High-throughput sequencing, also known as the "next generation sequencing technology", is characterized by being able to measure hundreds of thousands to millions of DNA molecules in parallel at one time, and generally has a short reading length.
According to the development history, influence and different sequencing principles and technologies, there are mainly the following types: massive parallel signature sequencing, MPSS, Polony sequencing, 454 pyrophosphate sequencing, Illumina (Solexa) sequencing, ABI solid-state sequencing, ionic semiconductor sequencing, DNA nanosphere sequencing and other gene analyzers (DNA sequencers) use capillary electrophoresis technology instead of traditional polyacrylamide plate electrophoresis, and use ddNTP labeled with four-color fluorescent dyes patented by the company. Therefore, through single primer PCR sequencing reaction, the generated PCR product is a single-stranded DNA mixture with four different fluorescent dyes at the 3' end, with a difference of 1 base, so that the sequencing PCR products of the four fluorescent dyes can be electrophoresed in a capillary tube, avoiding swimming lanes. Due to the different molecular sizes, the mobility in capillary electrophoresis is also different. When molecules pass through the capillary reading window, the CCD (Charge Coupled Device) camera detector in the laser detector window can detect fluorescent molecules one by one. The excited fluorescence is divided by grating to distinguish different colors of fluorescence representing different substrate information, and the images are synchronized on CCD camera. Analysis software can automatically convert different fluorescence into DNA sequence, so as to achieve the purpose of DNA sequencing. The analysis results can be output in various forms, such as gel electrophoresis map, fluorescence absorption peak map or base arrangement order.
It is an advanced precision instrument for measuring the base sequence, size and quantification of DNA fragments by computer automatic control, such as automatic glue filling, automatic sampling and automatic data collection and analysis. PE also provides gel polymers, including DNA sequencing gel (POP 6) and GeneScan gel (POP 4). The pore size of these gel particles is uniform, which avoids the influence of inconsistent gel preparation conditions on sequencing accuracy. It is mainly composed of capillary electrophoresis device, Macintosh computer, color printer and electrophoresis accessories. Computers include software for data collection, analysis and instrument operation. It uses the latest CCD camera detector to shorten DNA sequencing to 2.5h hours, and PCR fragment size analysis and quantitative analysis are 10 ~ 40 minutes.