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Gen Mutasyonunun Belirlenmesinde Rekombinaz Polimeraz Çoğaltım Tekniği Optimizasyonu Çalışmaları ve Sonuçları

Yıl 2023, , 1363 - 1372, 28.12.2023

Beste Çağdaş , Sebastian Kerstıng

https://doi.org/10.35414/akufemubid.1322267

Öz

İnsan genlerindeki tek nükleotid polimorfizmleri (SNP'ler) çok önemli genetik değişikliklerdir ve PCR (polimeraz zincir reaksiyonu) veya NGS (yeni nesil dizileme), SNP analizinde yaygın olarak kullanılır. Yeni teknolojilerin ilerlemesi üzerine yapılan çalışmalar sayesinde izotermal nükleik asit amplifikasyon yaklaşımına ilgi artmıştır. Bu yöntemlerden biri olarak, rekombinaz polimeraz amplifikasyonu (RPA), hasta başı nükleik asit ölçümünde çekici bir alternatif oluşturmaktadır. Çalışma kapsamında seçilen hedef SNP'ler, PIK3CA gen bölgesinde (E542K, E545K) tanımlanan mutasyonlardır ve PIK3CA mutasyonlarının değerlendirildiği DNA örnekleri, MCF7, BT474 ve ayrıca SKBr3 kanser hücre hatlarından izole edilmiştir. RPA reaksiyon koşulları için optimizasyon çalışmalarında analiz süresi, sıcaklık, primer ve ayrıca magnezyum asetat konsantrasyonu parametreleri değerlendirilmiştir. RPA ürünlerinin en verimli şekilde elde edilebildiği reaksiyon optimizasyon çalışmaları sonuçlarına göre reaksiyon süresi 20 dk, sıcaklık 40°C, primer konsantrasyonu 10 µM ve MgOAc konsantrasyonu ise 140 mM olarak değerlendirilmiştir.

Anahtar Kelimeler

Tek Nükleotid Polimorfizmi İzotermal Çoğaltım Polimeraz Zincir Reaksiyonu Rekombinaz Polimeraz Çoğaltım Gen Mutasyonu PIK3CA

