Homogeneous Assays

Under homogeneous conditions, mutation analysis is performed in solution and in a single sealed tube.100,101 These precautions reduce the risk of cross-contamination and enhance speed and simplicity. "Real-time" monitoring and quantitation are possible. Excellent discussions of this topic by Foy and Parkes100 and by Shi101 emphasize applications that are emerging in the clinical arena.100

Some homogeneous assay platforms incorporate ultrarapid thermocyclers that affect the time for analysis. For example, the thermocycler used in LightCycler equipment permits PCR to be performed within 20 minutes in glass capillaries, and that in the ABI Prism 7700 Sequence Detection System permits assays to be performed within 2 hours in microtiter plate-based systems. Options for non-PCR-based methods include BDProbeTecET®, which is a platform based on strand displacement and fluorescent detection,102 and Abbott LCx®, which is a platform that uses a ligase chain reaction for detection.103

Both nonspecific and specific homogeneous assay methods are available.100 Nonspecific methods detect the presence (or absence) of the amplified PCR product but provide no additional information about the product. They are prone to produce false-positive results attributed to undesired products that spuriously increase fluorescence. Specific methods that probe the amplified product are more desirable for molecular genetic diagnostics. Several of the most popular formats for specific methods are non-gel-based, and favor fluorescent signals for detection. Fluorescence resonance energy transfer (FRET)104 or fluorescence polarization105 has been utilized for signal generation. The signal appears rapidly and reliably. Examples of FRET-based technologies for specific high throughput detection of mutations include TaqMan assay, Molecular Beacons , Scorpion primers, and the Invader assay. The first three techniques use PCR-amplified fragments of DNA while the Invader system enables SNP genotyping without amplification. Fluorescence polarization combined with ARMS as noted above (see p. 195) has also been shown to be a highly sensitive technique for signal generation.93 The homogeneous assay methods described below are amenable to automation and high-throughput analysis.

Single-Base Primer Extension. The concept of single-base primer extension for mutation analysis, also termed ''minisequencing''80 and ''genetic bit analysis,''81 was developed several years ago. It has attracted a lot of attention and reference to this technique appears under many different names and acronyms. This concept is applicable to several different platforms including gel and capillary elec-trophoresis, numerous homogeneous assay formats such as ELISA among others, and solid phase assays performed on microarrays and in microtiter plates.106,107

The principle of this method in its simplest form involves single-base extension of an oligonucleotide primer annealed to single-stranded DNA at a location adjacent but not overlapping the polymorphic site. This is followed by the addition of DNA polymerase to catalyze extension of the primer in the presence of chain-terminating dideoxynucleotides.106 Single-base primer extension achieves its specificity through enzyme-based recognition instead of hybridization of the primer. The dideoxynucleotides are labeled by fluorophore or in various other ways to facilitate the identification of the single incorporated nucleotide. The low cost of reagents, robustness, accuracy, and flexibility with respect to platform and scalability from one-gene-at-a time assays to large-scale, high-throughput assays are among the advantages that recommend the method.

A homogeneous assay format using FRET to detect single-base pair exchanges in genomic DNA samples has been applied to detect variants of several genes including the cystic fibrosis gene, the HLA-H human leukocyte gene, and the tyrosine kinase protooncogene.108 The change in fluorescent intensity during primer extension can be monitored as time passes by a fluorescence spectrophotometer, or at its endpoint by a fluorescence plate reader.

A recent version of this methodology combines the advantages of mini-sequencing with those of a microarray format that permits highly multiplexed and parallel analysis. The methodology is described in detail by Lovamer and Syvanen.109

TaqMan® Assays. A fluorogenic probe consisting of an oligonucleotide labeled with a fluorophore in close proximity to a quencher dye is included in a typical PCR reaction. When TaqMan DNA polymerase encounters the probe-specific, PCR-amplified product, the 5'-nuclease activity cleaves the probe. Cleavage separates the dye from the quencher and results in an increase in fluorescent intensity.110 The entire assay is performed using a single thermocycling protocol and a standard method of analysis that enables automated genotyping. By using different reporter dyes, several allele-specific probes can be discriminated in a single analysis.

