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DNA bubble substrates were assembled from two partially complementary oligonucleotides with top and bottom strand sequences (Table 1). T-tailed Y-fork substrates were prepared by annealing two oligonucleotides (dT30 and dT60 series). To construct the arrested fork and 3′- or 5′-extension substrates, various combinations of oligonucleotides shown in Table 1 were annealed. Bub-82/lamin B2 containing the T-rich strand of the lamin B2 origin region was constructed as previously described (13). The bubble substrate, c-myc/DUE or c-myc/DUE-C, contains sequences (GenBank accession number K01908) from nucleotides 722 to 853 or those from 828 to 747 (complementary strand), respectively, in the central melted region. For each substrate, labeled oligonucleotide (3 pmol) was annealed with the unlabeled oligonucleotide (6 pmol) in a reaction mixture (50 μl) containing 20 mM Tris–HCl (pH 7.5), 10 mM MgCl2 and 25 mM NaCl, which were heated to 95°C, kept at 67°C for 1 h, and then allowed to slowly cool down to 37°C. The assembled substrates were purified from polyacrylamide gel by elution into TE buffer (24)."}

{"project":"craft-ca-core-ex-dev","denotations":[{"id":"T1909","span":{"begin":4,"end":7},"obj":"CHEBI_SO_EXT:DNA"},{"id":"T1910","span":{"begin":74,"end":90},"obj":"CHEBI_SO_EXT:oligonucleotide"},{"id":"T1911","span":{"begin":118,"end":127},"obj":"SO_EXT:biological_sequence"},{"id":"T1912","span":{"begin":150,"end":154},"obj":"GO:0005657"},{"id":"T1913","span":{"begin":183,"end":192},"obj":"GO:0097617"},{"id":"T1914","span":{"begin":197,"end":213},"obj":"CHEBI_SO_EXT:oligonucleotide"},{"id":"T1915","span":{"begin":264,"end":268},"obj":"GO:0005657"},{"id":"T1916","span":{"begin":273,"end":276},"obj":"_FRAGMENT"},{"id":"T1917","span":{"begin":283,"end":292},"obj":"GO:0006272"},{"id":"T1918","span":{"begin":280,"end":292},"obj":"GO:0006273"},{"id":"T1919","span":{"begin":329,"end":345},"obj":"CHEBI_SO_EXT:oligonucleotide"},{"id":"T1920","span":{"begin":368,"end":376},"obj":"GO:0097617"},{"id":"T1921","span":{"begin":385,"end":393},"obj":"PR_EXT:000009861"},{"id":"T1922","span":{"begin":409,"end":410},"obj":"CHEBI_EXT:thymine_nucleobase_or_nucleoside_or_nucleotide_molecular_entity_or_group"},{"id":"T1923","span":{"begin":430,"end":438},"obj":"PR_EXT:000009861"},{"id":"T1924","span":{"begin":439,"end":445},"obj":"SO_EXT:0000296"},{"id":"T1925","span":{"begin":521,"end":526},"obj":"PR_EXT:000000084"},{"id":"T1926","span":{"begin":534,"end":539},"obj":"PR_EXT:000000084"},{"id":"T1927","span":{"begin":556,"end":565},"obj":"SO_EXT:biological_sequence"},{"id":"T1928","span":{"begin":605,"end":616},"obj":"CHEBI_SO_EXT:nucleotide"},{"id":"T1929","span":{"begin":706,"end":712},"obj":"GO:0032508"},{"id":"T1930","span":{"begin":741,"end":748},"obj":"CHEBI_SO_EXT:molecular_label_or_mark_or_tag_process"},{"id":"T1931","span":{"begin":749,"end":764},"obj":"CHEBI_SO_EXT:oligonucleotide"},{"id":"T1932","span":{"begin":778,"end":786},"obj":"GO:0097617"},{"id":"T1933","span":{"begin":798,"end":805},"obj":"CHEBI_SO_EXT:molecular_label_or_mark_or_tag_process"},{"id":"T1934","span":{"begin":806,"end":821},"obj":"CHEBI_SO_EXT:oligonucleotide"},{"id":"T1935","span":{"begin":845,"end":852},"obj":"CHEBI:60004"},{"id":"T1936","span":{"begin":878,"end":882},"obj":"CHEBI:9754"},{"id":"T1937","span":{"begin":883,"end":886},"obj":"CHEBI:17883"},{"id":"T1938","span":{"begin":903,"end":908},"obj":"CHEBI:6636"},{"id":"T1939","span":{"begin":919,"end":923},"obj":"CHEBI:26710"},{"id":"T1940","span":{"begin":1064,"end":1078},"obj":"CHEBI_EXT:polyacrylamide"},{"id":"T1941","span":{"begin":1102,"end":1108},"obj":"CHEBI_CHMO_EXT:buffer_solution"}],"relations":[{"id":"R1120","pred":"_lexicallyChainedTo","subj":"T1917","obj":"T1916"}],"text":"The DNA bubble substrates were assembled from two partially complementary oligonucleotides with top and bottom strand sequences (Table 1). T-tailed Y-fork substrates were prepared by annealing two oligonucleotides (dT30 and dT60 series). To construct the arrested fork and 3′- or 5′-extension substrates, various combinations of oligonucleotides shown in Table 1 were annealed. Bub-82/lamin B2 containing the T-rich strand of the lamin B2 origin region was constructed as previously described (13). The bubble substrate, c-myc/DUE or c-myc/DUE-C, contains sequences (GenBank accession number K01908) from nucleotides 722 to 853 or those from 828 to 747 (complementary strand), respectively, in the central melted region. For each substrate, labeled oligonucleotide (3 pmol) was annealed with the unlabeled oligonucleotide (6 pmol) in a reaction mixture (50 μl) containing 20 mM Tris–HCl (pH 7.5), 10 mM MgCl2 and 25 mM NaCl, which were heated to 95°C, kept at 67°C for 1 h, and then allowed to slowly cool down to 37°C. The assembled substrates were purified from polyacrylamide gel by elution into TE buffer (24)."}

