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PMC:1140370 / 7424-8633
Annnotations
craft-sa-dev
The partial heteroduplex substrates on a single-stranded circular DNA with varied lengths of duplex regions were prepared by extending DNA chains from the 3′ end of the dT40-37mer (37mer region hybridizing at positions 6289–6326 of M13mp18 vector) annealed to the single-stranded circular DNA. DNA chains were elongated with Sequenase in the presence of [α-32P]dGTP, followed by extension after addition of ddGTP and all four dNTPs, resulting in substrates containing labeled duplex regions of varied lengths (13). The substrate (5 fmol) was first incubated with indicated amount of the Mcm4/6/7 complex at 37°C for 1 h in reaction mixture as described (11), and then reactions were stopped by addition of EDTA (20 mM), SDS (0.1%) and 2 μg proteinase K, followed by incubation for 20 min. Aliquots were electrophoresed on a 6.5% polyacrylamide gel in 1× TBE at 300 V for 2.5 h. The single-stranded circular DNAs used were M13mp18 and M13mp19+G-rich. The latter was constructed by inserting a G-rich repeat sequence (GGGGCGTGGGC)6, prepared by annealing of two oligonucleotides (5′-GATCC-[GGGGCGTGGGC]6-3′/5′-GATCC-[GCCCACGCCCC]6-3′) at the BamHI site of M13mp19 and the sequences of the insert were confirmed.
craft-ca-core-ex-dev
The partial heteroduplex substrates on a single-stranded circular DNA with varied lengths of duplex regions were prepared by extending DNA chains from the 3′ end of the dT40-37mer (37mer region hybridizing at positions 6289–6326 of M13mp18 vector) annealed to the single-stranded circular DNA. DNA chains were elongated with Sequenase in the presence of [α-32P]dGTP, followed by extension after addition of ddGTP and all four dNTPs, resulting in substrates containing labeled duplex regions of varied lengths (13). The substrate (5 fmol) was first incubated with indicated amount of the Mcm4/6/7 complex at 37°C for 1 h in reaction mixture as described (11), and then reactions were stopped by addition of EDTA (20 mM), SDS (0.1%) and 2 μg proteinase K, followed by incubation for 20 min. Aliquots were electrophoresed on a 6.5% polyacrylamide gel in 1× TBE at 300 V for 2.5 h. The single-stranded circular DNAs used were M13mp18 and M13mp19+G-rich. The latter was constructed by inserting a G-rich repeat sequence (GGGGCGTGGGC)6, prepared by annealing of two oligonucleotides (5′-GATCC-[GGGGCGTGGGC]6-3′/5′-GATCC-[GCCCACGCCCC]6-3′) at the BamHI site of M13mp19 and the sequences of the insert were confirmed.
craft-ca-core-dev
The partial heteroduplex substrates on a single-stranded circular DNA with varied lengths of duplex regions were prepared by extending DNA chains from the 3′ end of the dT40-37mer (37mer region hybridizing at positions 6289–6326 of M13mp18 vector) annealed to the single-stranded circular DNA. DNA chains were elongated with Sequenase in the presence of [α-32P]dGTP, followed by extension after addition of ddGTP and all four dNTPs, resulting in substrates containing labeled duplex regions of varied lengths (13). The substrate (5 fmol) was first incubated with indicated amount of the Mcm4/6/7 complex at 37°C for 1 h in reaction mixture as described (11), and then reactions were stopped by addition of EDTA (20 mM), SDS (0.1%) and 2 μg proteinase K, followed by incubation for 20 min. Aliquots were electrophoresed on a 6.5% polyacrylamide gel in 1× TBE at 300 V for 2.5 h. The single-stranded circular DNAs used were M13mp18 and M13mp19+G-rich. The latter was constructed by inserting a G-rich repeat sequence (GGGGCGTGGGC)6, prepared by annealing of two oligonucleotides (5′-GATCC-[GGGGCGTGGGC]6-3′/5′-GATCC-[GCCCACGCCCC]6-3′) at the BamHI site of M13mp19 and the sequences of the insert were confirmed.
2_test
The partial heteroduplex substrates on a single-stranded circular DNA with varied lengths of duplex regions were prepared by extending DNA chains from the 3′ end of the dT40-37mer (37mer region hybridizing at positions 6289–6326 of M13mp18 vector) annealed to the single-stranded circular DNA. DNA chains were elongated with Sequenase in the presence of [α-32P]dGTP, followed by extension after addition of ddGTP and all four dNTPs, resulting in substrates containing labeled duplex regions of varied lengths (13). The substrate (5 fmol) was first incubated with indicated amount of the Mcm4/6/7 complex at 37°C for 1 h in reaction mixture as described (11), and then reactions were stopped by addition of EDTA (20 mM), SDS (0.1%) and 2 μg proteinase K, followed by incubation for 20 min. Aliquots were electrophoresed on a 6.5% polyacrylamide gel in 1× TBE at 300 V for 2.5 h. The single-stranded circular DNAs used were M13mp18 and M13mp19+G-rich. The latter was constructed by inserting a G-rich repeat sequence (GGGGCGTGGGC)6, prepared by annealing of two oligonucleotides (5′-GATCC-[GGGGCGTGGGC]6-3′/5′-GATCC-[GCCCACGCCCC]6-3′) at the BamHI site of M13mp19 and the sequences of the insert were confirmed.