Pyrosequencing of BRAF, KRAS, and PIK3CA

Pyrosequencing of BRAF (codon 600), exon 3 of KRAS (codons 12 and 13), and PIK3CA (exon 9) was performed using the PyroMark Q96 ID QIAGEN software 2.5 system according to the manufacturer’s manual. For pyrosequencing, we used specific primers one of which was biotinylated to immobilize with streptavidin beads (GE healthcare) and to amplify each target region. The pyrosequencing reaction applied to Roche PCR 480 contained 20 ng of genomic DNA and results were analyzed by PyroMark Q24 Application Software 2.0 (v.2.0.6, Qiagen, Hilden, Germany). Finally, the results were double-checked by a High-Resolution Melting (HRM) analysis [21 (link)].

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Shariati A., Razavi S., Ghaznavi-Rad E., Jahanbin B., Akbari A., Norzaee S, & Darban-Sarokhalil D. (2021). Association between colorectal cancer and Fusobacterium nucleatum and Bacteroides fragilis bacteria in Iranian patients: a preliminary study. Infectious Agents and Cancer, 16, 41.

Publication 2021

PyroMark Q96 ID streptavidin beads PyroMark Q24 Application Software 2.0

Braf Codons Exon Genomic Immobilize Kras Pik3ca Primers Streptavidin

Corresponding Organization :

Other organizations : Iran University of Medical Sciences, Arak University of Medical Sciences, Imam Khomeini Hospital, University of Tehran, Biotechnology Research Center

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Variable analysis

independent variables

Pyrosequencing of BRAF (codon 600), exon 3 of KRAS (codons 12 and 13), and PIK3CA (exon 9)

dependent variables

DNA sequence of BRAF (codon 600), KRAS (codons 12 and 13), and PIK3CA (exon 9)

control variables

Specific primers used for amplification, one of which was biotinylated
Streptavidin beads (GE healthcare) used to immobilize the biotinylated primers
20 ng of genomic DNA used in the pyrosequencing reaction
PyroMark Q24 Application Software 2.0 (v.2.0.6, Qiagen, Hilden, Germany) used for data analysis
High-Resolution Melting (HRM) analysis used to double-check the results

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