Proje Numarası

1059B192001062

Kaynakça

  • Asiello, P.J. and Baeumner, A.J., 2011. Miniaturized isothermal nucleic acid amplification, a review. Lab on a Chip, 11(8), 1420-1430. https://doi.org/10.1039/C0LC00666A
  • Bader, A.G., Kang, S. and Vogt, P.K., 2006. Cancer-specific mutations in PIK3CA are oncogenic in vivo. Proceedings of the National Academy of Sciences, 103(5), 1475-1479. https://doi.org/10.1073/pnas.0510857103.
  • Banerji, S., Cibulskis, K., Rangel-Escareno, C., Brown, K.K., Carter, S.L., Frederick, A.M. and Meyerson, M., 2012. Sequence analysis of mutations and translocations across breast cancer subtypes. Nature, 486(7403), 405-409. https://doi.org/10.1038/nature11154
  • Coley, C.M., Barry, M.J., Fleming, C., Fahs, M.C. and Mulley, A.G., 1997. Early detection of prostate cancer. Part II: Estimating the risks, benefits, and costs. American College of Physicians. Annals of internal medicine, 126(6), 468-479. https://doi.org/10.7326/0003-4819-126-6-199703150-00010
  • Daddy Gaoh, S., Kweon, O., & Ahn, Y. 2023. Propidium Monoazide (PMAxx)-Recombinase Polymerase Amplification Exo (RPA Exo) Assay for Rapid Detection of Burkholderia cepacia Complex in Chlorhexidine Gluconate (CHX) and Benzalkonium Chloride (BZK) Solutions. Microorganisms, 11(6), 1401. https://doi.org/10.3390/microorganisms11061401
  • Dawson, S.J., Tsui, D.W.Y., Murtaza, M., Biggs, H., Rueda, O. M., Chin, S.-F., Dunning M. J., Gale D., Forshew T., Mahler-Araujo B., Rajan S., Humphray S., Becq J., Hallsal D., Wallis M., Bentley D., Caldas C., Rosenfeld, N., 2013. Analysis of Circulating Tumor DNA to Monitor Metastatic Breast Cancer. New England Journal of Medicine, 368(13), 1199-1209. https://doi.org/10.1056/NEJMoa1213261
  • Deng, N., Zhou, H., Fan, H. and Yuan, Y., 2017. Single nucleotide polymorphisms and cancer susceptibility. Oncotarget, 8(66), 110635–110649.https://doi.org/10.18632/oncotarget.22372.
  • Gorden, E. M., Sturk-Andreaggi, K. and Marshall, C. 2021. Capture enrichment and massively parallel sequencing for human identification. Forensic Science International: Genetics, 53, 102496. https://doi.org/10.1016/j.fsigen.2021.102496
  • Guo, Q., Zhou, K., Chen, C., Yue, Y., Shang, Z., Zhou, K., Fu Z., Liu, J., Lin, J., Xia, C., Tang, W., Cong, X., Sun X., and Hong, Y. 2021. Development of a recombinase polymerase amplification assay for Schistosomiasis Japonica diagnosis in the experimental mice and domestic goats. Frontiers in Cellular and Infection Microbiology, 1136. https://doi.org/10.3389/fcimb.2021.791997
  • Kersting, S., Rausch, V., Bier, F.F. and von Nickisch-Rosenegk, M., 2018. A recombinase polymerase amplification assay for the diagnosis of atypical pneumonia. Analytical biochemistry, 550, 54- 60. https://doi.org/10.1016/j.ab.2018.04.014
  • Kunze, A., Dilcher, M., Abd El Wahed, A., Hufert, F., Niessner, R., and Seidel, M., 2016. On-Chip Isothermal Nucleic Acid Amplification on Flow-Based Chemiluminescence Microarray Analysis Platform for the Detection of Viruses and Bacteria. Analytical Chemistry 88 (1), 898–905. https://doi.org/10.1021/acs.analchem.5b03540
  • Lillis, L., Lehman, D., Singhal, M.C., Cantera, J., Singleton, J., Labarre, P., Toyama, A., Piepenburg, O., Parker, M., Wood, R., Overbaugh, J. and Boyle, D.S., 2014. Non-instrumented incubation of a recombinase polymerase amplification assay for the rapid and sensitive detection of proviral HIV-1 DNA. PloS one, 9(9), e108189. https://doi.org/10.1371/journal.pone.0108189
  • Liu, Y., Lei, T., Liu, Z., Kuang, Y., Lyu, J. and Wang, Q., 2016. A novel technique to detect EGFR mutations in lung cancer. International journal of molecular sciences, 17(5), 792. https://doi.org/10.3390/ijms17050792
  • Lobato, I.M., O’Sullivan, C.K., 2018. Recombinase polymerase amplification: Basics, applications and recent advances. Trends Analytical Chemistry 98, 19–35. https://doi.org/10.1016/j.trac.2017.10.015
  • Ma, B., Li, J., Chen, K., Yu, X., Sun, C. and Zhang, M., 2020. Multiplex recombinase polymerase amplification assay for the simultaneous detection of three foodborne pathogens in seafood. Foods, 9(3), 278. https://doi.org/10.3390/foods9030278
  • Martorell, S., Palanca, S., Maquieira, Á., and Tortajada-Genaro, L.A., 2018. Blocked recombinase polymerase amplification for mutation analysis of PIK3CA gene. Analytical biochemistry, 544, 49-56. https://doi.org/10.1016/j.ab.2017.12.013
  • Munawar M., Martin F., Toljamo A., Kokko H. and Oksanen E., 2020. RPA-PCR couple: an approach to expedite plant diagnostics and overcome PCR inhibitors, BioTechniques, doi: 10.2144/btn-2020-0065. https://doi.org/10.2144/btn-2020-0065
  • Piepenburg, O., Williams, C.H., Stemple, D.L. and Armes, N.A., 2006. DNA detection using recombination proteins. PLoS biology, 4(7), e204. https://doi.org/10.1371/journal.pbio.0040204
  • Rathore, H., Biyani, R., Kato, H., Takamura, Y., & Biyani, M. 2019. Palm-size and one-inch gel electrophoretic device for reliable and field-applicable analysis of recombinase polymerase amplification. Analytical Methods, 11(39), 4969-4976. https://doi.org/10.1039/C9AY01476D
  • Sharma, N., Hoshika, S., Hutter, D., Bradley, K.M. and Benner, S.A., 2014. Recombinase-Based Isothermal Amplification of Nucleic Acids with Self-Avoiding Molecular Recognition Systems (SAMRS). ChemBioChem, 15, 2268 – 2274. https://doi.org/10.1002/cbic.201402250
  • Wang, R., Zhang, F., Wang, L., Qian, W., Qian, C., Wu, J. and Ying, Y., 2017. Instant, visual, and instrument-free method for on-site screening of GTS 40-3-2 soybean based on body-heat triggered recombinase polymerase amplification. Analytical chemistry, 89(8), 4413-4418. https://doi.org/10.1021/acs.analchem.7b00964
  • Wang, W., Xie, Y., Zhou, Z., Qin, Z., Wu, J. and He, J., 2013. PIK3CA hypomethylation plays a key role in activation of the PI3K/AKT pathway in esophageal cancer in Chinese patients. Acta Pharmacologica Sinica, 34(12), 1560-1567. https://doi.org/10.1038/aps.2013.163
  • Zhang, S., Sun, A., Wan, B., Du, Y., Wu, Y., Zhang, A., Jiang, D., Ji, P., Wei Z., Zhuang G. and Zhang, G., 2020. Development of a directly visualized recombinase polymerase amplification–sybr green i method for the rapid detection of African swine fever virus. Frontiers in microbiology, 11, 602709. https://doi.org/10.3389/fmicb.2020.602709
  • Zaghloul, H. and El-Shahat, M., 2014. Recombinase polymerase amplification as a promising tool in hepatitis C virus diagnosis. World Journal of Hepatology, 6(12), 916. https://doi.org/10.4254/wjh.v6.i12.916
  • Zanoli, L.M. and Spoto, G., 2013. Isothermal amplification methods for the detection of nucleic acids in microfluidic devices. Biosensors, 3(1), 18-43. https://doi.org/10.3390/bios3010018
  • https://blast.ncbi.nlm.nih.gov (Basic Local Alignment Search Tool) (01.03.2022)
  • https://www.who.int/news-room/fact-sheets/detail/cancer (World Health Organisation, 2020. (10.06.2023)
  • http://www.twistdx.co.uk/images/uploads/docs/ Appendix.pdf, TwistDx, Babraham, Cambridge. (30.03.2023).

