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Originally Published IVD Technology July/August 2005

Molecular Diagnostics

Expanding the scope of molecular analysis through a new genetic base pair

Platform technologies that use innovative chemistry can simplify genetic diagnostics.

James R. Prudent

The MultiCode-PLx system by EraGen Biosciences Inc. (Madison, WI).

Without question, biomolecular technologies are revolutionizing our healthcare system. This is clearly the case in the clinical testing segment, where molecular techniques are poised to replace some of the most standard and routine tests. Conceivably, by the end of this decade molecular testing will have become routine for infectious-disease screening and may replace standard culturing and staining processes.

Additionally, molecular platforms support tests that were once out of reach for reference laboratories. These laboratories are now performing, or considering performing, tests that allow complex analysis. Mixed-population genotyping for the early detection of drug-resistant infectious agents is one complex test that is on its way to the clinic, and transcriptional profiling for cancer diagnosis is another example. This trend is due to molecular tests that allow this level of sophistication to become simpler. The cost of these tests is coming down, and reimbursement is becoming more standard. These factors are important for driving the molecular testing market and suggest how molecular testing is changing healthcare.

Another factor driving development of the molecular testing market is the ever increasing number of targets whose analysis requires molecular techniques, such as human genetic aberrations that lead to disease or have other medical relevance. Perhaps the most well-known of these is the human cystic fibrosis transmembrane conductance regulator (CFTR) gene, which was first cloned in 1989.

Certain mutations in the CFTR gene are known to cause cystic fibrosis, a disease in which the body produces abnormally thick, sticky mucus that clogs the lungs and leads to life-threatening pulmonary infections. These thick secretions obstruct the pancreas as well, preventing digestive enzymes from reaching the intestines to help break down and absorb food.

CFTR multimutation screening is on the increase, with about 20% of pregnant women in the United States who receive prenatal care now being screened for cystic fibrosis. And, as the evidence supports the clinical utility of newborn screening for cystic fibrosis, the Centers for Disease Control and Prevention have suggested that states consider making such screening routine.¹

Cystic fibrosis testing appears to be the beginning of a trend: the American College of Obstetricians and Gynecologists recently published a committee opinion on carrier screening for additional genetic targets.² Yet the pace at which states and clinical testing laboratories adopt genetic screening and molecular testing may depend on the technologies at their disposal. For the benefits of molecular testing to be fully realized, the newer technologies will have to be more cost-effective, simpler to use, and provide benefits in addition to those previously available.

A new set of tools was developed recently that does simplify certain aspects of molecular testing and provides the desired additional benefits. Called MiltiCode (for multiple coding) by their creator, EraGen Biosciences Inc. (Madison, WI), these tools grew from the idea that genetic material was not necessarily limited to two base pairs.³ Indeed, other base pairs can exist chemically, and one additional base pair is now available commercially. The new commercial base pair consists of iso-guanine (iG) and 5'-methyl-isocytosine (iC). The components required to advance the commercialization potential of this base pair, such as triphosphates for enzymatic incorporation of iC:iG, have lately become available.

This article discusses current and potential areas of interest for this base pair as they pertain to molecular diagnostic testing.

Nucleic Acid Testing

There are two major testing formats for nucleic acid target examination. One is quantitative analysis, which can be used for infectious-disease load analysis or expression profiling. The other is end-point analysis, useful for genotyping or infectious-disease detection.

Quantitative polymerase chain reaction (QPCR) can be used for quantitation, but the technology requires expensive equipment, is somewhat limited in throughput, and can be complicated to develop. Nevertheless, the methodology is gaining in popularity because of its inherent benefits, which include quantitative accuracy, single-copy sensitivity, a high level of specificity, and speed.

Many QPCR formats use indirect detection mechanisms involving the hybridization of a probe or probes to the amplified sequences. The TaqMan system (Applied Biosystems; Foster City, CA) adds an additional enzymatic step of nuclease digestion of the probe in order to separate fluorescent reporters from quenchers. To develop probe-based QPCR assays requires a high level of expertise, especially when the assays are multiplexed. Understanding this, some companies are now providing predeveloped TaqMan assays.

