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Editorials |
a Author for correspondence. E-mail Carl_Wittwer{at}hlthsci.med.utah.edu
1 Department of Pathology, University of Utah School of Medicine, 50 North Medical Dr., Salt Lake City, UT 84132
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)(3)(4)(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
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 °C/s) provides qualitative information about the sequence of the target (3)(4)(5).
The probe melting temperature (Tm), 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 Tm 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 Tm 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 °C within runs (12). Thus, a small Tm shift 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 °C Tm 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
Tm. 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
1-antitrypsin gene, starting with the
amplification of genomic DNA. Additional applications are sure to
follow.
The power of multiplexing with both color and
Tm is the product of the number of
colors and the number of Tms that can
be differentiated. The 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 Tms 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
Tm 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.
Acknowledgments
C.T.W. holds equity interest in Idaho Technology. Idaho Technology has licensed hybridization probe and LightCycler® technology from the University of Utah and, in turn, licensed these technologies to Roche Molecular Biochemicals.
References
1-antitrypsin genotyping by multiplex real-time fluorescence PCR with the LightCycler. Clin Chem 1999;46:156-161.
The following articles in journals at HighWire Press have cited this article:
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