Pyrosequencing, a new enzyme cascade sequencing technology, is applicable to analyze known short sequence. Its repeatability and precision are comparable with Sanger DNA sequencing method, and the speed is greatly improved. Pyrosequencing technology is capable of sequencing large number of samples simultaneously. It also offers an ideal technology platform with the advantages of high-throughput and low cost for detecting single nucleotide polymorphisms (SNPs) intuitively. Being improved, this technology can meet the demand of nucleotide sequencing at one hundred and above samples. Then this technology also can be applied to the genotyping of important microorganisms, mutation detection and clone identification of specific DNA fragments and in other fields.
1.Theory Pyrosequencing technology is a series of enzyme cascade chemiluminescent reaction catalyzed by four enzymes in a reaction system. Principle of pyrosequencing technology lies in coupling the polymerization of each dNTP with the release of fluorescence signal at a time, after the primers and templates DNA annealing and under the synergies of DNA polymerase, ATP sulfurytase, 1uciferase and Apyrase. And then it can achieve the goal of DNA sequencing in real time through collecting the intensity of fluorescence. Reaction system of pyrosequencing technology is constituted by substrates, single chain to be detected, sequencing primers and four enzymes. The reaction substrates include adenosine- 5’-phosphosulfat and luciferin.
2.Reaction
In each round of sequencing reaction, only one type of deoxynucleotide triphosphate (dNTP) would be added to the reaction system. If dNTP happens to match with the next base of DNA template, it will be added to the 3 'end of the sequencing primer in the presence of DNA polymerase and release a molecule of pyrophosphate (PPi). PPi, produced by the help of ATP sulfate enzyme, can bind APS to generate ATP, which then can combine with luciferin to generate oxide luciferin and produce visible light under the catalysis of luciferase. A specific peak is available to observe through the feeble light detection device and software processing, the level of peak value is in proportion to the number of matched bases. If the dNTP can not match with the next base of DNA template, the reaction mentioned above will not happen and there will be no detectable peak at all. The remaining dNTP and small amount of residual ATP will be degraded in the presence of Apyrase eventually. After a complete reaction round, another type of dNTP would be added in the reaction system to repeat the above process. Then accurate DNA sequence information is obtained from the peak value results.
3.Application on gene analysis
Pyrosequencing technology is widely applied in SNP, genetic diversity, plant polymorphism analysis, molecular diagnosis of bacterial and viral genotyping, methylation analysis, forensic identification, pharmaceutical genomics and other fields. Gel electrophoresis, marking and staining for DNA samples are no longer required in this method, which has dominant characteristics, like high-throughput, low cost, short cycle and intuitive results. Compared with Sanger sequencing method, pyrosequencing technology has its specific advantages and plays a significant role in studying DNA analysis. Currently, pyrosequencing technology has been commonly used in molecular biology. Along with the continuous development and improvement of this technology, its application in practice will become more and more popular. Jonasson and other scientists used pyrosequencing technology to detect gene of pathogen 16S rRNA and achieved the rapid identification of bacteria against antibiotic in clinical specimens. Monstein and other scientists applied this technology to detect the variant V1 and V3 sequences of Helicobacter pylori 16S rRNA gene, which also proves that pyrosequencing technology is available for the rapid identification and classification of clinical specimens. Unnerstad utilized this technology to confirm genotypes of 106 different serotypes monocyto proliferated by Listeria, and finished a large number of samples sequencing in a short time with notable parallelism and high efficiency. And the workload will be great with conventional sequencing technology. Gharizadeh used pyrosequencing technology to identify HPV samples of 67 people, which can also prove that this technology can be utilized for the large-scale identification, genotyping and mutation of HPV and other pathogens. Uppsala University in Sweden used pyrosequencing technology to develop a new method to identify anthrax bacteria (bacillus anlhracis) and its pathogenicity status. They used this technique to analyze Ba813 gene(277bp length, it is a single copy in chromosome, which is a typically symbol that differs anthrax bacteria from other agrobacteriums) and determined a specific sequences of 20bp to identify anthrax bacteria. The accuracy rate is up to 99.6%. Pathogenic status of anthrax bacteria can be determined through analyzing the existence of two plasmids (lef gene identification of pX0l and cap gene of pX02) in strains. The accurate rate is up to 100‰. Edvinsson B applied this technology to classify the three subtypes of T toxoplasmosis. Based on the fragment amplification through Real-Time PCR technology, pyrosequencing technology was carried out to detect and confirm the polymorphism and its classification of two single nucleotides in GRA6 gene. The accuracy classification rate of detecting typical insect strains reaches up to 100%, and that of atypical insect strains is also up to 81%. What’s more, pyrosequencing technology is also applied to the rapid identification and classification of bordetella pertussis, parapertussis and other bacterias.
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