Advancements in Real-Time PCR Delivering … Experience, Expertise and Innovation in Every Test. ... covalent modification renders the enzyme ... Specificity Enhancement with Aptamer

Advancements in Real-Time PCR DeliveringExperience,Expertise andInnovationinEveryTest

The scientists of Roche Molecular Diagnostics, (RMD), are committed to the

advancement of the science of Polymerase Chain Reaction, (PCR), to meet

the challenges of ever-changing global medical needs. With innovation as

our motivation, RMD has pioneered the development of both standard

PCR and Real-Time PCR techniques. We have regularly introduced

new capabilities and improvements to Real-Time PCR over the earlier first

generation technologies. We have merged innovations in biochemistry with

the best of earlier PCR technologies to deliver a powerful combination:

robust test performance with full automation. This level of technological

advancement is unique in the industry, when compared with competitors within

the in vitro diagnostic, (IVD), field.

Pioneers of PCR Developing Gold Standards of Real-Time PCR

Leading the Advancement of Real-Time PCR

Improving Specificity

The power of PCR lies in its potential to detect small amounts of targets with extremely high sensitivity. Since PCR sensitivity and specific-ity are closely linked, much effort of the RMD scientific staff has been focused on increasing the specificity of PCR in order to deliver on this potential. Poor specificity of amplification can affect the sensitivity of detection of specific targets due to the generation of non-target products which can deplete amplifica-tion components such as primer and dNTPs.

Specific amplification is particularly important for multiplex reactions, even with the added specificity of probe-based detection, because the resultant competition for reagents from non-specific amplification can adversely affect

efficiency of target amplification, especially at low copy target input.

RMD Real-Time PCR assays employ several specific improvement approaches including:

• Enzyme Hot Start

• Nucleic Acid Chemistry

• Thermal Cycling

• Genotype Inclusivity

These methods invented and/or developed by RMD scientists have become the gold standard for Real-Time PCR, and are licensed by other companies in the molecular diagnostic field.

AdvancementsinReal-TimePCR 1

Improving Chemistry & Enzyme

RMD scientists have discovered and developed many of the foundational technologies that enable nucleic acid testing today, most notably; PCR, Taq Polymerase, and Real-Time detec-tion(1-8) . The development of Real-Time PCR, using dye- and probe-based assays to gener-ate specific detectable signal concomitantly with amplification, was a ground-breaking improvement that enabled quantitative analysis. RMD scientists continually refine the closely linked primer and probe chemistry and enzymology for the development of highly sensitive, specific, and robust assays amenable to automation and multiplexing.

Choosing the Right Primer Targets The choice of targets for our PCR primers and probes is carefully identified by an extensive screening of available viral and bacterial sequence public databases, in addition to RMD sequencing data across applicable genotypes and subtypes. The region for the Human Immunodeficiency Virus, (HIV-1), RNA

primer set and probe was chosen only after a thorough search of global databases to evalu-ate potential regions for PCR amplification.

As sequence databases expand and more genotypes and highly represented recombi-nant targets are identified, even better targets are likely to be found for future generation tests.

Designing Tolerance for Sequence Variation

Sequence variation in biological specimens creates an ongoing challenge for IVD technologies that exploit nucleic acid hybrid-ization test methods. RMD scientists employ multiple methods to build high-quality assays that exhibit tolerance for both known and unknown sequence variations. An example of the added value RMD brings to PCR based diagnostics is illustrated in the advance-ments for solutions that have been found to overcome sequence variation.

2 AdvancementsinReal-TimePCR

Figure 1Top bands 365bp and 272bp amplicon from P53 exon 4 and 5, respectivelyLower band is 117bp b-actin internal controlLanes 1, 2: EagleTaq Lanes 3, 4: Taq Lane 5: no enzyme controlLane 6: MW marker

Improving Specificity

EagleTaq Taq

p53 Exon 4 —

p53 Exon 5 —

b-actin —

bp

— 500 — 400 — 300

— 200

— 100


group of lysine side chains. This reversible covalent modification renders the enzyme inactive at low temperatures when non-specific amplicons are often produced. At higher temperatures, the covalent adducts are hydrolyzed, thus reactivating the polymerase so that PCR can proceed. This pioneering “hot start” of enzyme activity results in higher amplification specificity (see Figure 1).

