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Homogeneous amplification and variant detection by fluorescent hybridization probes.

Over the past 5 years, there has been substantial progress in sequencing the human genome and identifying clinically significant genes (1). Genes that are clinically significant are diagnostic or prognostic for disease and/or helpful in guiding treatment. Unknown gene mutations, resulting from germline or somatic DNA alterations, are initially defined by direct sequencing. Other methods that detect specific mutations can then be used for higher throughput.

Recently developed instrumentation and techniques for genotyping combine PCR and fluorescent hybridization probes for homogeneous amplification and product analysis within 1 h (2-5). The target is amplified from genomic DNA by rapid-cycle PCR (6) with all the reagents needed for genotyping present from the beginning of the reaction. After 15-20 min, PCR is completed and the instrument automatically begins a melting curve protocol. Fluorescence is acquired continuously as the reaction is slowly heated and genotypes are identified by their characteristic melting curves. Because amplification and genotyping occur in the same instrument in a closed tube format, there is no concern of contamination by previously amplified product.

Hybridization probes are oligonucleotides that are singly labeled with a donor or acceptor fluorophore. During probe/target hybridization, these fluorophores are brought into close proximity and fluorescence resonance energy transfer occurs. Two hybridization probe schemes for fluorescent resonance energy transfer have been developed (3,5). One method uses a 3'-labeled hybridization probe designed to anneal to a PCR strand extended by an internally labeled primer (3,4). This method requires that the fluorescently labeled primer be positioned near the mutation site, usually within 5 base pairs, to allow adequate resonance energy transfer with the complementary genotyping probe. The other method uses separate 3'- and 5'-labeled probes designed to hybridize to an unlabeled complementary PCR strand (5). This allows a pair of probes to be placed anywhere within a primer set. In this issue, von Ahsen et al. (7) use both the primer/probe and probe/probe schemes for genotyping mutations at two sites within the [[alpha].sub.1]-antitrypsin gene.

With hybridization probes, an increase in fluorescence resonance energy transfer is observed as a PCR reaction is cooled and probe/target annealing brings the donor and acceptor fluorophores into close proximity. Reciprocally, as the reaction is heated, the probe/target duplex is denatured, the fluoropohores are separated, and fluorescence resonance energy transfer drops to background. In PCR, once per cycle fluorescence acquisition during probe/target annealing provides quantitative information about the starting copy number (8). In addition, continuous monitoring during slow heating (0.1- 0.2[degrees]C/s) provides qualitative information about the sequence of the target (3-5).

The probe melting temperature ([T.sub.m]), defined as the point at which 50% of the probe has strand-separated from the target, can be determined from the inflection point of the melting curve or the center of the derivative melting curve (9). The [T.sub.m] is characteristic for a particular duplex and depends on such factors as length, G:C content, sequence order, and Watson-Crick pairing (10). Base-pair mismatches shift the stability of a duplex by varying amounts depending on the particular mismatch, the mismatch position, and neighboring base pairs (10,11). When a probe hybridizes over a sequence variant, a mismatch is formed and the duplex is destabilized. This is reflected by a shift in [T.sub.m] from the completely complementary duplex.

The derivative melting curve of a particular duplex generated under constant reaction conditions of heating rate, salt concentration, and probe/target concentrations is highly reproducible, with a standard deviation of only 0.1[degrees]C within runs (12). Thus, a small [T.sub.m] shift 0.2 from the expected melting curve profile suggests a new mutation. In one study in which 2100 samples were analyzed for the factor V Leiden mutation, a new base alteration was identified by only an 0.8[degrees]C [T.sub.m] shift from the expected Leiden mutation (12). Probe specificity also appears to be very high. When 200 heterozygous and homozygous factor V samples were analyzed with a probe complementary to the Leiden mutation, no additional alterations were identified. Unexpected variants have also been identified in the HFE and cystic fibrosis genes (5,13).