{"project":"craft-ca-core-dev","denotations":[{"id":"T1858","span":{"begin":150,"end":154},"obj":"GO:0005657"},{"id":"T1859","span":{"begin":183,"end":192},"obj":"GO:0097617"},{"id":"T1860","span":{"begin":264,"end":268},"obj":"GO:0005657"},{"id":"T1861","span":{"begin":273,"end":276},"obj":"_FRAGMENT"},{"id":"T1862","span":{"begin":283,"end":292},"obj":"GO:0006272"},{"id":"T1863","span":{"begin":280,"end":292},"obj":"GO:0006273"},{"id":"T1864","span":{"begin":368,"end":376},"obj":"GO:0097617"},{"id":"T1865","span":{"begin":385,"end":393},"obj":"PR:000009861"},{"id":"T1866","span":{"begin":430,"end":438},"obj":"PR:000009861"},{"id":"T1867","span":{"begin":439,"end":445},"obj":"SO:0000296"},{"id":"T1868","span":{"begin":521,"end":526},"obj":"PR:000000084"},{"id":"T1869","span":{"begin":534,"end":539},"obj":"PR:000000084"},{"id":"T1870","span":{"begin":706,"end":712},"obj":"GO:0032508"},{"id":"T1871","span":{"begin":778,"end":786},"obj":"GO:0097617"},{"id":"T1872","span":{"begin":845,"end":852},"obj":"CHEBI:60004"},{"id":"T1873","span":{"begin":878,"end":882},"obj":"CHEBI:9754"},{"id":"T1874","span":{"begin":883,"end":886},"obj":"CHEBI:17883"},{"id":"T1875","span":{"begin":903,"end":908},"obj":"CHEBI:6636"},{"id":"T1876","span":{"begin":919,"end":923},"obj":"CHEBI:26710"}],"relations":[{"id":"R1118","pred":"_lexicallyChainedTo","subj":"T1862","obj":"T1861"}],"text":"The DNA bubble substrates were assembled from two partially complementary oligonucleotides with top and bottom strand sequences (Table 1). T-tailed Y-fork substrates were prepared by annealing two oligonucleotides (dT30 and dT60 series). To construct the arrested fork and 3′- or 5′-extension substrates, various combinations of oligonucleotides shown in Table 1 were annealed. Bub-82/lamin B2 containing the T-rich strand of the lamin B2 origin region was constructed as previously described (13). The bubble substrate, c-myc/DUE or c-myc/DUE-C, contains sequences (GenBank accession number K01908) from nucleotides 722 to 853 or those from 828 to 747 (complementary strand), respectively, in the central melted region. For each substrate, labeled oligonucleotide (3 pmol) was annealed with the unlabeled oligonucleotide (6 pmol) in a reaction mixture (50 μl) containing 20 mM Tris–HCl (pH 7.5), 10 mM MgCl2 and 25 mM NaCl, which were heated to 95°C, kept at 67°C for 1 h, and then allowed to slowly cool down to 37°C. The assembled substrates were purified from polyacrylamide gel by elution into TE buffer (24)."}

{"project":"2_test","denotations":[{"id":"15917436-14609960-76775505","span":{"begin":494,"end":496},"obj":"14609960"}],"text":"The DNA bubble substrates were assembled from two partially complementary oligonucleotides with top and bottom strand sequences (Table 1). T-tailed Y-fork substrates were prepared by annealing two oligonucleotides (dT30 and dT60 series). To construct the arrested fork and 3′- or 5′-extension substrates, various combinations of oligonucleotides shown in Table 1 were annealed. Bub-82/lamin B2 containing the T-rich strand of the lamin B2 origin region was constructed as previously described (13). The bubble substrate, c-myc/DUE or c-myc/DUE-C, contains sequences (GenBank accession number K01908) from nucleotides 722 to 853 or those from 828 to 747 (complementary strand), respectively, in the central melted region. For each substrate, labeled oligonucleotide (3 pmol) was annealed with the unlabeled oligonucleotide (6 pmol) in a reaction mixture (50 μl) containing 20 mM Tris–HCl (pH 7.5), 10 mM MgCl2 and 25 mM NaCl, which were heated to 95°C, kept at 67°C for 1 h, and then allowed to slowly cool down to 37°C. The assembled substrates were purified from polyacrylamide gel by elution into TE buffer (24)."}