Other available QPCR formats detect the amplicon directly. Examples of these are SYBRGreen (Molecular Probes Inc.; Eugene, OR), LUX (Invitrogen Corp.; Carlsbad, CA), and Amplifluor (Intergen Co.; Burlington, MA). The major benefits of direct-detection methodology are ease of design and lower cost, because expensive probes are not required. Yet, many believe that probes are required to maintain specificity—a widely held notion that is simply incorrect.
The other major testing format, end-point analysis, is typically performed when only a positive or negative result is required. In order to increase throughput and reduce costs, tests can be multiplexed using solid-support capture to decode the complex reactions.

There are numerous chemistry formats for end-point multiplexed analysis, many using PCR for target amplification and solid supports to classify multiple detection products. The two predominant solid supports employed today are two-dimensional chips (slides or wafers) and microspheres. Both are just beginning to enter the molecular clinical testing market, but the instrumentation price tag can be relatively high.

This introduces the question of whether multiplexing capability will neutralize instrument costs. Clearly, the more tests performed on an instrument, the better. And, with testing for expanded panels increasing, menus for any given platform will also increase.

The New Base Pair

For the past five years, EraGen and its collaborators have been expanding the commercial potential of the base pair constructed from iC and iG.

What is unique and intriguing about the iC:iG base pair is that it behaves similarly yet orthogonally to the two base pairs found in nature.⁴ For example, it has been demonstrated that three of the major enzymes used in molecular diagnostics (polymerases, nucleases, and ligases) can recognize and carry out their specific activities on substrates that contain multiple iC:iG pairs.⁵ To show this, a DNA duplex model system for DNA break repair (consisting of multiple iC and iG bases) was created that required all three enzymes for complete repair. The experimental study also demonstrated that DNA targets containing both iC and iG could be correctly replicated and amplified with high fidelity under standard PCR amplification conditions.6 These findings helped improve understanding of the chemistry and enzymology associated with iC and iG as they pertain to the simplification of molecular diagnostic testing methodology.

Beyond enzymatic recognition, iC and iG allow for expanded specific molecular recognition—that is, hybridization of complementary DNA duplexes and, consequently, discrimination to natural-base mismatches within duplexes.⁷ It is well understood that duplex stability comes from both interstrand hydrogen bonding and intrastrand hydrophobic interactions between nucleo-bases. Specificity, on the other hand, is determined solely by the hydrogen bonding patterns. The hydrogen bond pattern used in the iC:iG pair has no equivalent in nature and is believed to contribute significantly to the pair’s specificity.

Software programs that calculate the melting-temperature stability of duplexes constructed solely of naturally occurring bases are available. However, such programs do not exist for duplexes that contain iC or iG. It has been observed that for each iC:iG base pair contained within a short duplex of 15–25 base pairs, the melting temperature is approximately 2°– 4°C higher than if that position were occupied by a C:G pair.⁸ The exact increase in stability is dependent on the nearest base pairs; that is, on nearest-neighbor effects. Though these effects have not been reported scientifically yet, they will become more important as more developers use the iC:iG pair to construct molecular coding systems.

Simplified Genetic Analysis in Application

Two platform technologies employing iC and iG have appeared: MultiCode RTx for solution-based quantitative analysis, and MultiCode PLx for solid-phase multiplexed end-point analysis. These platform technologies together will have applicability for more than 90% of the nucleic acid testing market.

They simplify genetic testing in a number of ways. Both technologies use site-specific enzymatic incorporation of labeled iG for signal generation. The PLx platform uses the iC:iG base pair for molecular recognition also. Both platforms can be multiplexed; however, the multiplexing capability of the RTx platform is more limited, owing to the instrumentation and the availability of spectrally addressable fluorescent dyes.

The RTx Platform. The MultiCode RTx system is a simplified QPCR technology.⁹ The system uses the iC:iG base pair to site-specifically incorporate the quencher molecule Dabcyl adjacent to a fluorophore during PCR.

Figure 1. MultiCode RTx uses PCR to exponentially amplify target sequences. During the cycling process, quenchers covalently attached to diGTP are enzymatically incorporated opposite an iC (placed on one of the two primers) and in close proximity to the fluorescent reporter. If target is present, the net fluorescence decreases with every cycle until the instrument begins to detect the decrease. The more target, the earlier this decrease is observed. The number of cycles in which a fluorescence change is observed is dependent on the number of initial targets added to the reaction (click to enlarge).

