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| Status |
Public on Sep 09, 2024 |
| Title |
Detection of 5mC and 5hmC in DNA templates generated by PCR using modified dCTPs by linear PCR using KTq DNA polymerase variant RIV A8 |
| Organism |
synthetic construct |
| Experiment type |
Other
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| Summary |
We have engineered the thermostable KlenTaq DNA polymerase variant called RIV A8 that produces error signatures specific for 5-methylcytosine (5mC) and 5-hydroxycytosine (5hmC) without prior chemical treatment of the DNA samples. These signatures are amplified during DNA amplicon library preparation and are detected by NGS. This method was applied to distinguish C from 5mC and C from 5hmC in DNA templates generated by PCR using modified dCTPs (unmodified by using dCTP, methylated by using d5mCTP, hydroxymethylated by using d5hmCTP).
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| Overall design |
DNA templates generated by PCR using modified dCTPs (unmodified by using dCTP, methylated by using d5mCTP, hydroxymethylated by using d5hmCTP) were linear amplified by RIV A8. Respective ssDNA product was used to prepare DNA amplicon libraries which were analyzed by NGS to detect 5mC and 5hmC by reading an increased error rate.
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| Contributor(s) |
Henkel M, Marx A, Motorin Y, Marchand V |
| Citation missing |
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| Submission date |
Jun 26, 2024 |
| Last update date |
Sep 10, 2024 |
| Contact name |
Melanie Henkel |
| E-mail(s) |
[email protected]
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| Organization name |
University of Konstanz
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| Department |
Department of Chemistry
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| Lab |
AG Marx
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| Street address |
Universitätsstraße 10
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| City |
Konstanz |
| State/province |
Baden-Württemberg |
| ZIP/Postal code |
78464 |
| Country |
Germany |
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| Platforms (1) |
| GPL32628 |
NextSeq 2000 (synthetic construct) |
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| Samples (3) |
| GSM8353450 |
RIV A8 354 bp C template 2 µM dGTP and 200 µM d(A/T/C)TP |
| GSM8353451 |
RIV A8 354 bp 5mC template 2 µM dGTP and 200 µM d(A/T/C)TP |
| GSM8353452 |
RIV A8 354 bp 5hmC template 2 µM dGTP and 200 µM d(A/T/C)TP |
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| Relations |
| BioProject |
PRJNA1128631 |
