An examination of critical parameters in hybridization‐based epigenotyping using magnetic microparticles
Gene-specific promoter methylation is involved in gene silencing and is an important cancer biomarker. Cancer-specific methylation patterns have been observed and clinically validated for numerous gene promoters, but the knowledge gleaned from this large body of work is currently under-utilized in t...
| Main Authors: | , , |
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| Other Authors: | |
| Format: | Article |
| Language: | English |
| Published: |
Wiley
2020
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| Online Access: | https://hdl.handle.net/1721.1/126193 |
| _version_ | 1826199325017899008 |
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| author | Tam, Brooke Elizabeth. Hao, Yining Sikes, Hadley D. |
| author2 | Massachusetts Institute of Technology. Department of Chemical Engineering |
| author_facet | Massachusetts Institute of Technology. Department of Chemical Engineering Tam, Brooke Elizabeth. Hao, Yining Sikes, Hadley D. |
| author_sort | Tam, Brooke Elizabeth. |
| collection | MIT |
| description | Gene-specific promoter methylation is involved in gene silencing and is an important cancer biomarker. Cancer-specific methylation patterns have been observed and clinically validated for numerous gene promoters, but the knowledge gleaned from this large body of work is currently under-utilized in the clinic. Methylation-specific PCR is currently the gold standard method for clinical methylation assessment, but several research groups have proposed hybridization-based techniques which could be simpler to implement and provide more accurate results. However, the sensitivity of this easier alternative must be improved dramatically in order to compete with methylation-specific PCR. Efficient sample capture is a key step in maximizing sensitivity, so here we investigate the key parameters involved in (i) maximizing the capture of gene-specific target DNA molecules at the surfaces of functionalized, magnetic microparticles and (ii) recognizing DNA methylation using an engineered methyl-CpG-binding domain (MBD) protein. The magnetic bead density, the probe concentration, and the MBD concentration were very important for maximizing detection, and other variables such as the hybridization time also impacted the target capture efficiency but had a smaller effect on the overall methylation assay. The effect of genomic DNA on the capture of the target sequence was also investigated, and model methylated vs. unmethylated target sequences could be distinguished in the presence of 1 ng/μL genomic DNA. The findings we report related to the underlying binding events involved in hybridization-based epigenotyping can be leveraged in combination with the many signal amplification and detection approaches that are currently being developed. |
| first_indexed | 2024-09-23T11:18:22Z |
| format | Article |
| id | mit-1721.1/126193 |
| institution | Massachusetts Institute of Technology |
| language | English |
| last_indexed | 2024-09-23T11:18:22Z |
| publishDate | 2020 |
| publisher | Wiley |
| record_format | dspace |
| spelling | mit-1721.1/1261932022-09-27T18:36:56Z An examination of critical parameters in hybridization‐based epigenotyping using magnetic microparticles Tam, Brooke Elizabeth. Hao, Yining Sikes, Hadley D. Massachusetts Institute of Technology. Department of Chemical Engineering Gene-specific promoter methylation is involved in gene silencing and is an important cancer biomarker. Cancer-specific methylation patterns have been observed and clinically validated for numerous gene promoters, but the knowledge gleaned from this large body of work is currently under-utilized in the clinic. Methylation-specific PCR is currently the gold standard method for clinical methylation assessment, but several research groups have proposed hybridization-based techniques which could be simpler to implement and provide more accurate results. However, the sensitivity of this easier alternative must be improved dramatically in order to compete with methylation-specific PCR. Efficient sample capture is a key step in maximizing sensitivity, so here we investigate the key parameters involved in (i) maximizing the capture of gene-specific target DNA molecules at the surfaces of functionalized, magnetic microparticles and (ii) recognizing DNA methylation using an engineered methyl-CpG-binding domain (MBD) protein. The magnetic bead density, the probe concentration, and the MBD concentration were very important for maximizing detection, and other variables such as the hybridization time also impacted the target capture efficiency but had a smaller effect on the overall methylation assay. The effect of genomic DNA on the capture of the target sequence was also investigated, and model methylated vs. unmethylated target sequences could be distinguished in the presence of 1 ng/μL genomic DNA. The findings we report related to the underlying binding events involved in hybridization-based epigenotyping can be leveraged in combination with the many signal amplification and detection approaches that are currently being developed. National Institute of Environmental Health Sciences (Grant P30-ES002109) National Cancer Institute (U.S.) (Grant P30CCA14051) 2020-07-15T12:35:47Z 2020-07-15T12:35:47Z 2018-11 2019-12-10T13:09:35Z Article http://purl.org/eprint/type/JournalArticle 8756-7938 1520-6033 https://hdl.handle.net/1721.1/126193 Tam, Brooke E. et al. “An examination of critical parameters in hybridization‐based epigenotyping using magnetic microparticles.” Biotechnology progress, vol. 34, no. 6, 2018, pp. 1589-1595 © 2018 The Author(s) en 10.1002/BTPR.2644 Biotechnology progress Creative Commons Attribution-Noncommercial-Share Alike http://creativecommons.org/licenses/by-nc-sa/4.0/ application/pdf Wiley PMC |
| spellingShingle | Tam, Brooke Elizabeth. Hao, Yining Sikes, Hadley D. An examination of critical parameters in hybridization‐based epigenotyping using magnetic microparticles |
| title | An examination of critical parameters in hybridization‐based epigenotyping using magnetic microparticles |
| title_full | An examination of critical parameters in hybridization‐based epigenotyping using magnetic microparticles |
| title_fullStr | An examination of critical parameters in hybridization‐based epigenotyping using magnetic microparticles |
| title_full_unstemmed | An examination of critical parameters in hybridization‐based epigenotyping using magnetic microparticles |
| title_short | An examination of critical parameters in hybridization‐based epigenotyping using magnetic microparticles |
| title_sort | examination of critical parameters in hybridization based epigenotyping using magnetic microparticles |
| url | https://hdl.handle.net/1721.1/126193 |
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