For the RTx, one of the two target-specific primers employed during PCR is manufactured to contain a fluorescent dye and an iC base on the 5' end. (If the fluorescent dye can be purchased in amidite form, the manufacturing process does not require postsynthetic labeling and is routine.) During the PCR, 2'-deoxy-isoguanosine triphosphate (diGTP) with covalently attached Dabcyl is used to incorporate the quencher into amplicons opposite the iC and in close proximity to the dye (see Figure 1). Each time another primer produces a new amplicon, the fluorescence of that primer is quenched by the quencher. As more and more of the labeled primers are used up, the fluorescence signal of the reaction overall diminishes. Therefore, placement of the quencher yields a net decrease in fluorescence, which is monitored in real time on any of a number of QPCR instruments.

Figure 2. MultiCode RTx data output. Tenfold changes in target input (107 to 101) were tested in seven reactions. For each reaction, the fluorescence falls below the threshold (dotted orange line) at a different PCR cycle. The more target added to the reaction, the sooner the fluorescence passes below the threshold (click to enlarge).

The PCR cycle with which the fluorescence passes below a predetermined threshold correlates to the number of initial target molecules present. The more target, the fewer cycles are necessary for the fluorescence to pass below the threshold (see Figure 2). The proximity of quencher and reporter allows the use of a wide range of fluorescent molecules, such as FAM, JOE, HEX, ROX, Cy3, Cy5, and most others.

The level of multiplexing possible is then dictated by the instrument’s capabilities, the spectral overlap between the dyes, and dye availability. An instrument- independent software package supplied by EraGen Biosciences assists in building standard curves and determining target number. The analysis software empowers users of different instruments to unify their data.

The RTx system streamlines design efforts considerably because it does not require probes and, more important, since it does not require changes in primer sequence beyond the single addition of an iC. Two good examples of this may be cited.

First, in an unpublished study, investigators at the Bernhard-Nocht-Institute of Tropical Medicine in Germany used existing PCR primer sequences specific to Trypanosoma cruzi to develop an RTx real-time system. Modifying only one of the primers to have a dye and an iC on the 5' end, they were able to detect as little kinetoplastid DNA as 10 copies. The other commercial nonprobe sys-tems tested, LUX and SYBRGreen, were not able to reach this level of sensitivity (see Figure 3).

Figure 3. Fluorescence data obtained in real time using three QPCR platforms: SYBRGreen (a), LUX (b), and MultiCode RTx (c). Primers for each platform were previously designed for a standard PCR assay for the detection of T. cruzi. Reagents to perform these assays were obtained from commercial suppliers (click to enlarge).

In the second notable example, primers specific for the human immu-nodeficiency virus (HIV) and first designed in 1988 were employed in an experiment.⁹ The RTx system was able to detect HIV-specific targets down to the single-copy level. This strongly suggests that, in many cases, primer sequence changes may not be required.

Besides simplification, MultiCode RTx offers other useful benefits. For example, a collaboration between EraGen and a group at Emory University has shown that the RTx system can be applied to mixed-population testing, or what is called needle-in-the-haystack analysis. Both the wild-type copies and the mutant copies caused by single-base changes are detected in the same reaction in these assays. In the case of 17 mutations, the data strongly suggest that for samples containing single-base mutants and wild-type copies in a ratio of 1:9999, the RTx system can accurately quantitate both populations simultaneously. For two important mutations in particular—the M184V and the K65R, both of which confer resistance to certain reverse transcriptase inhibitors—standard curves with linearity approaching an R2 value of 0.99 were constructed for both mutant and wild-type targets from the mixed samples.¹⁰

Another collaboration, whose results await publication, revealed a different benefit: the ability to test three targets with three separate dyes in a single reaction using the LightCycler 1 from Roche Diagnostics Corp. (Indianapolis). This instrument has a single-emission light-emitting diode. The largest number of channels used to detect individual targets in a single assay previously had been two. The sensitivity and specificity of the assays were equivalent to the best published real-time dedicated (nonmultiplexed) assays for identical targets. The extra level of multiplexing here allows for additional controls and higher throughput.