Optimization Studies and Results of Recombinase Polymerase Amplification Technique for Gene Mutation Detection

Yıl 2023, , 1363 - 1372, 28.12.2023

Beste Çağdaş , Sebastian Kerstıng

https://doi.org/10.35414/akufemubid.1322267

Öz

Single nucleotide polymorphisms (SNPs) in human genes are very significant genetic changes and PCR (polymerase chain reaction) or NGS (next-generation sequencing) are extensively employed in SNP analysis. Thanks to the studies on the progress of new technologies, interest in the isothermal nucleic acid amplification approach has increased. As one of these methods, recombinase polymerase amplification (RPA) represents an attractive option for point-of-care nucleic acid quantification. The target SNPs selected within the scope of the study are mutations identified in the PIK3CA gene region (E542K, E545K), and DNA samples which were evaluated about PIK3CA mutations were isolated from the cancer cells MCF7, BT474, and also SKBr3. The optimization studies for the RPA reaction conditions were carried out for parameters such as assay time, temperature, primer, and also magnesium acetate concentration. According to the results of the reaction optimization studies, in which the RPA products can be obtained in the most efficient way, the assay time was determined as 20 min; the temperature as 40°C; the primer concentration as 10 µM and the MgOAc concentration as 140 mM.

Anahtar Kelimeler

Single Nucleotide Polymorphism Isothermal Amplification Polymerase Chain Reaction Recombinase Polymerase Amplification Gene Mutation PIK3CA

Destekleyen Kurum

TÜRKİYE BİLİMSEL VE TEKNOLOJİK ARAŞTIRMA KURUMU

Proje Numarası

1059B192001062

Teşekkür

We acknowledge the Scientific and Technological Research Council of Turkey (Tubitak 2219 Award # 1059B192001062) for Dr. Beste ÇAĞDAŞ. We are also grateful to the Fraunhofer Institute For Cell Therapy and Immunology for supporting this study at Dr. Markus von Nickisch-Rosenegk’s laboratory.