1. Hot Start Enzymes To enhance assay specificity, many methods begin with a “hot start”, where critical reaction components are made unavailable or inactive until sufficiently high reaction temperatures are reached. A critical invention to control non-specific amplification utilizes covalent modification of the DNA polymerases(9,10) (TaqGold, EagleTaq, ZO5 Gold). These enzymes are DNA polymerases that are chemically modified at the e-amino

P53 Multiplex Amplification Improvements with EagleTaq

Figure 2 Aptamer Hot Start System. At low temperatures the aptamer forms a hairpin structure that binds to and inactivates the DNA polymerase, preventing amplification.

At high temperatures the aptamer hairpin structures melts out and is released from the DNA poly-merase, allowing amplificationto occur.

2. Novel Aptamer Hot Start Method Another hot start method to control non- specific amplification relies on aptamers. Aptamers are small oligonucleotides whose unique secondary and tertiary structures allow them to bind with high affinity to the active sites of enzymes at low temperatures. At higher temperatures, the secondary and tertiary structures are denatured, allowing the aptamer to dissociate from the complex (see Figure 2).

In collaboration with scientists from NeXstar Pharmaceuticals (later acquired by Gilead), RMD scientists have developed aptamers that tightly bind to and inhibit polymerases com-

monly used in PCR. The aptamers originally identified were further characterized at RMD and their sequence modified to achieve optimal temperature control of polymerase inhibition. In contrast to covalently modified enzymes, aptamers do not require extended incubation at high temperature (e.g., 10 min. at 95ºC) for activation. This is particularly advantageous when working with thermo- labile RNA targets such as HIV and HCV.

Both the enzymatic and aptamer hot-start methods make RMD’s Real-Time PCR assays extremely robust, thus enabling the automa-tion of assay set-up (see Figure 3).

Hot Start PCR: Specificity Enhancement with Aptamer

Figure 3 RT-PCR of HIV template with Z05 DNA polymerase +/- aptamer following 6 hr incubation at 37ºC to mimic onboard stability of a completed reaction. The benefit of aptamer to growth curves in both yield and Ct value is clearly evident.


3. Primer Chemistry for Improving Amplification Specificity primer dimer formation and by making ampli-fication of non-target templates exceedingly unlikely to occur. These features are especially critical for multiplexing.

The specificity enhancement achieved via enzyme and aptamer hot start, as well as alkylated primers, enables a more sensitive PCR together with a robust amplification across an increased, very large dynamic range. The reduction of PCR artifacts (e.g., “primer-dimers”) that occurs when these new technical advances are utilized is remarkable, and an example of added value that RMD brings to PCR based diagnostics (see Figure 5).

Figure 4 Left Example of modified primer: tert-butyl benzyl group attached to N6 atom of dA. Such modified bases would be placed at the 3' end of the primer.

Figure 4 Right Structural model of modified primer bound to Taq polymerase. The bulky moiety points into the major groove of the DNA, away from the active site of the enzyme. Extension of a modified primer annealed to matched template is unaffected. A previously initiated primer dimer, as modeled here, can not be extended due the steric hindrance of two tert-butyl benzyl groups on both the priming and templating bases. This leads to a marked enhancement of specificity and thus sensitivity of PCR.

Figure 5 Performance enhancement of HCV TaqMan® amplification and detection of HCV RNA with 3' alkylated primers. Red growth curves represent reactions performed using modified primers. Blue growth curves represent reactions per-formed without using modified primers.

TaqMan® Growth Curves With and Without Modified Primers


Alkylated Primers: Structure

An additional method to prevent non-specific amplification involves covalent modifications to the primer terminus. RMD scientists discov-ered that, by placing alkyl side groups on the major groove side of the base at or near the 3'-end of primers, the specificity of PCR can be improved(11, 12) . By adding “bulkiness” to the 3'-end of primers, alkyl modifications render primer dimer artifacts nearly impossible to propagate (see Figure 4).

