IVD Technology
Magazine
IVDT Article Index
Originally Published May 2000
Technologies for mutation detection
Methods used to detect genetic mutations linked to various diseases will be the bases of a myriad of new molecular diagnostic tests. Manufacturers must consider many factors when deciding which of these technologies to use.
Angela Ryan, Richard Schifreen, John Shultz, Isobel MacIver, and Dan Kephart
As the Human Genome Project nears completion and more is understood about the relationship between genes, genetic mutations, and disease, many more assays will be developed to detect these specific conditions. Genetic-mutation detection is one of the fastest growing areas in clinical laboratories.
Choosing a mutation-detection method requires careful consideration of many factors. Clinical laboratory directors are seeking precise, dependable technologies to use in their assays. The tests must also be easy to use and cost-effective in relation to the amount that insurance companies will reimburse the laboratory. Accuracy in the 99.9% range and day-to-day reliability are the basic requirements for the acceptance of new technologies, but attributes such as cost and convenience are also important.
Accuracy
Genetic tests can have a profound impact on patients, who may receive results indicating that they are susceptible to cancer or other diseases. The symptoms of their diseases may be treatable but the underlying genetic mutation is permanent and can be transmitted to the next generation. Testing conducted many years prior to the onset of disease may negatively affect a patient's access to health and life insurance.
Incorrect diagnosis of a genetic mutation can have devastating consequences, so accuracy of 99.99% or higher is essential for such assays. To determine the accuracy of a technology, the new method is validated on thousands of samples in which the genotype has been previously determined with a "gold standard" method such as Southern blot or polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). If the results disagree, the sample is sent to be sequenced to determine which method gives the correct result.
Even with the most accurate method of analysis, sample preparation and amplification methods must be optimized to eliminate any potential inaccuracies. Published reports describe situations in which preferential amplification of one allele caused miscalls.1 Other publications document the selective in vivo degradation of mutant mRNA that occurred while the wild-type message remained intact, thereby masking the presence of the mutation.2
It is preferable to have a no-call result, which would require the test to be repeated, than a miscall that provides incorrect results that are then reported to the patient. Some methods of testing require the performance of duplicate assays to ensure accuracy, but this process also doubles the costs for reagents and labor.
Data interpretation can also affect accuracy. Wild-type, heterozygous, and homozygous mutant results must be clearly distinguished from one another.
Data-handling software should be a design priority and should incorporate protection against mixing up sample identification numbers and reporting erroneous results.
Reliability
No laboratory director wants to be in a situation in which he or she has to contact a physician to request another patient blood sample because the mutation-detection method in the assay failed. Day-to-day performance reliability is required because repeating tests increases cost and delays reporting of the results. The worst-case scenario is one in which the reagent or method has a limited failure with only one sample in a run and causes erroneous, but believable, results. Troubleshooting complex tests can be difficult unless both expert internal staff and reagent-supplier technical support are available.
Convenience
Simplicity, convenience, and ease of use are interchangeable and equivalent terms that represent the cornerstone of any suitable mutation-detection method for the clinical laboratory. A convenient assay reduces costs by minimizing hands-on time, eliminates the possibility of operator error causing erroneous results or run failure, and increases reliability. Potential for human error is determined to a large extent by assay design. For example, a poorly designed assay may require the user to conduct multiple pipetting steps or to add very low volumes of reagents. These tasks are both inconvenient and associated with high error rates.
Many methods are adapted from research protocols and are not configured for the workflow of a clinical laboratory. Research laboratories may use methods that require radioactivity, have multiple components that require manipulation, and use equipment not readily available in clinical laboratories. Also, the data that result from research efforts require interpretation, rather than providing clear-cut determinations.
Cost-Effectiveness
In today's clinical laboratories, cost-effectiveness is always a factor. The cost of the reagents used, the amount of time a laboratory employee spends performing the test, the cost of the equipment used, the size of the batch to be processed and the speed at which it can be analyzed, and the cost of the royalty burden imposed by the company that owns a technology patent (such as polymerase chain reaction [PCR]) can all contribute significantly to the overall cost-effectiveness of the technology.
Reagent price may only be a small component of total test cost. For example, a technology that uses inexpensive reagents may not be cost-effective if the labor required to perform the test is substantially increased by doing so. Conversely, the promise of reduced labor can be a significant benefit for a new technology. Minimizing hands-on steps in the test procedure, increasing batch size, and using true walkaway automation are all factors that reduce labor time.
