Nuclease Assay

A further currently used allele-specific hybridization method is performed in a homogenous, solution-phase reaction. In the 5' nuclease, or TaqMan® assay introduced by

Figure 4 Primer extension using microarrays. Abbreviations: PCR, polymerase chain reaction; ddNTPs, dideoxy nucleotides.

Applied Biosystems, alleles are discriminated using the 5' nuclease activity of Taq polymerase to detect a fluorescent reporter signal generated during PCR (Fig. 4).

In addition to PCR primers, one pair of TaqMan probes, consisting of an oligonu-cleotide labeled with both a 5' fluorescent reporter dye, such as FAM, and a 3' quencher dye, such as TAMRA, is included in a typical PCR. This is illustrated in Figure 5A. The TaqMan probes are designed to be complementary to the wild type and to the variant allele, which is located between the two PCR primers. During annealing of PCR primers, the probe hybridizes to the polymorphic template. Subsequent amplification of the probe-specific product causes cleavage of the probe by the 5' nuclease activity of the Taq polymerase, generating an increase in reporter fluorescence because the reporter dye is liberated from the quenching activity of TAMRA. By using different reporter dyes, cleavage of allele-specific probes can be detected in a single tube. Each unique TaqMan probe binds preferentially to one of the alleles. As a consequence, binding of the TaqMan probe to the unmatched target sequence is highly reduced or even completely abrogated. Additionally, TaqMan probes modified with minor groove binder features provide better allelic discrimination. This modification allows shorter probes that enhance the affinity to the allele-specific target.

An alternative hybridization method for SNP genotyping uses wavelength shifting Molecular Beacon probes with a stem-loop structure. When not bound to the target, the hairpin stem keeps the fluorophore so close to the quencher that fluorescence does not occur. However, when the probe anneals to its target sequence, the fluorophore is separated from the quencher, and fluorescence restored. The increase in fluorescence during amplification can be monitored in real-time or at least after completion of the PCR. A sequence detection system (SDS) algorithm for allelic discrimination generates three clusters, and genotypes are inferred based on the fluorescence readout.

Figure 5 (A) 5'-Exonuclease assay (TaqMan®); (B) Bead array (Illumina®). Abbreviation: PCR, polymerase reaction chain.

Because costs of modified probes with fluorescent and quenching features is a limiting factor in application of the TaqMan approach this technology is most suitable for analyzing a limited number of SNPs in large samples. TaqMan assays can be performed in 96- or 384-well formats. Due to the incorporation of allele-specific probes in the PCR any post-PCR processing is avoided, thus, TaqMan technology is robust with low-error rates and has an user-friendly interface. Furthermore, the investment for instrumentation is relatively low compared with mass spectrometry.

The main disadvantage of TaqMan is that the design of probes and the establishment of assays require more labor. Accuracy is highly dependent on hybridization conditions influenced by buffer composition or primer concentrations. Therefore, the vendor offers assays on demand that provide design and validation of assays. Thus, TaqMan-based technology also makes HT assignment of genotypes feasible in daily routines.

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