Correctly annealed and modified primers can be extended as efficiently as unmodified primers. The 3' alkylation of primers thereby increases PCR specificity by suppressing

Designing Tolerance for Detecting Sequence Variation

Figure 6 The thermal cycling conditions are modified so that the desired outcome of each phase of the PCR is matched to the specific cycling conditions applied. Following UNG sterilization, the RT step begins with a lower, tolerant temperature (yet still high enough to melt out secondary structure) then ramps up to a higher, more stringent tem-perature. Amplification occurs in distinct phases, begin-ning with a few cycles of a ramped extension from higher

Phase Matched Thermal Cycling for Mismatch Tolerance

The converse of specificity is the ability to detect target templates with mismatches to the primer or probe sequences. The genomes of viruses, especially RNA viruses, evolve rapidly, as for example HIV-1 Group M with over 9 subtypes. The result is viral populations with significant sequence heterogeneity. This presents a challenge to any nucleic acid detection assay since sequence diversity in the primer/probe binding regions can affect the ability with which assays can detect all viral isolates. RMD scientists have developed a number of strategies to achieve mismatch tolerance without sacrificing specificity.

RMD scientists have engineered unique thermal cycling conditions that work in concert with reagents to optimize both the sensitivity and specificity of our Real-Time PCR tests. By utilizing the novel concept of phase-matched thermal cycling, the desired overall balance between specificity and genotype inclusivity can be achieved.


1. Phase Matched Thermal Cycling for Mismatch Tolerance

Phase matched thermal cycling breaks the uniformity of cycling and optimizes the thermal parameters in separate sections to achieve the desirable attributes for each phase of PCR. Cycling phases with lower temperatures improve mismatch tolerance, while phases with higher temperatures improve specificity, and hence sensitivity (see Figure 6).

to lower temperatures, allowing extension of targets mismatched to primers. The next phase of amplifica-tion occurs at a uniform and stringent temperature, so that only specific product is efficiently amplified. The final phase of amplification is at relatively low temperatures to allow ef f icient hybridization and cleavage of probes to mismatched genotypes.


The mismatch tolerance of an assay can be further improved by incorporating nucleotide analogs. These analogs, which offer enhanced thermodynamic stability in duplex DNA structures, can significantly increase the Tm of probe/template hybrid structures. In this way, the destabilization of any mismatch to a probe can be overcome through the extra stability that these base modifiers confer (see Figure 7).

The use of such modifiers also allows relatively short probes to be utilized, which cover less sequence space of the intended target, and are therefore less likely to impinge on multiple mismatches within any one target.

The degree of stabilization conferred by chem-ical modification is sufficient to overcome the destabilization of the probe when annealed to mismatched templates, and has been optimized in conjunction with the phase matched PCR thermal cycling profiles as described above (see Figure 8).

2. Probe Chemistry for Mismatch Tolerance

Figure 7 Comparison of TaqMan® detection of various HIV-1 subtypes with unmodified or modified probes. In this case the modified probe contains 21 stabilizing 5-methyl-deoxycytidine or 5-propynyl-deoxyuridine modifications.

The modified probe allows efficient detection even with the heavily mismatched subtype. Our probes have been tailored in this manner to ensure both specificity and mismatch tolerance to allow genotype coverage.

Figure 8 Comparison of melting curves of unmodified or modified probes against oligonucleotides with the same sequence as the HIV-1 subtypes in the above growth curves.

In a modified probe, the use of 5-methyl-deoxycytidine confers about 0.4ºC extra stabilization for every substitution. A 5-propynyl-deoxyuridine can be used as a substitute for thymidine in probes, whereby a similar degree of stabilization is possible. The most aggressive stabilizer used is 5-propynyl-deoxycytidine, which gives about 0.9ºC stabilization over conventional cytidine residues. By judicious use of these stabilizing bases, the Tm of probes can be boosted by as much as 10ºC.