Figure 4. MultiCode PLx is a three-step process. Targets are amplified by PCR using primers that contain single iCs on their 5' end (a). Then, target-specific extenders are added to the reaction and extended in the presence of biotin covalently attached to diGTP (b). Only when the correct target is present does the extension product contain both tag and label. Finally, microspheres, to which are attached complements to the tags, are added (c). Each microsphere corresponds to a particular target; when fluorescent, it indicates the presence of that target in the initial sample (click to enlarge).

The PLx Platform. The other technology for discussion uses the iC:iG pair for both site-specific incorporation of a reporter and molecular recognition. MultiCode PLx is a three-step platform technology that simplifies solid-phase end-point analysis by eliminating a number of standard and technically challenging steps. The iC:iG nucleobase pair is used in each step of the PLx process: PCR, extension labeling, and liquid decoding (see Figure 4).

PCR primers are first designed to be target-specific and contain single iCs. The amplicons act as labeling templates for the target-specific extension (TSE) step. During the TSE step, labels attached to diGTP are incorporated site-specifically onto tagged target-specific extenders. The tags are short sequences, typically eight nucleotides long, assembled using a mix of natural and nonnatural bases. The tags are designed to hybridize only to their perfect complements (called EraCode), which are covalently attached to color-addressed microspheres. In the final step, decoding of the extension reactions is accomplished at room temperature by capturing the tagged extenders onto the EraCode-addressed microspheres.

Without further manipulation, the microspheres are then injected into the instrument and the multiplexed reactions are read for fluorescence. PLx is now deployed on the LabMAP instrument from Luminex Corp. (Austin, TX). Because multiple targets are queried in a single reaction, MultiCode is a very cost-effective, high-throughput multiplexed system. Moreover, all steps are carried out in the same reaction well without transfers or washings.

PLx was used for cystic fibrosis multimutation detection in a collaborative study carried out with the University of Wisconsin and the Wisconsin State Laboratory of Hygiene to demonstrate the simplicity of the platform in a clinical setting. Specifically, the platform was shown to be able to detect a panel of mutation types—deletions, single base changes, insertions—that can bring about cystic fibrosis.¹¹ PLx was automated on a robotic workstation in that study. Advances in simplicity reported include the elimination of transfers, solid-support washings or aspirations, high-temperature hybridizations, and certain enzymatic steps. Since labeling is accomplished by means of iG and not one of the four natural bases, the standard triphosphates do not interfere and thus do not require removal. Also, since the coding is achieved through iC:iG base pairing, the codes do not recognize any naturally occurring sequences. This development allows room-temperature hybridization that makes washing unnecessary. Therefore, PLx is several steps closer to the goal of a walkaway system.

A number of other PLx testing panels have been developed or are in development for various genetic targets. For instance, the Blood Center of South Eastern Wisconsin has developed a test for human platelet antigen polymorphism screening.

PLx is also being applied to the detection and screening of infectious-disease targets, for example, in investigative efforts pertaining to the development of panel tests for viruses responsible for many types of respiratory diseases. EraGen is collaborating with a group at the University of Wisconsin Medical School to develop a practical and accurate assay that will be used initially to improve understanding of the contribution made by viral respiratory infections in infancy to the increasing occurrence of inner-city asthma. This panel has more than 10 targets, including subtypes of influenza, parainfluenza, and respiratory syncytial viruses, as well as all the picornaviruses. Initial results from testing more than 500 patient samples indicate that the panel is specific and sensitive relative to results obtained by standard culturing methodology.

Assay development for coronaviruses, adenoviruses, and avian flu is also under way.

Potential Future Applications

Both platform technologies, RTx and PLx, involve polymerase incorporation of labels attached to diGTP. Where other methods are not available, MultiCode’s ability to incorporate labels at specific sites may find utility, such as 3'-end labeling during PCR or specific internal labeling. Exponential amplification of iC and iG in the nonpriming regions of PCR amplicons (that is, the interior areas of the primers) also has been observed.⁶ Experimental results demonstrated high efficiency of incorporation of MultiCode bases opposite their MultiCode base pair (96% ± 3%).

