Pyro sequencing is a DNA sequencing technology based on the principle of sequencing-by-synthesis. The method consist of series of four enzymatic steps to detect sequence of nuclei acid during synthesis. Pyro sequencing can be easily automated and provides highly accurate results. The method is highly suitable for analysis of single nucleotide polymorphism and sequencing of small fragment of DNA. Pyro sequencing techniques consist of series of sequential steps for detection of DNA sequence.
The DNA fragment whose sequence needs to be identified is isolated and amplified. The template strand is labelled with biotin. Denaturation of double stranded DNA into single strand results in biotinylated single stranded PCR amplicon, which is isolated and hybridized with sequencing primer. The hybridized primer and template strand is incubated with enzyme DNA polymerase, luciferin, luciferase, apyrase, ATP sulfurylase, with substrates adenosine 5' phosphosulfate (APS). ATP sulfurylase converts PPi to ATP in the presence of adenosine 5' phosphosulfate (APS).
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The luciferin is converted into oxyluciferin by ATP driven luciferase-mediated reaction that generates visible light of amounts that are proportional to the amount of ATP. The light is illuminated and detected by a charge-coupled device (CCD) camera and seen as a peak in the raw data output. Apyrase degrades those nucleotides that are not incorporated and ATP. Once the degradation is accomplished, next nucleotide is added. This series of reactions take place sequentially. As the reaction continues, the complementary DNA strand is synthesized and the sequence of nucleotide is determined from the pyrogram trace.
Pyro sequencing technology enables quick, accurate, and real time quantification of sequence variation. Pyro sequencing has emerged as a versatile technology, with a number of applications in the field of modern biology. The technique was earlier used for SNPs analysis and determination of short fragment of DNA sequence. However, with technological advancements, pyro sequencing is expected to achieve long read length, reduced time frame, and improvement in automation process.
Increase in demand for clinical diagnosis, improving technology with decreasing costs of DNA sequencing, and higher research spending by biotechnology companies are anticipated to drive the pyro sequencing market. High cost of molecular diagnostic tests and devices, rising complexity of diseases, and impact of alternative sequencing and diagnostic procedures act as restraints of the pyro sequencing market.
The global pyro sequencing market has been segmented based on application, end-user, and region. In terms of application, the market has been segmented into DNA methylation analysis, mutation analysis and genotyping, microbial identification and drug resistance typing, and others. The development of therapeutic agents needs information on how gene polymorphism has impact on metabolism, determination of gene-mutation related diseases, analysis of forensic DNA which relies on detection of sequence variation are factors attributed to increase in use of mutational analysis and genotypic studies. Increase in number of mutational diseases and rise in forensic DNA analysis with new advancement in technology for clinical diagnosis are projected to augment the mutational analysis and genotyping segment.
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Based on end-user, the pyro sequencing market has been segmented into research centers & academic/government institutes, hospitals & clinics, pharmaceutical & biotechnology companies, and others. Demand for DNA sequencing is increasing in research and academic institutes. It is projected to rise during the forecast period.
In terms of region, the pyro sequencing market has been segmented into North America, Europe, Asia Pacific, Latin America, and Middle East & Africa. North America dominates the global market due to advanced health care infrastructure, high health care expenditure, and upcoming advance technology. Collaboration of key players with academic research institutes for advanced technology development for clinical diagnosis application is anticipated to boost the pyro sequencing market in Asia Pacific during the forecast period.
Key players operating in the pyro sequencing market are Roche Diagnostics, Pyrobett Pte Ltd., QIAGEN, Seqomics, Macrogen, Illumina, Eurofins Genomics, and Microsynth AG, among others.
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