Kaynakça

  • Asiello, P.J. and Baeumner, A.J., 2011. Miniaturized isothermal nucleic acid amplification, a review. Lab on a Chip, 11(8), 1420-1430. https://doi.org/10.1039/C0LC00666A
  • Bader, A.G., Kang, S. and Vogt, P.K., 2006. Cancer-specific mutations in PIK3CA are oncogenic in vivo. Proceedings of the National Academy of Sciences, 103(5), 1475-1479. https://doi.org/10.1073/pnas.0510857103.
  • Banerji, S., Cibulskis, K., Rangel-Escareno, C., Brown, K.K., Carter, S.L., Frederick, A.M. and Meyerson, M., 2012. Sequence analysis of mutations and translocations across breast cancer subtypes. Nature, 486(7403), 405-409. https://doi.org/10.1038/nature11154
  • Coley, C.M., Barry, M.J., Fleming, C., Fahs, M.C. and Mulley, A.G., 1997. Early detection of prostate cancer. Part II: Estimating the risks, benefits, and costs. American College of Physicians. Annals of internal medicine, 126(6), 468-479. https://doi.org/10.7326/0003-4819-126-6-199703150-00010
  • Daddy Gaoh, S., Kweon, O., & Ahn, Y. 2023. Propidium Monoazide (PMAxx)-Recombinase Polymerase Amplification Exo (RPA Exo) Assay for Rapid Detection of Burkholderia cepacia Complex in Chlorhexidine Gluconate (CHX) and Benzalkonium Chloride (BZK) Solutions. Microorganisms, 11(6), 1401. https://doi.org/10.3390/microorganisms11061401
  • Dawson, S.J., Tsui, D.W.Y., Murtaza, M., Biggs, H., Rueda, O. M., Chin, S.-F., Dunning M. J., Gale D., Forshew T., Mahler-Araujo B., Rajan S., Humphray S., Becq J., Hallsal D., Wallis M., Bentley D., Caldas C., Rosenfeld, N., 2013. Analysis of Circulating Tumor DNA to Monitor Metastatic Breast Cancer. New England Journal of Medicine, 368(13), 1199-1209. https://doi.org/10.1056/NEJMoa1213261
  • Deng, N., Zhou, H., Fan, H. and Yuan, Y., 2017. Single nucleotide polymorphisms and cancer susceptibility. Oncotarget, 8(66), 110635–110649.https://doi.org/10.18632/oncotarget.22372.
  • Gorden, E. M., Sturk-Andreaggi, K. and Marshall, C. 2021. Capture enrichment and massively parallel sequencing for human identification. Forensic Science International: Genetics, 53, 102496. https://doi.org/10.1016/j.fsigen.2021.102496
  • Guo, Q., Zhou, K., Chen, C., Yue, Y., Shang, Z., Zhou, K., Fu Z., Liu, J., Lin, J., Xia, C., Tang, W., Cong, X., Sun X., and Hong, Y. 2021. Development of a recombinase polymerase amplification assay for Schistosomiasis Japonica diagnosis in the experimental mice and domestic goats. Frontiers in Cellular and Infection Microbiology, 1136. https://doi.org/10.3389/fcimb.2021.791997
  • Kersting, S., Rausch, V., Bier, F.F. and von Nickisch-Rosenegk, M., 2018. A recombinase polymerase amplification assay for the diagnosis of atypical pneumonia. Analytical biochemistry, 550, 54- 60. https://doi.org/10.1016/j.ab.2018.04.014
  • Kunze, A., Dilcher, M., Abd El Wahed, A., Hufert, F., Niessner, R., and Seidel, M., 2016. On-Chip Isothermal Nucleic Acid Amplification on Flow-Based Chemiluminescence Microarray Analysis Platform for the Detection of Viruses and Bacteria. Analytical Chemistry 88 (1), 898–905. https://doi.org/10.1021/acs.analchem.5b03540
  • Lillis, L., Lehman, D., Singhal, M.C., Cantera, J., Singleton, J., Labarre, P., Toyama, A., Piepenburg, O., Parker, M., Wood, R., Overbaugh, J. and Boyle, D.S., 2014. Non-instrumented incubation of a recombinase polymerase amplification assay for the rapid and sensitive detection of proviral HIV-1 DNA. PloS one, 9(9), e108189. https://doi.org/10.1371/journal.pone.0108189
