Gene sequencing method realized by detecting pyrophosphoric acid charges

Abstract

The invention discloses a gene sequencing method capable of increasing the signal-to-noise ratio by detecting pyrophosphoric acid charges. According to the method, by adding dNTP, polymerases and catalysts to a reaction system, pyrophosphoric acid is released through base pairing, pyrophosphoric acid is hydrolyzed to generate hydrogen phosphate ions, and the hydrogen phosphate ions are combined with sensor surface receptors to generate electrical signals which are further translated into corresponding bases. Pyrophosphoric acid generated during amplification can be detected, the diffusion rate of pyrophosphoric acid is much slower than that of hydrogen ions because the relative molecular mass of pyrophosphoric acid is 174, electrical signals which can be detected last for a long time, and accuracy can be effectively improved. Besides, pyrophosphoric acid has four positive charges, diffusion is slowed down, effective surface concentration is high, the converted electric signals are strong, and the detected signal-to-noise ratio is high. The high signal-to-noise ratio allows the use of smaller sensors and a sensor array with higher density and larger scale, so that sequencing throughput can be further improved and sequencing cost can be reduced.

Description

technical field [0001] The invention belongs to the field of gene sequencing, in particular to a method for gene sequencing by detecting pyrophosphate charge. technical background [0002] Acquiring the genetic information of organisms quickly and accurately has always been of great significance to life science research. For each organism, the genome contains the genetic information of the entire organism. Sequencing technology can truly reflect the genetic information on genomic DNA, and then more comprehensively reveal the complexity and diversity of the genome, so it plays a very important role in life science research. [0003] Sequencing technology can be traced back to the 1950s at the earliest. As early as 1954, there were reports on early sequencing technology, that is, Whitfeld et al. used chemical degradation methods to determine polyribonucleotide sequences; in 1977, Sanger et al. invented double The deoxynucleotide terminal termination method (Sanger gene seque...

AI Technical Summary

Problems solved by technology

The cost of first-generation sequencing technology is high. According to estimates, it will cost US$3 billion to complete the Human Genome Project with this method; the amount of data analysis is large; the degree of automation is not high or manual operations are required; some polymerase chain reaction (PCR) products cannot It needs to be analyzed to prepare a single clone; and this method is slow and takes a long time for sequencing. It is estimated that it will take at least 3 years to complete the sequencing of the human genome with this method

Relatively speaking, the workload of second-generation sequencing is still relatively large, and the cost is still relatively high. It is not suitable for mutation detection of single genes with known sequences; the key is that the read length is relatively short, the time is not fast enough, and the amount of template required is still low There are many, so it is impossible to detect at the level of single cell and single molecule

The third-generation sequencing technology is suitable for genome-wide determination with a small amount of input, high throughput, and a high degree of automation. Therefore, it is not suitable for the detection of single gene loci that are not demanding, such as genetic diagnosis of single-gene diseases. In other words, the cost performance is reduced; in addition, the third-generation sequencing technology still needs to focus on reducing background noise, improving accuracy, and reducing sequencing costs; in addition, how to maintain the ductility of DNA in terms of DNA immobilization without dimer structures , there are also places to be solved and improved

Next-generation sequencing technology, such as Ion Torrent's semiconductor sequencing technology, uses semiconductor ion-sensitive field-effect sensors, which can continuously reduce sensor size, increase array size, increase sequencing throughput, and reduce costs with the help of Moore's Law in the semiconductor industry; However, one of the major challenges facing next-generation gene sequencing is how to improve the signal-to-noise ratio, which decreases dramatically as sensors get smaller and smaller, while maintaining high accuracy

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