Stabilized TaqMan® Probes Enable Mismatch Tolerance

Examples of designer polymerases developed by RMD scientists are enzymes with better fidelity, higher reverse-transcriptase activity and improved efficiency in incorporating nucleotide analogs, including dideoxy nucleotides for DNA sequencing(18-21). For example, the enzyme used to sequence the human genome was originally developed by RMD scientists(27-28) . The use of designer DNA polymerases enable RMD to continually improve and develop novel diagnostic assays.

Improving the Chemistry and Enzyme

and amplification step(17) . The benefits of single enzyme, single buffer RT-PCR are the robustness of a closed tube reaction combined with the specificity conferred by the high temperature (see Figure 8).

Current research efforts focus on the design of polymerases with the features needed for specific applications using structure-based mutagenesis and domain swapping, as well as by screening and selection of mutagenic libraries of DNA polymerases (see Figure 9).

Figure 9 Designer DNA polymerases tailored for high fidelity, long PCR and RT-PCR. Molecular modeling approaches were used to create chimeric DNA polymerases with the 5' nuclease and polymerase domain and the 3' exo domain of ZO5 and Tma polymerases, respectively. The molecular model for the 3' exo proofreading domain of the chimeric polymerase bound to DNA is shown in the figure above. The wild type residues involved in metal binding (left) and DNA binding (right) are shown. These residues were individually mutated to alanine in order to down modulate the proofreading activity(20) .

Designer Polymerases


These enzymes have the right balance of polymerase and proofreading activity. Such polymerases are utilized in AmpliChip assays with long targets.

The protein is shown as a colored ribbon according to secondary structure. Three bases of the DNA substrate in the 3' exonuclease active site and two bound metal ions are also shown.

In the early days of PCR, amplification of RNA molecules required the use of an additional enzyme, a reverse transcriptase generally derived from a mesophilic virus, for the synthesis of DNA copies (cDNA) of the RNA template (reverse transcription or RT). Once RT was complete, PCR amplification could proceed. This was a labor-intensive process susceptible to contamination due to the need to open tubes for enzyme addition. RMD scientists developed efficient RT-PCR assays that use a single thermoactive DNA polymerase to carry out both cDNA synthesis and PCR. A recombinant DNA polymerase from the thermophilic eubacterium Thermus thermophilus (Tth) and the distinct but related Thermus sp. Z05 were found to possess efficient reverse transcriptase activity in the presence of Mn+2 (13-16) . The use of a thermoactive reverse transcriptase allows for increased specificity of primer binding and extension as well as the alleviation of the secondary structures present in the RNA template. Additionally, metal binding buffers were discovered that provide the optimal free Mn+2 concentration for both the RT

Selecting the Right Primer Target

RMD scientists have also developed unique bioinformatics tools and have ongoing collaborations to determine thermodynamic parameters for our proprietary nucleotide modifications.

PathogenNumber ofSequences

HIV-1 9926

HCV 3500

HBV 1800

WNV 250

Figure 10Key pathogen sequences acquired by the Roche Global Surveillance Program.

Global Surveillance Program

Num

ber

of V

aria

nts

Sequence Variation Frequency: HIV-1


The Global Surveillance Program and Bioinformatics Tools The continuing and expanding mutational sequence variation that is inevitable in global viral and bacterial infectious diseases require constant vigilance. Rapidly evolving retroviruses are especially prone to mutate their genomes in response to the selective pressure in the form of antiretroviral therapy. This is of particular concern in nucleic acid testing if the amplified target sequence of a test encodes a drug target(22) . Since the founding of RMD, a dedicated research team has focused its efforts on the ongoing work of identifying sequence variants in public databases, as well as our own extensive sequencing efforts of patient specimens (see Figure 10).

The choice of targets for our PCR primers and probes is carefully identified by an extensive screening of available public viral and bacterial sequence databases plus our own sequencing of regions across the applicable genotypes and subtypes. Extensive research has gone into the choice of the region used for HIV-1 RNA primer set and probe. This included a global database search of other potential regions for PCR amplification (see Figure 11). As more genotypes and highly represented recombinant targets are identified, and as the available sequence database expands, even better targets are likely to be found for future generation tests.

