Allele-Specific PCR for PIK3CA Mutation Detection Using Phosphoryl Guanidine Modified Primers Научная публикация
| Журнал |
Diagnostics
ISSN: 2075-4418 |
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| Вых. Данные | Год: 2023, Том: 13, Номер: 2, Номер статьи : 250, Страниц : DOI: 10.3390/diagnostics13020250 | ||
| Ключевые слова | mutation detection; PIK3CA mutations; allele-specific PCR; modified oligonucleotides; phosphoryl guanidine oligonucleotide | ||
| Авторы |
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| Организации |
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Реферат:
Phosphoryl guanidine (PG) is the novel uncharged modification of internucleotide phosphates of oligonucleotides. Incorporating PG modification into PCR primers leads to increased discrimination between wild-type and mutated DNA, providing extraordinary detection limits in an allele-specific real-time polymerase chain reaction (AS-PCR). Herein, we used PG-modification to improve the specificity of AS primers with unfavorable Pyr/Pur primer’s 3′-end mismatch in the template/primer complex. Two mutations of the PIK3CA gene (E542K, E545K) were chosen to validate the advantages of the PG modification. Several primers with PG modifications were synthesized for each mutation and assessed using AS-PCR with the plasmid controls and DNA obtained from formalin-fixed paraffin-embedded (FFPE) tissues. The assay allows the detection of 0.5% of mutated DNA on the wild-type DNA plasmid template′s background with good specificity. Compared with ddPCR, the primers with PG-modification demonstrated 100% specificity and 100% sensitivity on the DNA from FFPE with mutation presence higher than 0.5%. Our results indicate the high potential of PG-modified primers for point mutation detection. The main principle of the developed methodology can be used to improve the specificity of primers regardless of sequences.
Библиографическая ссылка:
Chubarov A.S.
, Oskorbin I.P.
, Novikova L.M.
, Filipenko M.L.
, Lomzov A.A.
, Pyshnyi D.V.
Allele-Specific PCR for PIK3CA Mutation Detection Using Phosphoryl Guanidine Modified Primers
Diagnostics. 2023. V.13. N2. 250 . DOI: 10.3390/diagnostics13020250 WOS Scopus РИНЦ OpenAlex
Allele-Specific PCR for PIK3CA Mutation Detection Using Phosphoryl Guanidine Modified Primers
Diagnostics. 2023. V.13. N2. 250 . DOI: 10.3390/diagnostics13020250 WOS Scopus РИНЦ OpenAlex
Даты:
| Опубликована в печати: | 6 янв. 2023 г. |
Идентификаторы БД:
| Web of science: | WOS:000914632700001 |
| Scopus: | 2-s2.0-85146781102 |
| РИНЦ: | 60388616 |
| OpenAlex: | W4317352012 |