The fixed costs associated with the instrumentation required for total automation must be less than the value of the labor saved by switching from a manual method of testing. Some suppliers will arrange reagent-rental agreements with the test developer, allowing the capital cost of the system to be amortized over the number of tests run. This will eliminate the drain on a limited capital-equipment budget and allow for clear cost comparisons between differing technologies.
Analyzing data and entering results manually into a laboratory information management system (LIMS) can require hours of technician time followed by further review by the laboratory director. In addition, manual entry can be prone to errors. An instrument that integrates effectively with the LIMS can both reduce cost and improve laboratory service.
Clinical labs are experiencing substantial cost increases due to the royalty burden associated with licensed technologies, especially PCR. Laboratories are reportedly forced to accept a royalty burden of between 9 and 15% of the total reimbursement (including sample preparation and interpretation) whenever PCR is used in performing home-brew assays for detecting clinically significant mutations. Technologies that eliminate the PCR royalty will be attractive if the savings are passed on to the customer.
Emerging Mutation-Detection Technologies
The choice of mutation-detection technologies is quickly expanding. New methods that claim to meet the needs of the clinical laboratory seem to be announced almost weekly. Of course, these claims do not always turn out as promised. Clinical laboratories must validate new technologies against their individual needs, but this is expensive in terms of real costs and technologist time, both always in short supply. Many labs will not even consider trying a new technology until it has been proven in the research community and has a published track record showing robustness and accuracy in clinical studies.
Two of the following technologies have been widely tested in the research community. The third is currently being developed for diagnostic applications.
TaqMan. TaqMan (PE Biosystems; Foster City, CA) is a real-time PCR method that allows one-step mutation detection. In PCR, forward and reverse primers hybridize to a specific sequence of the target DNA in order to amplify the target sequence. The TaqMan probe, with its bound fluorophore and quencher, hybridizes to a second target sequence within the amplified product. When the PCR product is further amplified in subsequent cycles, the AmpliTaq enzyme cleaves the TaqMan probe (5' nuclease activity) so that it can continue to copy its target sequence.
The reporter dye and quencher dye are separated, resulting in increased fluorescence of the reporter. This process occurs in every amplification cycle and does not interfere with the exponential accumulation of product. Because release of the reporter dye is associated with the amplification of the specific gene DNA, the fluorescent signal is generated only if the gene sequence is present in the sample.
The TaqMan methodology can detect as few as five copies of target in a background of 500 ng of DNA. The risk of PCR contamination is eliminated with this technology because detection occurs within the amplification reaction, thus also eliminating postamplification analysis costs.
In order to be used for diagnostic tests, TaqMan must be reconfigured since it is currently only used in the research community. New tests will have to be developed and validated.
Invader. The Invader technology from Third Wave Technologies (Madison, WI) eliminates the need for PCR and works with either DNA or RNA targets. Applications include single nucleotide polymorphism (SNP) and mutation detection in genomic DNA. The Invader assay detects specific DNA and RNA sequences by using structure-specific Cleavase enzymes to cleave a complex formed by the hybridization of overlapping oligonucleotide probes. Elevated temperature and an excess of one of the probes enable multiple probes to be cleaved for each target sequence present without temperature cycling. These cleaved probes then direct cleavage of a labeled probe.
The secondary probe oligonucleotide can be 5'-end labeled with fluorescein or another fluorophore that is quenched by an internal dye. Upon cleavage, the fluorescein-labeled product can be detected using a standard fluorescent plate reader. These sequential reactions produce 106 to 107 labeled cleavage products per target sequence per hour.
In beta-site testing, the Factor V Leiden Invader assay was determined to be 99.5% accurate when compared with PCR and 99.01% accurate when compared with PCR-RFLP.
With Invader, the amount of DNA or RNA added to the reaction must be quantified, which takes away from the ease of use. Invader assays are compatible with standard manual or automated detection systems, and work is under way to adapt the entire procedure to a completely automated format, according to the company. The clear cost advantage of this methodology is that PCR is not required, therefore no royalties are added to the assay price.