Allele-specific Taqman probes have been used extensively to detect polymorphisms in drug metabolizing enzymes.111-113

Molecular Beacon® Assays. Molecular Beacons are hairpin-shaped fluorophore-labeled oligonucleotide probes that fluoresce on hybridization.114 The fluoro-phore is attached to one end and a quencher is attached to the other end of the hairpin that keeps these two molecules in close proximity, quenching the emitted fluorescence. In the presence of a complementary DNA target, the Molecular Beacon undergoes a conformational change, separating the fluorophore from the quencher, permitting fluorescence to occur. Hairpin probes are advantageous for mutation analysis because they are more specific than linear DNA probes. Molecular Beacons labeled with different fluorophores that emit signals at different wavelengths permit the analysis of more than one allelic combination in the same PCR reaction.

Molecular Beacon probes have been employed to detect the C-to-T point mutation in the methylenetetrahydrofolate reductase gene that is associated with cardiovascular disease.114

Scorpion® Primer Assays. Another method for efficient detection of PCR product uses fluorescent molecules that combine the primer and the probe functions into a single molecule called a ''Scorpion'' primer.115 The Scorpion primer is a hairpin-shaped probe element attached via a non-amplifiable segment to the 5'-end primer that carries a fluorophore/quencher pair (similar in principle to the Molecular Beacon). In isolation, the Scorpion primer is fully quenched and does not fluoresce. As the primer extends during PCR, the probe element hybridizes to a target on the newly formed strand from which the hairpin loop opens (but is not cleaved) to separate the fluorophore from the quencher to emit the fluorescent signal.

Application of this system to the analysis of the BRCA2 gene115 and the cystic fibrosis gene116 detected variant forms of these genes in wild-type and variant samples. Comparative fluorescence accumulation patterns showed the sensitivity of Scorpion primers to be approximately equal to that achieved with TaqMan and greater by several-fold under standard conditions than with Molecular Bea-cons.115 Another direct comparison between Scorpion, TaqMan, and Molecular Beacons on the Roche-LightCycler indicates that Scorpions performed better, particularly under the fast cycling condition.116 Though experience with the Scorpion primers is limited, it appears that probing of target DNA sequences with unimolecular primers may provide an alternative approach that is beneficial in assay design, reliability, and speed in the analysis of variants.

InvaderTM System Assays. The Invader system relies on naturally occurring and engineered enzymes, referred to as Cleavases® (Third Wave Technologies, Madison, WI), that cleave DNA molecules at particular locations in response to structure rather than sequence.117 A typical Invader assay includes two downstream allele-specific oligonucleotide signal probes (wild type or variant) plus an upstream Invader probe. The 5'-end of the downstream signal probes contains a ''flap'' that is not complementary to the DNA target sequence. The Invader system involves sequential primary and secondary reactions. When the 3'-end of the upstream probe overlaps (or invades) the hybridization site of the 5'-end of a downstream probe by at least one base, the ''flap'' is cleaved by the 5'-exonuclease activity of Cleavase®. The primary reaction occurs only when the 3'-end of the upstream probe invades the end of the duplex formed between the downstream probe and the DNA target by at least one base. The ''flap'' that is cleaved serves as an Invader probe that directs the secondary reaction. The secondary reaction uses a synthetic DNA sequence as a target; the second signal probe is hairpin shaped and is labeled with a fluorophore in close proximity to a quencher. Cleavage of the secondary signal probe by the Cleavase® generates the fluorescence signal. Because the sequences of the 5'-flap of the primary signal probe and the secondary signal probes are independent of the target, a universal detector system could be designed.117,118

The Invader system thus achieves its specificity by combining hybridization with enzyme recognition but without product amplification. It can use genomic DNA directly to discriminate variant from wild-type genotypes in mixed populations at very low ratios of (1/1000 or lower) of variant to wild type. The technique is rapid and adaptable to large-scale analysis of SNPs that use high-throughput methods of detection such as FRET and matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS). Applications of the Invader system to Factor V Leiden, factor II (prothrombin), cystic fibrosis, and apolipoprotein E, and to measurement of mRNA levels of ubiquitin demonstrate its versatility and validity for analysis of variants, SNP profiling, and gene expression analysis.118

The Invader assay has been combined with multiplex PCR in an automated high-throughput procedure that is capable of genotyping 300,000- 400,000 SNPs in 1 day. The amount of DNA needed to assay one SNP is 0.4 ng.119

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