These tools allow the genetic variability of the thousands (or even hundreds of thousands) of sequences in the public and in-house databases to be analyzed and performance, particularly specificity and genotype coverage, of existing assays to be predicted. These same tools, in conjunction with the Global Surveillance Program, are being used to identify potential target sequences for next generation primers and probes to further improve genotype coverage of our viral tests. Thus, our next generation PCR tests will benefit from this vital activity, just as our current tests already have.

Global Surveillance Program & Bioinformatics Tools for Target Selection.

Figure 11 The natural and human-induced selective pressure in the form of drug treatment, for example HAART for HIV, causes extensive mutations and genetic variability in the genome of a virus. This is a graphical view of the statistics of variability across a given region of a viral genome. ~2,300 complete HIV-1 genome sequences were aligned. Bars indicate the number of mutations, deletions and insertions at each position relative to a reference sequence. Regions of low variability are ideal targets in primer and probe design.

Internal Quantitation Standard (IQS)/Internal Control (IC)


Figure 13 The HIV-1 probe and the QS probe share the same primers and are equivalent in length but have a different target sequence that is used to differentiate the amplification products.

The QS is added to the reaction mixture during specimen preparation at a known copy number. The concentration of the unknown target can be calculated based on the concentration of the QS.

Real-Time PCR assays are inherently quantitative since the rate at which signal accumulates is proportional to the starting target concentration. Thus, Real-Time PCR offers a convenient method to determine viral titer in clinical samples. The IQS and IC are artificially constructed nucleic acids that are added to each amplification reaction at known copy numbers and are co-amplified with target nucleic acids. The relative signals between target and IQS are used to calculate

the target concentration in the sample. In this way, the IQS corrects for any change in amplification efficiency due to the presence of inhibitors or variance in reagents, instrumentation or operator error(23, 24) .

The IC serves to assure that the PCR chemistry is working in each qualitative test and provides greater assurance of a target negative result.

Structure of HIV-1 and HIV-1 Quantitation Standard.

Gold Standard for the Prevention of Carry-Over Contamination

The power of PCR is its ability to detect a single molecule by synthesizing billions of copies of target nucleic acids. This extreme sensitivity may also be a disadvantage unless some simple precautions are taken. RMD scientists successfully applied a method to eliminate false-positive results from carry-over contamination by differentiating amplification products from target molecules(25, 26) .

This is accomplished by incorporating dUTP into amplicons. In the case of carry-over contamination from a previous PCR, the dU residues in any contaminating amplicons would be removed from the amplicons by the included enzyme Uracil-N-glycosidase (UNG or AmpErase). Removal of dU residues creates abasic sites in amplicons, which

Figure 14Selective Amplification Using AmpErase Enzyme.

Inactivated Amplicon DNA

Heat(above 55ºC)Alkaline pH

Inactivated Amplicon DNAAmpErase EnzymeCarry-Over Amplicon DNA

Unmodified Specimen/Target DNA

InactivatedAmpErase

Enzyme

Specimen/Target DNA Unmodified Speciman/Target DNA


are susceptible to backbone breakage when heat is applied, rendering the treated amplicons incapable of serving as targets for amplifications.

Target nucleic acids, which contain TTP or rUTP are not affected by AmpErase enzyme. Thus, the incorporation of dUTP means amplicons are differentiated from target nucleic acids and allow AmpErase enzyme to selectively render them non-amplifiable. The dUTP/UNG sterilization has been established as the gold standard of carry-over prevention in the molecular diagnostics industry and the technology has been licensed by many other PCR test developers (see Figure 14).

Complete Real-Time PCR Automation

• Hands-off processing, including overnight runs

• Entire process controlled by a single software interface which provides automatic sample handling and tracking from specimen input to result reporting (data analysis) all in one room

• Continuous load capabilities, providing flexibility with run size (continuous access)

• No recalibration of assays or instrument necessary

• Multiple assays (currently, up to 4 test types possible) run simultaneously

• Barcoded reagent cassettes (on board reagent surveillance, lot number, expiration date, remaining volume).