READIT. The Reversed Enzyme Activity DNA Interrogation Test (READIT) mutation-detection system from Promega (Madison, WI) detects the presence or absence of a particular DNA allele in a reaction. This molecular-analysis technology can analyze a variety of sequence variations, including insertions, deletions, SNPs, and chromosomal translocations. READIT can also provide multiplexed PCR and reverse transcriptase–PCR product detection in a high-throughput, arrayed-plate format. Instrumentation for both medium (up to 48) and large (more than 96) batch sizes is under development.
Figure 1. The READase polymerase and kinase act in concert to produce ATP. The coupled reaction is complete in 10 minutes.
The READIT technology is based on driving enzymatic reactions in the reverse direction by mass action. The system uses three different enzymes, the READase polymerase, READase kinase, and luciferase. In the presence of high levels of pyrophosphate, the polymerase catalyzes the addition of inorganic pyrophosphate across the terminal phosphodiester bond of duplex DNA (i.e., depolymerization or pyrophosphorolysis), resulting in the release of high-energy deoxynucleoside triphosphates (dNTPs). In a coupled reaction, the kinase transfers the gamma phosphate from the released dNTPs onto adenosine diphosphate (ADP) that is present in the reaction mixture. This results in the formation of adenosine triphosphate (ATP; see Figure 1). Once generated, the ATP is used by luciferase present in a second reaction to produce light (see Figure 2).
Figure 2. The luciferase/luciferin reaction generates a light signal proportional to the amount of ATP produced in the first reaction. The reaction and measurement take approximately 15 seconds using a single-tube luminometer.
Two interrogation probes are used to analyze a biallelic polymorphism (see Figure 3). The first probe is perfectly homologous to allele one (e.g., wild-type allele) and is positioned such that its 3'-end is in close proximity to the site to be interrogated. The second interrogation probe hybridizes to the same template position, but its 3'-end corresponds to allele two (e.g., mutant allele).
Figure 3. The specificity of the READIT reaction resides in the ability of the READase polymerase to differentiate between a probe/template that is perfectly matched and one that is mismatched near the 3'-end.
The perfect hybrid formed by the wild-type and mutant interrogation probes and their respective templates serves as the substrate for READase polymerase–mediated pyrophosphorolysis, and leads to the generation of light in the READIT reaction. The terminal 3' mismatch formed between the wild-type interrogation probe and the mutant template, or the mutant interrogation probe and the wild-type template, is not a substrate for the polymerase, so minimal light is generated.
When a heterozygous reaction is analyzed, dNTPs will be released in the presence of either interrogation probe. In this way, the 3'-end of the probe can interrogate the DNA target at single-nucleotide resolution for the determination of homozygous (wild-type and mutant) and heterozygous reactions (see Figure 4).
Figure 4. Clotting factor mutation clinical data set. READIT analysis shows greater than 6 S separation between allelic cells. This enables statistical confidence in assignments. For this study, 400 samples were analyzed and three distinct populations are easily observed: wild-type samples with a mean ratio of approximately 1.0, heterozygous samples with a mean ratio of approximately 0.5, and 3 homozygous mutant samples.
Studies demonstrating the accuracy and reliability of the system are being conducted in clinical laboratories. The main benefits of the READIT technology will be the flexibility for users to develop their own assays—or use assays developed by others--and in cost reductions through flexible automation. Promega expects that several companies will be offering applications and instrumentation to expand the utility of the technology to a range of different laboratory needs.
The READIT technology reagents will be priced competitively and labor costs will be minimized by appropriate use of automation. Promega has announced that there will be no end-user royalties for the use of its technology in diagnostic applications. The technology is expected to be commercially available by the end of this year.
Conclusion
Ultimately, the right technology for a laboratory is dependent on the individual needs of the laboratory, the test volume of the laboratory, and the type of testing that will be performed.
References
1. I Findlay et al, "Allelic Drop-out and Preferential Amplification in Single Cells and Human Blastomeres: Implications for Preimplantation Diagnosis of Sex and Cystic Fibrosis," Human Reproduction 10, no. 6 (1995): 1609–1618.
2. SM Powell et al, "Molecular Diagnosis of Familial Adenomatous Polyposis," New England Journal of Medicine 329, no. 27 (1993): 1982–1987.
Angela Ryan is the marketing manager and Richard Schifreen is the business unit leader for molecular diagnostics; John Shultz is an R&D group manager; Isobel MacIver is the manager for technical writing; and Dan Kephart is the scientific applications services manager at Promega Corp. (Madison, WI).
