Methods of prenatal diagnosis using digital pcr
A digital and chromosomal technology, applied in laboratory containers, chemical instruments and methods, biochemical equipment and methods, etc.
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Embodiment 1
[0046] Example 1: Selection of control genes and target genes, as well as design of primer pairs and probes
[0047]For genetic analysis of fetal genes, e.g., trisomy and the like, such as analysis of genetic abnormalities, chromosome 21 selected (due to trisomy causes Down's syndrome) as a target chromosome, chromosome selected chromosome 1 and 2 as a control chromosome . Then, in order to detect genes, four genes selected region on chromosome 21 as the target gene, to select a region of a gene on chromosome 1 and chromosome 2 gene region of a gene as a control. According to these genes, primers designed to amplify each gene and a fluorescently labeled probe can be confirmed the amplification reaction product. These primers and probes, respectively. (Daejeon, Korea) and Thermo Fisher Scientific (USA) synthesized from Bioneer Co., Ltd. Chromosome 21, chromosome 1 and chromosome 2 as the selected region figure 1 As shown in the primer and probe sequences and design as shown in Tab...
Embodiment 2
[0054] Example 2: Digital PCR for normal or Down syndrome
[0055] gDNA normal or Down's syndrome (T21 of) purchased from Coriell, to obtain normal or Down's syndrome (T21 of) digital values of gDNA PCR results.
[0056] The gDNA was used as a template, PCR QX200 digital system (Bio-Rad, USA) digital PCR. In order to establish the appropriate concentration for the samples of the digital PCR, gDNA 1-5000 fold serially diluted samples in the experiment. Preparing a reaction solution containing 0.5-1.0μM 0.1-0.25μM primers and probes as 20μl of PCR master mix digital (master mix), adjusted to a concentration of 2-10ng of sample injection (see Table 3), and assigned to the respective PCR tube. The PCR tube containing the solution is installed in the droplet generator DG8 cartridge, and dispensing the sample into the sample wells 20μl and 70μl of oil droplet generator (gasket (Gasket)) assigned to the oil hole. Subsequently, the gasket is mounted in the cartridge, and the droplet ge...
Embodiment 3
[0063] Example 3: Number of amniotic fluid samples of pregnant women with normal or Down syndrome
[0064] Extracting the fetus from the amniotic fluid sample cfDNA normal harbor or Down's syndrome (T21) fetuses of pregnant women. CfDNA as a template using fetal samples, digital PCR.
[0065] First, circulating nucleic acids using a QIAamp kit (Qiagen Cat. No. 55114, Qiagen, Germany), gDNA extracted from the cells. GDNA extraction carried out according to the manufacturer's manual. Specifically, the cells were lysed with lysis buffer protein, and 100μl of Proteinase K was added to 50ml centrifuge tube, to remove the protein component, thereby improving the purity of a sample. Then, a solution containing proteinase K, 50ml centrifuge tubes ACL buffer containing 1.0μg of vector RNA, followed by vortexing and the cell lysate buffer ACL. The mixture was incubated at 60 ℃ 30 minutes and then 3.6 ml of buffer ACB (lysate buffer) was added to the centrifuge tube, followed by vortexing t...
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