Small molecule microarrays for drug residue detection in foodstuffs.

Microarrays have been used as tools for analyzing biological compositions at different levels. In this study, we proposed a small molecule microarray (SMM) method for detection of three veterinary drug residues, chloramphenicol, clenbuterol, and tylosin, in foodstuffs simultaneously and quantitatively. The small drug molecules were immobilized on the surface of the modified glass slides. Then the mixture of drug corresponding antibodies and standards or samples was added to the reaction area. After incubation, the antigen-antibody binding was detected using cy5 labeled secondary antibody. The calibration curves of the residues were drawn, and they indicated the lowest detection limit the linearity range. The detectable concentrations of the three residues are lower than the maximum residue levels (MRLs). No cross reactivity was found among the three residues. The coefficient of variation of the spot intensities was below 5% in a subarray, and below 15% among subarrays. The spike sample test and the comparison of detection results by SMMs and ELISA demonstrated the accuracy of the proposed SMMs method.

Simultaneous genotyping of DRB1/3/4/5 loci by oligonucleotide microarray.

Matching of the HLA antigens for donor-recipient in transplantation, disease predisposition or protection, population studies, and forensic testing requires accurate but simple typing methods. Here, we describe a DNA-based tissue-typing assay that determines the haplotype of the DRB1/3/4/5 loci in hy-bridization of oligonucleotide array after sample amplification. Using this multianalyte DNA hybridization system, we analyzed seven regions of exon 2 of DRB loci that have single-base discrimination. Thirty-six oligonucleotide probes complementary to the alleles of interest were immobilized on each microslide. The efficiency and specificity of identifying DRB genotypes using the oligonucleotide arrays was evaluated by blinded analysis of 147 samples from reference standards and subjects. The established method provides a rapid and inexpensive DRB "low-resolution" typing tool for prescreening a large number of samples.

Estimation of relative allele frequencies of single-nucleotide polymorphisms in different populations by microarray hybridization of pooled DNA.

Single-nucleotide polymorphisms (SNPs) are considered useful polymorphic markers for genetic studies of polygenic traits. A new practical approach to high-throughput genotyping of SNPs in a large number of individuals is needed in association study and other studies on relationships between genes and diseases. We have developed an accurate and high-throughput method for determining the allele frequencies by pooling the DNA samples and applying a DNA microarray hybridization analysis. In this method, the combination of the microarray, DNA pooling, probe pair hybridization, and fluorescent ratio analysis solves the dual problems of parallel multiple sample analysis, and parallel multiplex SNP genotyping for association study. Multiple DNA samples are immobilized on a slide and a single hybridization is performed with a pool of allele-specific oligonucleotide probes. The results of this study show that hybridization of microarray from pooled DNA samples can accurately obtain estimates of absolute allele frequencies in a sample pool. This method can also be used to identify differences in allele frequencies in distinct populations. It is amenable to automation and is suitable for immediate utilization for high-throughput genotyping of SNP.

Simultaneous detection of alpha-thalassemia and beta-thalassemia by oligonucleotide microarray.

In this study, we describe a reliable microarray-based assay for the simultaneous detection of alpha/beta-globin genotypes. The efficiency and specificity of this method were evaluated by blinded analysis of 1,880 samples. The assay provides unambiguous detection of complex combinations of heterozygous, compound heterozygous and homozygous alpha/beta-thalassemia genotypes.

Rapid detection of common Chinese glucose-6-phosphate dehydrogenase (G6PD) mutations by microarray-based assay.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzymopathy, affecting more than 200 million people worldwide. To date more than 123 mutations in the G6PD gene have been discovered, among which 12 point mutations are found in the Chinese. Setting up a simple and accurate method for detecting these mutations is not only useful for diagnosing G6PD deficiency under some circumstances that it is difficult to measure the activity of the enzyme, but also for studying the frequency of the G6PD genotypes. The purpose of this study was to develop a simple, inexpensive and accurate method for detecting these common mutations. Microarray-based assay was described in this study. Samples from 198 G6PD-deficient persons were investigated. The DNA sequencing data supported the results obtained by microarray-based assay. Thus, we concluded that the microarray-based assay is a rapid, simple, inexpensive, and accurate method for detecting the most common G6PD gene mutations among the Chinese. This method involves the selective amplification of human G6PD gene with specific oligonucleotide primers, fragmentation and labeling of PCR products, followed by hybridization with allele-specific oligonucleotide (ASO) probes on chip.