ABSTRACT
Single nucleotide polymorphisms (SNPs) are important markers which can be used in the association study for searching for associated genes of complex diseases. High-throughput methods in a large number of samples are needed for SNP genotyping. In this study, we applied polyacrylamide gel-based microarray combined with universal dual-color hybridization for trios-based associative study of BDNF polymorphisms with autism in Chinese. All four SNPs in both patients and their parents could be analyzed quickly and correctly. In single SNP TDT analysis, C270T showed preferential transmission of the T allele compared to the C allele (TDT p < 0.001) in autism. In haplotype TDT analysis, C270T polymorphism also existed in the haplotype combination which showed significant association (TDT p < 0.05). These results suggest a potential association between BDNF and autism in the Chinese population. The study also show that the polyacrylamide gel-based microarray combined with universal dual-color detectors is a rapid, simple, high-throughput method for SNPs genotyping, and can be very effective and cost effective in association study of susceptible gene with disorders in large samples.
Subject(s)
Autistic Disorder/genetics , Brain-Derived Neurotrophic Factor/genetics , DNA Mutational Analysis/instrumentation , Electrophoresis, Polyacrylamide Gel/instrumentation , Genetic Predisposition to Disease/genetics , Oligonucleotide Array Sequence Analysis/instrumentation , Polymorphism, Single Nucleotide/genetics , Autistic Disorder/diagnosis , Child, Preschool , Equipment Design , Equipment Failure Analysis , Female , Humans , MaleABSTRACT
In this paper we describe a novel method for detecting many DNA fragments through efficient amplification by using an emulsion system based on "on-chip" PCR instead of conventional multiplex polymerase chain reaction (PCR). During the preparation of on-chip PCR, a set of primers were immobilized on a slide and other sets were in an emulsion system. Different emulsion phase primers and other related PCR components were dispersed in different droplets of the emulsion system, and then, due to the thermal instability of emulsion droplets, they would be released onto the surface of the slide after preheating in the first PCR step. To test the above method, we used plasma DNAs from pregnant women who was carrying a male fetus for gender identification. Four different Y chromosome DNA fragments were selected. Results showed that different DNA fragments could be simultaneously amplified with satisfactory results. It is suggested that a simple, convenient and inexpensive on-chip PCR method has been developed.
Subject(s)
Emulsions/chemistry , Polymerase Chain Reaction/methods , DNA/blood , Female , Fetus , Humans , Pregnancy , Reproducibility of Results , Sex Determination Analysis , TemperatureABSTRACT
Deletions in Y chromosome are thought to be pathologically involved in some cases of male infertility associated with azoospermia or oligozoospermia. An emulsion-based multiplex PCR method was developed for detecting Y chromosome microdeletions in infertile men and a plasma sample of pregnant women carrying a male fetus. The sensitivity of multiplex PCR in emulsion was evaluated. Conventional PCR was also carried out for comparison. A total of 13 sequence-tagged sites (STSs) distributed in the AZF region were analyzed simultaneously with this method. The SRY gene was also detected as the inner control. Results showed that Y chromosome microdeletions were found in 4 of 19 infertile patients. Also, in 1 of 63 samples collected from pregnant women, microdeletions were found in some of the detected sites. It is suggested that the emulsion PCR assay was proven to be a promising diagnostic tool and could be widely used in further clinical and academic research.
