29 dna polymerase

All DNA oligonucleotides were obtained from Takara Biotechnology Co. (Dalian, China) and purified by standard 10% denaturing (8 M urea) polyacrylamide gel electrophoresis (dPAGE) or high-performance liquid chromatography (HPLC). T4 DNA ligase, dNTP, and T4 polynucleotide kinase (PNK) were purchased from Takara Biotechnology Co. (Dalian, China). ϕ29 DNA polymerase was acquired from Thermo Scientific (Waltham, MA, USA). Pullulan was obtained from Sangon Biotech (Shanghai, China). BCA Protein Assay Kit was acquired from Beyotime Biotechnology (Shanghai, China). Triton X-100 cell lysis buffer SS0890 was obtained from NOVON (Beijing, China). Nitrocellulose paper (HF180), Whatman filter paper 1# and Whatman 3MM CHR chromatography paper were purchased from GE Healthcare (Chicago, IL, USA). All other chemicals were purchased from Sangon Biotech (Shanghai, China) and used without further purification. Water was purified with a Greate Fun SUMMER-S-10 water purification system.
The sequences of the DNA molecules were as follows:
CDT1 (anti-EC1): GATATCATAT CACACAACTG TAAAGAAATC CATCCCCACA CAGTGTAGTG TTCCGGTCGC AGGTCTCGAC AACGCACATC (5′→3′)
TP1: ATATGATATC GATGTGCGTT (5′→3′)
DT1: ACGCACATCG ATATCATATC AC (5′→3′)
CDT2: TAGCTAGGAA GAGTCCCAAC CCGCCCTACC CAAAATGTCT CGGAT (5′→3′)
TP2: TTCCTAGCTA ATCCGAGACA (5′→3′)
F-RS28: ACTCTTCCTA GCFrAQGGTTC GATCAAGA-invert dT (5′→3′)

Multiply primed rolling-circle amplification (RCA) was performed as previously described [10 (link),13 (link)]. Briefly, 100 ng of total DNA (2 μL of 50 ng/μL solution) from papillomatous tissue was denatured at 95°C for 5 minutes and immediately cooled on ice. Twenty-three microlitres of a previously prepared solution containing 1.5 mM of each dNTP (Invitrogen), 6.2 mM random exonuclease-resistant hexanucleotides (Thermo), 2 U of ϕ29 DNA polymerase (Thermo) and 2.5 μL of reaction buffer [50 mM Tris/HCl pH 7.5, 10 mM MgCl₂, 10 mM (NH₄)₂SO₄, 4 mM dithiothreitol] were added to denatured DNA. The amplification solution was incubated for 18 hours at 30°C, followed by 10 min at 65°C to inactivate the enzyme. The amplicon was electrophoresed in a 0.8% agarose gel and visualized on a UV light source after ethidium bromide staining. The RCA products were purified with a commercial kit (GFX^™ Purification Kit; Amersham Biosciences).