RMD scientists developed the first fully automated Real-Time, continuous load IVD platform, featuring sample-in, result-out capability - The COBAS® AmpliPrep/COBAS® TaqMan® System. Designed to increase efficiency and productivity with automated sample preparation, amplification and quantitation of RNA or DNA, this system provides high throughput for virology testing including HIV-1, HCV, and HBV tests. Assay reagents have been designed and optimized for robust performance when used with the COBAS® AmpliPrep/COBAS® TaqMan® Systems and accompanying software.

Therefore, from assay feasibility to research and development optimization, tests created for this instrument include all aspects of specimen stability, target purification, target stability, reagent stability, PCR reliability, detection optimization, software compatibility, user friendliness and more. This “all inclusive” RMD management of systems development results in unmatched capabilities which include:

State-of-the-Art Product Development and Manufacturing

In addition to cutting edge assay technology, system design and performance, reproducible manufacturing processes and a continuous supply of product are highly critical to success.

The foundation of RMD's strategy has been to manufacture the majority of kits and kit components in-house. This strategy has effectively allowed RMD to utilize its internal knowledge and expertise in bulk formulation, fill, and packaging to produce the highest quality and cutting-edge products.

Additionally, RMD's proprietary knowledge in the manufacture of highly complex and specially modified oligonucleotides and enzymes is key to providing more accurate diagnostic information.

Roche's manufacturing plant in Branchburg, New Jersey is the world's largest PCR manufacturing facility. It incorporates state-of-the-art quality management which means ISO 13485 certification, operation in accordance with GMP, and compliance with FDA and other international regulatory bodies. Our quality systems meet all the international standards and are readily in line with changes to internal workflows and processes.

This in-house manufacturing expertise is complemented by a divisional global supply chain that ensures on time delivery of products to customers around the world.

Conclusion

The creative advances in PCR-based tests developed by RMD scientists

contribute to three key areas which together offer unmatched value: quality,

robustness and innovation.

The quality of RMD PCR tests rests upon 17 years of consistent advances which include:

1. Improved sensitivity and dynamic range as a result of increased automation combined with homogenous quantitative Real-Time assays.

2. Quality results, reassurance and relief from ongoing standard curve generation is possible with run controls (low and high titer viral standards) and internal (Qualitative Standard, QS) standards.

3. One of the first to adopt global analytical standards (e.g. the WHO HIV- 1 RNA standard directly used by RMD to calibrate the COBAS® AmpliPrep/COBAS® AMPLICOR HIV-1 Test), which allows users to be confident of test- to-test, lot-to-lot consistency of results across analyzers including comparing test results from other manufacturers who use the same standards.


The robustness of RMD PCR tests can be attributed to extensive optimization trials including buffer components, primer and probe design and synthesis, and thermostable enzyme molecular engineering. This blending of the deep experience of our staff PCR design professionals combined with software and hardware developed by RMD engineers smoothly unite COBAS® analyzers with Real-Time test biochemistry.

Innovations incorporated into RMD PCR in vitro diagnostic tests are illustrated by an extensive patent portfolio. RMD holds over 200 patents in the PCR field including Real-Time PCR. Additionally, RMD has a validated global leadership in state-of-the-art manufacturing facilities and the singular ability to integrate test chemistry, software, and hardware into a system that is RMD designed, RMD manufactured, and RMD serviced.

As the world leader in in vitro diagnostics, Roche Molecular Diagnostics supplies a wide range of rapid, reliable instruments and tests for disease screening and diagnosis for the laboratory. Roche Molecular Diagnostics leverages these innovative products and combined strengths to meet your molecular testing needs.

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Advancements in Real-Time PCR Delivering … Experience, Expertise and Innovation in Every Test. ... covalent modification renders the enzyme ... Specificity Enhancement with Aptamer