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A METHOD OF GENE SEQUENCING BASE ON SINGLE MOLECULE PCR LIBRARY PREPARATION ON MICROWELL ARRAY CHIP

Abstract:

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.