This breakthrough that uses standard PCR conditions and the triphosphates of iC and iG allows scientists to amplify iC- and iG-containing sequences for a number of purposes. Among these are tagging in diagnostics and use in the oligoligation assay, which builds tags into ligation products that are amplified by common PCR primers. These amplified tags can be captured on solid-phase arrays for analysis. DNA tags, or molecular codes, also are used in nanotechnology, as well as to mark explosives and valuable art pieces.

Yet a further application for MultiCode technology is in the field of aptamers. Aptamers are single-stranded and highly folded DNA or RNA molecules that, like antibodies, can bind target molecules with high affinity and specificity. They are generated through multiple rounds of selection that begin with large, random libraries of chemically synthesized oligonucleotides.¹² Aptamers have been attracting growing interest in the field of molecular testing because, unlike with antibodies, selection and cloning are rapid, aptamers can be selected to have catalytic properties that can be turned on or off after target recognition and binding, and they do not require animal immunization. However, aptamers made of standard nucleic acids are somewhat limited in enzymatic capability. Modified nucleic acids therefore are being explored. To expand on this, additional functional groups on iG may be useful. The demonstrated high fidelity of iC or iG incorporation is well within the range essential to aptamer production.

Conclusion

The expansion of the genetic alphabet represented by the introduction of the iC:iG base pair could lead to a paradigm shift in the way molecular diagnostic tests are developed. MultiCode technologies that supplement nature’s chemistry are just beginning to find practical uses. As scientists, manufacturers, and clinicians become more comfortable with these chemistries, additional uses will surely be discovered.

References

1. Centers for Disease Control and Prevention, U.S. Department of Health and Human Services, “Newborn Screening for Cystic Fibrosis: Evaluation of Benefits and Risks and Recommendations for State Newborn Screening Programs,” Morbidity and Mortality Weekly Report vol. 53, no. RR-13 (Atlanta: CDC, 2004).

2. ACOG Committee Opinion #298, August 2004, “Prenatal and Preconceptional Carrier Screening for Genetic Diseases in Individuals of Eastern European Jewish Descent,” Obstetrics & Gynecology 104, no. 2 (2004): 425–428.

3. SA Benner, “Enhanced: Redesigning Genetics,” Science 306 (2004): 625–626.

4. JA Piccirilli et al., “Enzymatic Incorporation of a New Base Pair into DNA and RNA Extends the Genetic Alphabet,” Nature 343 (1990): 33–37.

5. MJ Moser and JR Prudent, “Enzymatic Repair of an Expanded Genetic Information
System,” Nucleic Acids Research 31 (2003): 5048–5053.

6. SC Johnson et al., “A Third Base Pair for the Polymerase Chain Reaction: Inserting IsoC and IsoG,” Nucleic Acids Research 32 (2004): 1937–1941.

7. T Horn, C Chang, and ML Collins, “Hybridization Properties of the 5-Methyl-Isocytidine/Isoguanosine Base Pair in Synthetic Oligodeoxynucleotides,” Tetrahedron Letters 36 (1995): 2033–2036.

8. CR Geyer, TR Battersby, and SA Benner, “Nucleobase Pairing in Expanded Watson-crick-like Genetic Information Systems,” Structure 11, no. 12 (2003): 1485–1498.

9. CB Sherrill et al., “Nucleic Acid Analysis Using an Expanded Genetic Alphabet
to Quench Fluorescence,” Journal of the American Chemical Society 126 (2004): 4550–4556.

10. MJ Moser et al., “Quantifying Mixed Popluations of Drug-Resistant Human Immunodeficiency Virus Type 1,” Antimicrobial Agents and Chemotherapy 49, no. 8 (2005):—.

11. SC Johnson et al., “Multiplexed Genetic Analysis Using an Expanded Genetic Alphabet,” Clinical Chemistry 50 (2004): 2019–2027.

12. AD Ellington and JW Szostak, “In Vitro Selection of RNA Molecules That Bind Specific Ligands,” Nature 346 (1990): 818–822.

James R. Prudent is chief scientific officer at EraGen Biosciences Inc. (Madison, WI). He can be reached at jprudent@eragen.com.

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