  • Liu, Y., Lei, T., Liu, Z., Kuang, Y., Lyu, J. and Wang, Q., 2016. A novel technique to detect EGFR mutations in lung cancer. International journal of molecular sciences, 17(5), 792. https://doi.org/10.3390/ijms17050792
  • Lobato, I.M., O’Sullivan, C.K., 2018. Recombinase polymerase amplification: Basics, applications and recent advances. Trends Analytical Chemistry 98, 19–35. https://doi.org/10.1016/j.trac.2017.10.015
  • Ma, B., Li, J., Chen, K., Yu, X., Sun, C. and Zhang, M., 2020. Multiplex recombinase polymerase amplification assay for the simultaneous detection of three foodborne pathogens in seafood. Foods, 9(3), 278. https://doi.org/10.3390/foods9030278
  • Martorell, S., Palanca, S., Maquieira, Á., and Tortajada-Genaro, L.A., 2018. Blocked recombinase polymerase amplification for mutation analysis of PIK3CA gene. Analytical biochemistry, 544, 49-56. https://doi.org/10.1016/j.ab.2017.12.013
  • Munawar M., Martin F., Toljamo A., Kokko H. and Oksanen E., 2020. RPA-PCR couple: an approach to expedite plant diagnostics and overcome PCR inhibitors, BioTechniques, doi: 10.2144/btn-2020-0065. https://doi.org/10.2144/btn-2020-0065
  • Piepenburg, O., Williams, C.H., Stemple, D.L. and Armes, N.A., 2006. DNA detection using recombination proteins. PLoS biology, 4(7), e204. https://doi.org/10.1371/journal.pbio.0040204
  • Rathore, H., Biyani, R., Kato, H., Takamura, Y., & Biyani, M. 2019. Palm-size and one-inch gel electrophoretic device for reliable and field-applicable analysis of recombinase polymerase amplification. Analytical Methods, 11(39), 4969-4976. https://doi.org/10.1039/C9AY01476D
  • Sharma, N., Hoshika, S., Hutter, D., Bradley, K.M. and Benner, S.A., 2014. Recombinase-Based Isothermal Amplification of Nucleic Acids with Self-Avoiding Molecular Recognition Systems (SAMRS). ChemBioChem, 15, 2268 – 2274. https://doi.org/10.1002/cbic.201402250
  • Wang, R., Zhang, F., Wang, L., Qian, W., Qian, C., Wu, J. and Ying, Y., 2017. Instant, visual, and instrument-free method for on-site screening of GTS 40-3-2 soybean based on body-heat triggered recombinase polymerase amplification. Analytical chemistry, 89(8), 4413-4418. https://doi.org/10.1021/acs.analchem.7b00964
  • Wang, W., Xie, Y., Zhou, Z., Qin, Z., Wu, J. and He, J., 2013. PIK3CA hypomethylation plays a key role in activation of the PI3K/AKT pathway in esophageal cancer in Chinese patients. Acta Pharmacologica Sinica, 34(12), 1560-1567. https://doi.org/10.1038/aps.2013.163
  • Zhang, S., Sun, A., Wan, B., Du, Y., Wu, Y., Zhang, A., Jiang, D., Ji, P., Wei Z., Zhuang G. and Zhang, G., 2020. Development of a directly visualized recombinase polymerase amplification–sybr green i method for the rapid detection of African swine fever virus. Frontiers in microbiology, 11, 602709. https://doi.org/10.3389/fmicb.2020.602709
  • Zaghloul, H. and El-Shahat, M., 2014. Recombinase polymerase amplification as a promising tool in hepatitis C virus diagnosis. World Journal of Hepatology, 6(12), 916. https://doi.org/10.4254/wjh.v6.i12.916
  • Zanoli, L.M. and Spoto, G., 2013. Isothermal amplification methods for the detection of nucleic acids in microfluidic devices. Biosensors, 3(1), 18-43. https://doi.org/10.3390/bios3010018
  • https://blast.ncbi.nlm.nih.gov (Basic Local Alignment Search Tool) (01.03.2022)
  • https://www.who.int/news-room/fact-sheets/detail/cancer (World Health Organisation, 2020. (10.06.2023)
  • http://www.twistdx.co.uk/images/uploads/docs/ Appendix.pdf, TwistDx, Babraham, Cambridge. (30.03.2023).
Toplam 28 adet kaynakça vardır.

