solution. These molecules possess partial Bacillus stearothermophilus derived restriction enzyme (BsoBI) recognition sites (designated by thick red lines) at either end (S1-amp and S2-amp; nicked/partial BsoBI recognition sites are shown by double red lines). They feed into the exponential phase of SDA, which is depicted in Figure 3.

The displaced single-stranded molecules S1-amp and S2-amp generated by the sequence of events just described hybridize to SDA primers S1 and S2 (the first event in Figure 3). DNA polymerase extension, nicking, displacement, and regeneration of the nick site occur next. Then, displaced single-stranded molecules with partial BsoBI sites at either end circulate back into step 1 of the Figure 3 sequence to bring about exponential amplification.

Figure 5. The universal detection format for the discrimination of two alleles in a single reaction. Hybridization of a correctly matched adapter primer ([a] and [c]) leads to efficient extension and generation of an allele-specific fluorescent signal (click to enlarge).

Universal Detection. The universal detection format of the ProbeTec ET system enables discrimination of multiple SNPs by means of a single pair of fluorescently labeled detector probes. An important feature of this detection method is that the probes, which are relatively expensive and time-consuming to make by comparison with unlabeled primers, do not have to have any homology with the target nucleic acid sequence (see Figures 4 and 5).

The detection format for homogeneous SDA-based SNP analysis involves the following sequence of events, with reference to Figure 4: An adapter primer hybridizes to the amplified target downstream of an SDA primer (step 1). This adapter primer comprises an allele-specific 3' sequence (A in the figure) 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). That product is then captured by a complementary SDA primer (3).

Simultaneous extension from the 3' ends of the SDA primer and the adapter primer extension product generates the complement of the 5' adapter primer tail sequence and a double-stranded restriction recognition site (4). Then, nicking of the restriction site followed by 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, step 6, leads to opening of the detector probe hairpin, separation of the fluorophore and quencher, and generation of target-specific fluorescence, step 7. Finally, additional fluorescent signal is produced by cleavage of the double-stranded detector probe restriction site (8).

Figure 6. Representative SDA amplification curves reflect genotyping of the –367 b2AR SNP locus performed on three blood samples, characterized by a homozygous C allele (sample 1), a homozygous T allele (sample 2), and a heterozygous C/T allele (sample 3), and a control that contained water instead of a blood sample (click to enlarge).

Single-Reaction Discrimination. Each SDA reaction contains two adapter primers and two universal detector probes. Each adapter primer is specific to one of two allelic variants, and possesses a 5' tail sequence homologous to one of the universal detector probes. Hybridization of an adapter primer correctly matched to the target sequence, followed by extension, generates allele-specific fluorescent signal. The diagnostic nucleotide is located one base from the 3' end of the adapter primer; this is to maximize discriminatory power when it is mismatched with the target sequence. A mismatch at that N -1 position results in less-efficient extension than when the adapter primer sequence is perfectly matched. This process of single-reaction discrimination of two alleles is schematized in Figure 5. The ratio of signals obtained from each optical channel over the course of the reaction is used to determine the identity of the nucleotide at the targeted locus.

The use of universal detector probes can significantly reduce the cost and complexity of developing novel assays because a single pair of labeled oligonucleotides can be employed across multiple analytes. In the examples to be discussed, the development of each assay was simplified further by means of employing generic buffer conditions and using the same concentrations of SDA enzymes for each analyte.

Allelic Discrimination and Data Analysis

To determine the utility of the BD ProbeTec ET system for genetic analysis, novel SDA-based assays were developed for the detection of SNPs at six different loci within the human b2-adrenergic receptor (b2AR) gene. This gene is associated with the response of asthma patients to treatment with b-agonists.3 Such haplotype analysis has become increasingly important in the emerging field of pharmacogenomics. Phenotypic responses frequently involve the interaction of several loci throughout the genome, and individual SNPs can predict them only poorly.

Figure 7. Results of SDA-based SNP analysis conducted on the b2AR –654 SNP locus using whole unprocessed blood from three individuals in sample volumes ranging from 0.14 to 20 µl (equivalent to 0.1 to 13% of final reaction volume). Homozygous alleles (G/G and A/A) have r-Max values above +1 and below –1, while r-Max values for the heterozygous allele G/A fall between +1 and –1. The bar graphs at right indicate the frequency with which particular r-Max scores were obtained (click to enlarge).

All of the new assays were designed to employ a common pair of universal fluorescent-energy-transfer detector probes that allow discrimination of the two alternative alleles at each locus. For each microwell, two readings were taken every minute during the course of the reaction: one corresponding to the rhodamine (ROX)-labeled detector probe (allele A) and another corresponding to the fluorescein (FAM)-labeled detector probe (allele B). A maximum-density function was then applied to determine the most likely log-ratio value for the two signals over time. This number was output as the r-Max score.

For all six of the b2AR assays, a high positive r-Max value (above +1.5) was considered to indicate homozygous allele A, whereas a low negative r-Max value (below –1.5) was considered to represent homozygous allele B. Values close to zero, that is, between –1.5 and +1.5, were deemed to indicate a heterozygous allele A/B genotype.

