Pinpoint assay

Several assays have been patented for mutation detection these are mainly designed for single-nucleotide polymorphism (SNP) analysis and use MALDI-TOF spectrometry (Invader , Sequazyme-PinPoint assay, MassARRAY , GOOD assay) all of which use PCR amplification, or require a high DNA concentration in the sample (Invader ). 

Figure 7. Allele Quantitation with Modified ddNTP. Mixtures of ratios of homozygous and heterozygous PCR products for the APOE E locus were prepared and genotyped with ddCTP and ddTTP (regular Sequazyme-PinPoint assay), or a mixture ofddCTP and biotinylated ddUTP. The measured fraction of A allele, as assayed by the peak area offraction ofddT or biotinylated ddU incorporation, was plotted vj. the known fraction of the A allele in the...

Single base extension methods such as the PinPoint assay have an advantage for multiplexed SNP marker analysis. Notably, they can combine aU assays irrespective of the alleles to be genotyped simply by using the four dideoxynucleotides... 

A variation of the pharmacophore library was seen in an approach used to determine CD4+ T cell epitopes.31 In a 14-mer peptide, the researchers keep residues in positions 1, 4, and 6 constant since they functioned as anchor positions for binding receptor, and proceeded to vary the other residues in the peptide. This allowed the use of a shorter synthetic route compared to one that would be needed if all positions were to be varied. Limiting the size of the library allowed the authors to obtain better assays. By doing a partial release of the peptides, the authors were able to find the final peptide that represented the epitope of the T cell receptor. In another example, the epitope to inhibit stimulation of the thyrotropin receptor also was found via combinatorial libraries.32 Since the synthesis of a totally random hexapeptide library was deemed impractical, the authors opted to hold one position constant while the other five residues were randomized. This method was repeated for each residue in the peptide. The residues that were determined to be the most active were used as a basis for a second-generation library. The only limitation of the library was not the quantity of product synthesized, but to properly pinpoint the peptides in an assay. 

Such range finding may frequently straddle the optimal enzyme concentration for assay. Thus, a second series of enzyme dilutions is assayed to pinpoint the enzyme concentration over a narrower range. Usually four to five dilutions over a 10- to 20-fold concentration range are adequate. 

The abortive initiation assay (130) was used to pinpoint the Hg(ll)-responsive step in transcriptional initiation of the genes responsible for detoxifying Hg(II). This analysis provides information on the specific site... 

SNP genotyping by MALDI-TOF MS takes advantage of mass differences between allele-specific primer extension products. At present, three related assays are used, the PROBE— primer oligo base extension assay, which was further developed to the MassExtend assay by SEQUENOM— the PinPoint, and the GOOD assay (8). A representative scheme is depicted in Fig. 1. 

An LDH assay is a good initial diagnostic test because the isoenzyme forms of LDH are so widespread in the human system. Thus, the locations of diseases can be pinpointed. 

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