Trapping DNA Replication Origins from the Human Genome
Abstract
:1. Introduction
2. Results and Discussion
2.1. Scheme for Isolation and Cloning of Nascent DNAs from Origins of Replication
2.2. Trapping Nascent DNAs in Human Cells by UV Irradiation and Chemical Crosslinking
2.3. Trapping Newly Synthesized DNAs in Cells Arrested in Early S Phase with Aphidicolin and Hydroxyurea
2.4. Isolation of Nascent DNAs and Evaluation of Enrichment of the Origin-Derived DNAs Using Competitive PCR
Fraction | 3H total count (cpm) | 14C total count (cpm) | 3H/14C ratio | 3H-labeled DNA abundance (fold) |
---|---|---|---|---|
Total DNA | 12,997 | 127,230 | 0.102 | 1 |
Pooled DNA | 4,811 | 293 | 16.4 | 161 |
Immunopurified DNA | 3,320 | <10 | >332 | >3,250 |
2.5. Cloning of BrdU-Labeled Nascent DNA and Sequence Analysis of the DNA Clones
Library I | Library II | |
---|---|---|
DNA source | UV-treated proliferating cells | Synchronized cells in early S phase |
No. of determined sequences | 133 | 221 |
No. of independently sequenced inserts | 132 | 187 |
Total length of the sequences (bp) | 85,168 | 207,895 |
Mean length of the sequences (bp) | 640 ± 328 | 941 ± 279 |
2.6. Competitive PCR Assays for Library II Loci
2.7. Sequence Analyses of the DNA Clones from Libraries I and II
2.8. In Silico Mapping of the Putative Replication Origin Sequences to the Human Genome and Analysis of their Flanking Sequences
3. Experimental Section
3.1. Cell Culture
3.2. UV Irradiation Experiments
3.3. Chemical Crosslinking Experiments
3.4. BrdU-Labeling of Nascent DNA Synthesized in Early S Phase
3.5. Purification of BrdU-Labeled DNA
3.6. Cloning of BrdU-Labeled DNA
3.7. DNA Sequencing
3.8. Competitive PCR
3.9. Sequence Data Analyses and In Silico Mapping to the Human Genome
4. Conclusions
Acknowledgments
References
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Eki, T.; Murakami, Y.; Hanaoka, F. Trapping DNA Replication Origins from the Human Genome. Genes 2013, 4, 198-225. https://doi.org/10.3390/genes4020198
Eki T, Murakami Y, Hanaoka F. Trapping DNA Replication Origins from the Human Genome. Genes. 2013; 4(2):198-225. https://doi.org/10.3390/genes4020198
Chicago/Turabian StyleEki, Toshihiko, Yasufumi Murakami, and Fumio Hanaoka. 2013. "Trapping DNA Replication Origins from the Human Genome" Genes 4, no. 2: 198-225. https://doi.org/10.3390/genes4020198