SDA amplification curves for the –367 b2AR SNP locus were generated from 3-µl samples of unprocessed whole blood taken from three individuals (see Figure 6). It should be noted that the SDA amplicon for this locus has a G+C nucleotide content of 78%. The graphs show the increase in fluorescence over time in each of the two optical channels, FAM and ROX. Blood sample 1 is homozygous for nucleotide C at the –367 position, with a strong ROX signal and low background fluorescence in the FAM channel. By contrast, blood sample 2 is homozygous for nucleotide T at this locus, with a strong FAM signal but only background signal in the ROX channel. The third sample is heterozygous at the –367 locus, having C at this position on one chromosome and T on the other. With sample 3, signal of approximately equal intensity was detected in both the FAM and ROX channels. The negative control contained no human DNA target and therefore generated no signal above background in either optic.

Typically, SDA-based SNP assays can be performed using 3 µl of whole, unprocessed blood spiked directly into the reaction mixture without prior extraction or purification of the DNA. Data similar to those shown in Figure 6 have also been obtained with the –654 b2AR SNP assay using 0.14 to 20 µl of whole, unprocessed blood per reaction, representing as much as 13% of the total reaction volume (see Figure 7). These data illustrate the reproducibility and robustness of the SDA-based SNP technology for genotyping clinical specimens without the need for costly and time-consuming nucleic acid isolation. In the experimental samples, blood specimens were collected using K2EDTA as the anticoagulant. However, samples collected with heparin or citrate anticoagulant have also been shown through experiment to exhibit similar compatibility with SDA.

Genotyping from Alternative Specimen Matrices

Sha-Sha Wang, PhD, is a project scientist, Keith E. Thornton, PhD, is a senior scientist, Andrew M. Kuhn, PhD, is statistics manager, James G. Nadeau, PhD, is a research fellow, and Tobin J. Hellyer, PhD, is R&D manager for molecular diagnostics at BD Diagnostics (Sparks, MD). The authors can be reached at sha-sha_wang@bd.com,  keith_thornton@bd.com,  max_kuhn@bd.com,   jnadeau@bd.com, and tobin_ hellyer@bd.com,  respectively.

While whole blood is the most common specimen for routine genetic analysis, its collection requires an invasive procedure. Either trained members of staff or the patients themselves must use lancets to collect blood and then properly employ filter paper cards for preservation and transport of the specimens. But recent studies suggest that sample types that are less difficult to obtain, such as buccal swabs, mouthwashes, and urine, also contain sufficient human DNA for routine genotyping.

To investigate this potential, matched purified DNA, frozen blood, dried blood, buccal swab, mouthwash, and urine specimens from 10 different donors were genotyped using the –654 and –367 b2AR SNP assays. Matched samples from each individual yielded identical b2AR genotypes that were in complete concordance with DNA sequence analysis (see Table II). Ther-Max values and genotyping results displayed in Table II were generated with a 30-minute amplification period. Including the length of time required for specimen preparation, SDA-based genotyping results can therefore be obtained within 1 to 1.5 hours depending on the level of template DNA, the specimen type, and the specific mutation under investigation.

Conclusion

SDA-based SNP analysis is a simple, fast, and accurate method of genotyping that precludes the need for extensive sample processing and postamplification manipulation of reactions. With a universal detection format that accommodates many different analytes, SDA offers the opportunity for a simple, standardized protocol for genotypic analysis that is applicable to the vast majority of SNPs and across a broad range of clinical samples. A system for SDA-based genotyping has been shown to provide a high-throughput, user-friendly assay format for the clinical laboratory.

References
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10. D Akduman et al., “Evaluation of a Strand Displacement Amplification Assay (BD ProbeTec-SDA) for Detection of Neisseria gonorrhoeae in Urine Specimens,” Journal of Clinical Microbiology 40 (2002): 281–283.

11. B Van Der Pol et al., “Multicenter Evaluation of the BD ProbeTec ET System for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae in Urine Specimens, Female Endocervical Swabs, and Male Urethral Swabs,” Journal of Clinical Microbiology 39 (2001): 1008–1016.

12. RP Verkooyen et al., “Reliability of Nucleic Acid Amplification Methods for Detection of Chlamydia trachomatis in Urine: Results of the First International Collaborative Quality Control Study among 96 Laboratories,” Journal of Clinical Microbiology 41 (2003): 3013–3016.

13. J Maugein et al., “Evaluation of the BD ProbeTec ET DTB Assay for Direct Detection of Mycobacterium tuberculosis Complex from Clinical Samples,” Diagnostic Microbiology and Infectious Disease 44 (2002): 151–155.

14. G Mazzarelli et al., “Evaluation of the BD ProbeTec ET System for Direct Detection of Mycobacterium tuberculosis in Pulmonary and Extrapulmonary Samples: A Multicenter Study,” Journal of Clinical Microbiology 41 (2003): 1779–1782.

15. GE Pfyffer et al., “Performance Characteristics of the BD ProbeTec System for Direct Detection of Mycobacterium tuberculosis Complex in Respiratory Specimens,” Journal of Clinical Microbiology 37 (1999): 137–140.

16. JG Nadeau et al., “Real-Time, Sequence-Specific Detection of Nucleic Acids during Strand Displacement Amplification,” Analytical Biochemistry 247 (1999): 130–137.  

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