Ayrıntılar

Birincil Dil İngilizce
Konular Biyomühendislik (Diğer)
Bölüm Makaleler
Yazarlar

Beste Çağdaş 0000-0003-2534-382X

Sebastian Kerstıng Bu kişi benim 0000-0003-0474-7825

Proje Numarası 1059B192001062
Erken Görünüm Tarihi 22 Aralık 2023
Yayımlanma Tarihi 28 Aralık 2023
Gönderilme Tarihi 4 Temmuz 2023
Yayımlandığı Sayı Yıl 2023

Kaynak Göster

APA Çağdaş, B., & Kerstıng, S. (2023). Optimization Studies and Results of Recombinase Polymerase Amplification Technique for Gene Mutation Detection. Afyon Kocatepe Üniversitesi Fen Ve Mühendislik Bilimleri Dergisi, 23(6), 1363-1372. https://doi.org/10.35414/akufemubid.1322267
AMA Çağdaş B, Kerstıng S. Optimization Studies and Results of Recombinase Polymerase Amplification Technique for Gene Mutation Detection. Afyon Kocatepe Üniversitesi Fen Ve Mühendislik Bilimleri Dergisi. Aralık 2023;23(6):1363-1372. doi:10.35414/akufemubid.1322267
Chicago Çağdaş, Beste, ve Sebastian Kerstıng. “Optimization Studies and Results of Recombinase Polymerase Amplification Technique for Gene Mutation Detection”. Afyon Kocatepe Üniversitesi Fen Ve Mühendislik Bilimleri Dergisi 23, sy. 6 (Aralık 2023): 1363-72. https://doi.org/10.35414/akufemubid.1322267.
EndNote Çağdaş B, Kerstıng S (01 Aralık 2023) Optimization Studies and Results of Recombinase Polymerase Amplification Technique for Gene Mutation Detection. Afyon Kocatepe Üniversitesi Fen Ve Mühendislik Bilimleri Dergisi 23 6 1363–1372.
IEEE B. Çağdaş ve S. Kerstıng, “Optimization Studies and Results of Recombinase Polymerase Amplification Technique for Gene Mutation Detection”, Afyon Kocatepe Üniversitesi Fen Ve Mühendislik Bilimleri Dergisi, c. 23, sy. 6, ss. 1363–1372, 2023, doi: 10.35414/akufemubid.1322267.
ISNAD Çağdaş, Beste - Kerstıng, Sebastian. “Optimization Studies and Results of Recombinase Polymerase Amplification Technique for Gene Mutation Detection”. Afyon Kocatepe Üniversitesi Fen Ve Mühendislik Bilimleri Dergisi 23/6 (Aralık 2023), 1363-1372. https://doi.org/10.35414/akufemubid.1322267.
JAMA Çağdaş B, Kerstıng S. Optimization Studies and Results of Recombinase Polymerase Amplification Technique for Gene Mutation Detection. Afyon Kocatepe Üniversitesi Fen Ve Mühendislik Bilimleri Dergisi. 2023;23:1363–1372.
MLA Çağdaş, Beste ve Sebastian Kerstıng. “Optimization Studies and Results of Recombinase Polymerase Amplification Technique for Gene Mutation Detection”. Afyon Kocatepe Üniversitesi Fen Ve Mühendislik Bilimleri Dergisi, c. 23, sy. 6, 2023, ss. 1363-72, doi:10.35414/akufemubid.1322267.
Vancouver Çağdaş B, Kerstıng S. Optimization Studies and Results of Recombinase Polymerase Amplification Technique for Gene Mutation Detection. Afyon Kocatepe Üniversitesi Fen Ve Mühendislik Bilimleri Dergisi. 2023;23(6):1363-72.


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