The high specificity of fluorescent hybridization probes is complemented by their high stability (14), making them optimal for the clinical laboratory. Hybridization probe genotyping assays that use a single acceptor color include factor V Leiden (3,15), methylenetetrahydrofolate reductase (4, 15), prothrombin (15), HFE (5,16), apolipoprotein (apo) E (17), apo B-3500 (17), human platelet antigen 1 (18), N-acetyltransferase 2 (19), plasminogen activator inhibitor-1 (20), BRCA1 (21), and antiviral resistance-associated mutations in the hepatitis B virus (22). The feasibility of using two acceptor colors for multiplexing was recently demonstrated in a synthetic system for variants of the apo E gene, where new solution color compensation techniques were introduced (9).

Homogeneous PCR and mutation detection can be done with other types of fluorescently labeled oligonucleotides, such as exonuclease probes or hairpin probes (1). However, these probes are technically more difficult to optimize and synthesize. For example, the probes must be designed to anneal only to the perfectly matched target for proper scoring during amplification. Furthermore, each probe needs to be dual-labeled, which is more challenging than single labeling, and a new probe must be synthesized for each allele of interest.

Systems developed for variant analysis strive to increase the power of the assay by multiplexing. Because exonuclease and hairpin probes can report fluorescence only on the perfectly matched allele, additional probes with different fluorescent emissions are designed for each allele. Currently, as many as six different fluorescent dyes have been combined with a common quencher (23). These assays are limited to one dimension (i.e., color). In contrast, hybridization probes can identify multiple alleles by using both color and [T.sub.m]. In this issue of the journal, von Ahsen et al. (7) successfully apply this two-color technique to the simultaneous genotyping of two sites within the [[alpha].sub.1]-antitrypsin gene, starting with the amplification of genomic DNA. Additional applications are sure to follow.

The power of multiplexing with both color and [T.sub.m] is the product of the number of colors and the number of [T.sub.m]s that can be differentiated. The [[alpha].sub.1]-antitrypsin genotyping described here uses two acceptor colors for reporting on two allelic sites (7), giving a total multiplex of four. Because at least four [T.sub.m]s can be differentiated in a single melting curve profile (5), a multiplex of eight with two acceptor colors is easily within reach. Furthermore, because up to six colors can be distinguished (23), it should be possible to use color and [T.sub.m] to reach a multiplex at least as high as 24. Although multiplexed hybridization probes do not currently provide the information content of sequencing or the throughput of solid-phase hybridization arrays, they do present a practical option for simple, rapid genotyping in the clinical laboratory.

C.T.W. holds equity interest in Idaho Technology. Idaho Technology has licensed hybridization probe and Light-Cycler[R] technology from the University of Utah and, in turn, licensed these technologies to Roche Molecular Biochemicals.


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(3.) Lay MJ, Wittwer CT. Real-time fluorescence genotyping of factor V Leiden during rapid-cycle PCR. Clin Chem 1997;43:2262-7.

(4.) Bernard PS, Lay MJ, Wittwer CT. Integrated amplification and detection of the C677T point mutation in the methylenetetrahydrofolate reductase gene by fluorescence resonance energy transfer and probe melting curves. Anal Biochem 1998;255:101-7.

(5.) Bernard PS, Ajioka RS, Kushner JP, Wittwer CT. Homogenous multiplex genotyping of hemochromatosis mutations with fluorescent hybridization probes. Am J Pathol 1998;153:1055-61.

(6.) Wittwer CT, Garling DJ. Rapid cycle DNA amplification: time and temperature optimization. Biotechniques 1991;10:76-83.

(7.) von Ahsen N, Oellerich M, Schutz E. Use of two reporter dyes without interference in a single-tube rapid cycle PCR: [[alpha].sub.1]-antitrypsin genotyping by multiplex real-time fluorescence PCR with the LightCycler. Clin Chem 2000; 46:156-61.

(8.) Wittwer CT, Herrmann MG, Moss AA, Rasmussen RP. Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques 1997;22: 130-8.

