公开(公告)号：US12059677B2

公开(公告)日：2024-08-13

申请号：US16971967

申请日：2019-04-12

申请人： ZHANGJIAGANG ONE-CHIP BIOTECHNOLOGY CO., LTD.

发明人： Yabao Liu ,  Yaofei Yue ,  Zhifeng Zhang

IPC分类号： C12Q1/6806 ,  B01L3/00 ,  C12N15/10 ,  C12Q1/6874

CPC分类号： B01L3/5027 ,  C12N15/1096 ,  C12Q1/6806 ,  C12Q1/6874 ,  B01L2300/0829 ,  C12Q1/6874 ,  C12Q2531/113 ,  C12Q2563/159 ,  C12Q2565/301 ,  C12Q2565/519 ,  C12Q1/6806 ,  C12Q2531/113 ,  C12Q2563/159 ,  C12Q2565/301 ,  C12Q2565/519

摘要： Provided is a method of gene sequencing on a microwell array chip, including: Step 1: adding a PCR amplification system containing DNA fragments to be sequenced into the microwell array chip, allowing microwells to each individually form reaction spaces and allowing one DNA fragment to be contained in one microwell; Steps 2 to 3: subjecting the microwell array chip to PCR amplification, and denaturing amplified double-stranded DNAs in individual microwells; and Steps 4 and 5: sequencing the DNA fragments with sequencing primer S2 molecules and dNTPs in each microwell with a sensor at the bottom of the microwell.

公开(公告)号：US20210077995A1

公开(公告)日：2021-03-18

申请号：US16971967

申请日：2019-04-12

申请人： ZHANGJIAGANG ONE-CHIP BIOTECHNOLOGY CO., LTD.

发明人： Yabao Liu ,  Yaofei Yue ,  Zhifeng Zhang

IPC分类号： B01L3/00 ,  C12Q1/6806 ,  C12Q1/6869 ,  C12N15/10

摘要： Provided is a method of gene sequencing based on a single molecule library preparation on a microwell array chip, the single molecule library being further amplified by PCR on the same microwell array chip, the method including: Step 1: adding a PCR amplification system containing DNA fragments to be tested into the microwell array chip, allowing microwells on the microwell array chip to each individually form reaction spaces and allowing one DNA fragment to be contained in one microwell, Step 1: subjecting the sealed microwell array chip after step 1 to PCR amplification reaction on a PCR machine, Step 3: denaturing amplified double-stranded DNA molecules of the DNA fragment in individual microwell to single-stranded DNA molecules, Step 4: allowing sequencing primer S2 molecules to be paired with the single-stranded DNA molecules of the DNA fragment in individual microwell via annealing, Step 5: the dNTP added into microwells in sequence of dGTP, dCTP, dATP and dTTP is paired with a base under sequencing, with hydrogen ions or pyrophosphate PPi ions released and thus charges of DNA backbones increased, resulting in signal response of a sensor at bottom of the microwell, the signal is recorded and converted into gene sequence information, and Step 6: repeating step 5.