RESUMO
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.
Assuntos
Bioensaio/métodos , Análise de Sequência/métodos , Actinas/genética , Animais , Sequência de Bases , Quinase 4 Dependente de Ciclina/genética , Camundongos , Coloração e RotulagemRESUMO
To avoid sequencing error resulting from use of apyrase in conventional 4- enzyme pyrosequencing system, a non-apyrase 3-enzyme pyrosequencing system with a better performance of quantitative analysis was established. The method is to immobilize biotinylated DNA template, ATP sulfurylase and luciferase on streptavidin-coated magnetic beads for pyrosequencing. After pyrosequencing, ATP produced from the pyrosequencing reaction and excess dNTPs were removed by magnetic separation technique; another dNTP was then dispensed for sequencing reaction, and the components interfering with the next circle of pyrosequencing reaction were removed by the same way, achieving the circular sequencing. This new system can accurately measure base sequences of a target DNA template, and also can quantitatively determine the relative ratio of two alleles. The allele ratios in two SNPs (rs1042917 and rs4818219) having a higher heterozygote rate on chromosome 21 were successfully detected for 16 normal samples and 8 clinical samples from Down's syndrome patients. The results can accurately demonstrate whether or not the target sample has equal copies of chromosome 21 from mother and father. This paper established a non-apyrase 3-enzyme pyrosequencing method, which owns a good perform-ance of quantitative analysis. The method is especially suitable to allelic quantification of an SNP, enabling the rapid diagnosis of Down's syndrome by analyzing allele ratio of SNPs on chromosome 21.
Assuntos
DNA/genética , Síndrome de Down/diagnóstico , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA/métodos , Alelos , Apirase/metabolismo , Cromossomos Humanos Par 21/genética , DNA/química , DNA/metabolismo , Síndrome de Down/genética , Frequência do Gene , Humanos , Luciferases/metabolismo , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Sulfato Adenililtransferase/metabolismoRESUMO
OBJECTIVE: To establish a method to detect Down's syndrome through quantitative pyrosequencing of the heterozygous single nucleotide polymorphisms (SNPs) on the chromosome 21. METHODS: An improved allele-specific-amplification was used to screen heterozygous SNPs on the chromosome 21 from 84 normal samples. Pyrosequencing was used to quantitatively determine the ratio between the two alleles of a heterozygote, and the diagnosis of Down's syndrome was thus carried out based on the ratio. RESULTS: By genotyping 84 genomic DNA samples from normal Chinese population, 6 SNPs with a relatively high level of heterozygosity were screened out. Heterozygote coverage of 92.9% was achieved by using a panel of 6 SNPs on the chromosome 21. Ten clinical samples from Down's syndrome patients were quantitatively determined by pyrosequencing, and 9 samples were accurately diagnosed by comparing the ratio of the two alleles. The pyrosequencing results showed that the ratio of the two alleles were 2:1 or 1:2 for the Down's syndrome patients. CONCLUSION: The method has the advantage of a low cost, simple process, and time-saving operation and could be potentially applicable to the rapid diagnosis of Down's syndrome.