(9.) Bernard PS, Pritham GH, Wittwer CT. Color multiplexing hybridization probes using the apolipoprotein E locus as a model system for genotyping. Anal Biochem 1999;273:221-8.

(10.) SantaLucia J Jr, Allawi HT, Seneviratne PA. Improved nearest-neighbor parameters for predicting DNA duplex stability. Biochemistry 1996;35: 3555-62.

(11.) Guo Z, Liu Q, Smith LM. Enhanced discrimination of single nucleotide polymorphisms by artificial mismatch hybridization. Nat Biotechnol 1997;4: 331-5.

(12.) Lyon E, Millson A, Phan T, Wittwer CT. Detection and identification of base alterations within the region of factor V Leiden by fluorescent melting curves. Mol Diagn 1998;3:203-10.

(13.) Gundry CN, Bernard PS, Herrmann MG, Reed GH, Wittwer CT. Rapid F508del, F508C assay using fluorescent hybridization probes. Genet Test 1999;3:365-70.

(14.) Mitchell RS, Stevenson E, Mouritsen CL, Bohling S, Lyon E. A comparison of storage conditions for PCR mixtures with fluorescently labeled probes [Abstract]. J Mol Diagn 1999;1:60.

(15.) von Ahsen N, Schutz E, Armstrong VW, Oellerich M. Rapid detection of prothrombotic mutations of prothrombin (G20210), factor V (G1691A), and methylenetetrahydrofolate reductase (C677T) by real-time fluorescence PCR with the LightCycler. Clin Chem 1999;45:694-6.

(16.) Mangasser-Stephan K, Tag C, Reiser A, Gressner AM. Rapid genotyping of hemochromatosis gene mutations on the LightCycler with fluorescent hybridization probes. Clin Chem 1999;45:1875-8.

(17.) Aslanidis C, Schmitz G. High-speed apolipoprotein E genotyping and apolipoprotein B3500 mutation detection using real-time fluorescence PCR and melting curves. Clin Chem 1999;45:1094-7.

(18.) Nauck MS, Gierens H, Nauck MA, Marz W, Wieland H. Rapid genotyping of human platelet antigen 1 (HPA-1) with fluorophore-labelled hybridization probes on the LightCycler. Br J Haematol 1999;105:803-10.

(19.) Blomeke B, Sieben S, Spotter D, Landt O, Merk HF. Identification of N-acetyltransferase 2 genotypes by continuous monitoring of fluorogenic hybridization probes. Anal Biochem 1999;275:93-7.

(20.) Nauck M, Wieland H, Marz W. Rapid, homogeneous genotyping of the 4G/5G polymorphism in the promoter region of the PAII gene by fluorescence resonance energy transfer and probe melting curves. Clin Chem 1999;45:1141-7.

(21.) Pals G, Pindolia K, Worsham MJ. A rapid and sensitive approach to mutation detection using real-time polymerase chain reaction and melting curve analysis, using BRCA1 as an example. Mol Diagn 1999;4:241-6.

(22.) Cane PA, Cook P, Ratcliffe D, Multimer D, Pillay D. Use of real-time PCR and fluorimetry to detect lamivudine resistance-associated mutations in hepatitits B virus. Antimicrob Agents Chemother 1999;43:1600-8.

(23.) Lee LG, Livak KJ, Mullah B, Graham RJ, Vinayak RS, Woudenberg TM. Seven-color, homogenous detection of six PCR products. Biotechniques 1999;27:342-9.

Philip S. Bernard

Carl T. Wittwer *

Department of Pathology

University of Utah School of Medicine

50 North Medical Dr.

Salt Lake City, UT 84132

* Author for correspondence. E-mail Carl_Wittwer@hlthsci. Bernard and Wittwer: Editorial
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Title Annotation:Editorial
Author:Bernard, Philip S.; Wittwer, Carl T.
Publication:Clinical Chemistry
Date:Feb 1, 2000
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