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Aim: Screening of an easily detectable biomarker to replace the HLA allele B variant (HLA-B*58:01) testing for predicting allopurinol-induced severe cutaneous adverse reactions (SCARs) in eastern Chinese patients. Methods: Six SNPs and the HLA-B*58:01 were analyzed in 17 patients with allopurinol-induced SCARs and in 151 control patients. SNPs were analyzed by pyrosequencing, and HLA-B*58:01 was evaluated by sequencing-based techniques. Consistency between sequencing-based HLA-B*58:01 testing and pyrosequencing-based rs9263726 testing was investigated in 262 individuals. Results: A significant association with allopurinol-induced SCARs was found at rs9263726 (odds ratio: 108.8) and HLA-B*58:01 (odds ratio: 108.8). The kappa values between sequencing-based HLA-B*58:01 testing and pyrosequencing-based rs9263726 testing were 0.96 (>0.75), demonstrating they were well coincident with each other. Conclusion: rs9263726 was a useful surrogate of HLA-B*58:01 testing for prescreening allopurinol-induced SCARs in eastern Chinese patients.

Pyrosequencing has been one of the most commonly used methods for genotyping; however, generally it needs single-stranded DNA (ssDNA) preparation from PCR amplicons as well as purified genomic DNA extraction from whole blood. To simplify the process of a pyrosequencing protocol, we proposed an improved linear-after-the-exponential (LATE)-PCR by employing whole blood as the starting material. A successful LATE-PCR was achieved by using a common Taq DNA polymerase in high pH buffer (HpH-buffer). As amplicons from LATE-PCR contain a large amount of ssDNA, pyrosequencing can be performed on the amplicons directly. Since DNA extraction and ssDNA preparation are omitted, the labor, cost and cross-contamination risk is decreased compared to conventional pyrosequencing-based genotyping protocols. The results for typing three polymorphisms related to personalized medicine of fluorouracil indicate that the proposed whole-blood LATE-PCR can be well coupled with pyrosequencing, thus becoming a potential tool in personalized medicine.

A 3-dimensional (3-D) polyacrylamide gel microarray based on dual-color fluorescence hybridization was an efficient SNP typing method with a high-throughput, but it is expensive to use dual dye-labeled allele-specific probes to type various SNPs. To lower the typing cost on 3-D polyacrylamide gel microarray, we propose a novel method by incorporating Cy5-dCTP into label-free allele-specific probes hybridizing to gel-immobilized targets. The method is much simple. At first, raw PCR products without any purification was spotted on the acryl-modified slides to copolymerize with acrylamide monomers. Then a pair of allele-specific probes were respectively added into two different areas of a hydrogel chip to hybridize with the single-stranded DNA targets immobilized in the gel-pads. Before extension reaction with Cy5-dCTP, electrophoresis was performed on the gel chip to remove non-specific allele-specific probes, and a high specificity was thus obtained. After the extension reaction, electrophoresis was used once more to remove the unincorporated Cy5-dCTP absorbed in the gel pads, and a low background image was achieved. The method was successfully employed to type the SNP (C14417G) in the OLR-1 gene for 40 different samples, and the typing results were consistent with those by pyrosequencing, indicating that the proposed method is accurate and specific in SNP typing. As no use of dye-modified probes, the typing cost is significantly decreased in comparison with the conventional typing method based on dual-color fluorescence hybridization, in particular when typing multiple SNPs. In addition to the low cost, our method has a low risk of cross-contamination from PCR amplicons due to no need of purification step of PCR products. Although only proof-of-concept results were given, we believe that the proposed method should be very useful for screening the biomarkers related to disease-susceptibility and personalized medicine where detection of many SNPs in different genes is required.

Presently most techniques for gene expression analysis are based on a dye label. Here we describe a novel method for comparing gene expression levels among various tissues or cells by sequence-tagged reverse-transcription PCR coupled with pyrosequencing (termed "SRPP"). This method includes three steps: (i) reverse transcription of mRNA with sources-specific RT primers consisting of a tail at the 5'-end for supplying a common PCR priming site, a source-specific sequence in the middle, and a poly-T stretch plus several degenerate bases at the 3'-end for annealing the mRNA strand. (ii) PCR amplification of the templates produced by pooling sequence-labeled cDNAs equally from different sources. (iii) Decoding and quantification of the source-specific sequences tagged in the amplicons by pyrosequencing. The signal ratio in the pyrogram is proportional to the amounts of mRNAs among different sources. As the signal is detected by observing bioluminescence, neither dye, nor electrophoresis, or laser source was used. The expression levels of six kinds of genes (Cdk2ap2, Vps4b, Fas, Fos, Cdk4, and Actb) among the kidney, the brain, and the heart tissues of a mouse were accurately detected, suggesting that the new method is promising in quantitatively comparing gene expression levels among different sources at a low cost.

Pyrosequencing is a powerful tool widely used in genetic analysis, however template preparation prior to pyrosequencing is still costly and time-consuming. To achieve an inexpensive and labor-saving template preparation for pyrosequencing, we have successfully developed a single-tube multiplex PCR including a pre-amplification and a universal amplification. In the process of pre-amplification, a low concentration of target-specific primers tagged with universal ends introduced universal priming regions into amplicons. In the process of universal amplification, a high concentration of universal primers was used for yielding amplicons with various SNPs of interest. As only a universal biotinylated primer and one step of single-stranded DNA preparation were required for typing multiple SNPs located on different sequences, pyrosequencing-based genotyping became time-saving, labor-saving, sample-saving, and cost-saving. By a simple optimization of multiplex PCR condition, only a 4-plex and a 3-plex PCR were required for typing 7 SNPs related to tamoxifen metabolism. Further study showed that pyrosequencing coupled with an improved multiplex PCR protocol allowed around 30% decrease of either typing cost or typing labor. Considering the biotinylated primer and the optimized condition of the multiplex PCR are independent of SNP locus, it is easy to use the same condition and the identical biotinylated primer for typing other SNPs. The preliminary typing results of the 7 SNPs in 11 samples demonstrated that multiplex PCR-based pyrosequencing could be promising in personalized medicine at a low cost.

A novel dye-free labeling method for a multiplex bioassay was proposed by using short sequence-based barcodes consisting of a reporter base and repeats of two stuffer bases; then, the barcodes were quantitatively decoded by a single pyrosequencing assay without any pre-separation.

Pyrosequencing is an ideal tool for verifying the sequence of amplicons. To enable pyrosequencing on amplicons from nucleic acid sequence-based amplification (NASBA), asymmetric NASBA with unequal concentrations of T7 promoter primer and reverse transcription primer was proposed. By optimizing the ratio of two primers and the concentration of dNTPs and NTPs, the amount of single-stranded cDNA in the amplicons from asymmetric NASBA was found increased 12 times more than the conventional NASBA through the real-time detection of a molecular beacon specific to cDNA of interest. More than 20 bases have been successfully detected by pyrosequencing on amplicons from asymmetric NASBA using Human parainfluenza virus (HPIV) as an amplification template. The primary results indicate that the combination of NASBA with a pyrosequencing system is practical, and should open a new field in clinical diagnosis.