CONTEXT: DNA sequencing is critical to identifying many human genetic disorders caused by DNA mutations, including cancer. Pyrosequencing is less complex, involves fewer steps, and has a superior limit of detection compared with Sanger sequencing. The fundamental basis of pyrosequencing is that pyrophosphate is released when a deoxyribonucleotide triphosphate is added to the end of a nascent strand of DNA. Because deoxyribonucleotide triphosphates are sequentially added to the reaction and because the pyrophosphate concentration is continuously monitored, the DNA sequence can be determined. OBJECTIVE: To demonstrate the fundamental principles of pyrosequencing. DATA SOURCES: Salient features of pyrosequencing are demonstrated using the free software program Pyromaker ( http://pyromaker.pathology.jhmi.edu ), through which users can input DNA sequences and other pyrosequencing parameters to generate the expected pyrosequencing results. CONCLUSIONS: We demonstrate how mutant and wild-type DNA sequences result in different pyrograms. Using pyrograms of established mutations in tumors, we explain how to analyze the pyrogram peaks generated by different dispensation sequences. Further, we demonstrate some limitations of pyrosequencing, including how some complex mutations can be indistinguishable from single base mutations. Pyrosequencing is the basis of the Roche 454 next-generation sequencer and many of the same principles also apply to the Ion Torrent hydrogen ion-based next-generation sequencers.
CONTEXT: DNA sequencing is critical to identifying many humangenetic disorders caused by DNA mutations, including cancer. Pyrosequencing is less complex, involves fewer steps, and has a superior limit of detection compared with Sanger sequencing. The fundamental basis of pyrosequencing is that pyrophosphate is released when a deoxyribonucleotide triphosphate is added to the end of a nascent strand of DNA. Because deoxyribonucleotide triphosphates are sequentially added to the reaction and because the pyrophosphate concentration is continuously monitored, the DNA sequence can be determined. OBJECTIVE: To demonstrate the fundamental principles of pyrosequencing. DATA SOURCES: Salient features of pyrosequencing are demonstrated using the free software program Pyromaker ( http://pyromaker.pathology.jhmi.edu ), through which users can input DNA sequences and other pyrosequencing parameters to generate the expected pyrosequencing results. CONCLUSIONS: We demonstrate how mutant and wild-type DNA sequences result in different pyrograms. Using pyrograms of established mutations in tumors, we explain how to analyze the pyrogram peaks generated by different dispensation sequences. Further, we demonstrate some limitations of pyrosequencing, including how some complex mutations can be indistinguishable from single base mutations. Pyrosequencing is the basis of the Roche 454 next-generation sequencer and many of the same principles also apply to the Ion Torrent hydrogen ion-based next-generation sequencers.
Authors: Vasiliy P Mishin; Tatiana Baranovich; Rebecca Garten; Anton Chesnokov; Anwar I Abd Elal; Michelle Adamczyk; Jennifer LaPlante; Kirsten St George; Alicia M Fry; John Barnes; Stephanie C Chester; Xiyan Xu; Jacqueline M Katz; David E Wentworth; Larisa V Gubareva Journal: J Clin Microbiol Date: 2016-12-28 Impact factor: 5.948
Authors: Irina G Minko; Lauriel F Earley; Kimberly E Larlee; Ying-Chih Lin; R Stephen Lloyd Journal: Environ Mol Mutagen Date: 2014-06-24 Impact factor: 3.216
Authors: Liang Cheng; Antonio Lopez-Beltran; Francesco Massari; Gregory T MacLennan; Rodolfo Montironi Journal: Mod Pathol Date: 2017-11-17 Impact factor: 7.842