Rapid DNA amplification in a capillary tube by natural convection with a single isothermal heater

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1 Supplementary Materials For: Rapid DNA amplification in a capillary tube by natural convection with a single isothermal heater Wen Pin Chou 1, Ping Hei Chen 1, Jr Ming Miao 2, Long Sheng Kuo 1, Shiou Hwei Yeh 3, and Pei Jer Chen 4 1 Department of Mechanical Engineering, National Taiwan University, Taipei, Taiwan, 2 Department of Materials Engineering, National Pingtung University of Science and Technology, Pingtung, Taiwan, 3 Graduate Institute of Microbiology, National Taiwan University, Taipei, Taiwan, and 4 Graduate Institute of Clinical Medicine, National Taiwan University, Taipei, Taiwan BioTechniques 50:52-57 ( January 2011) doi / Keywords: natural convection; DNA amplification; nucleic acids testing. Supplementary Figure S1. Two heating blocks are placed in a dry bath maintained at 95 C. First, the entire length of the glass capillary tubes (LightCycler capillaries, 100 μl, Roche) containing the samples are heated by conduction in the right heating block for 10 min to activate the Taq polymerase. After that, tubes are transferred to the left block with a plastic holder and then heated only at the bottom to create natural convection for DNA amplification by CCPCR in 30 min. 1

2 Supplementary Table S1. The list of CCPCR primer sequences. Name Length (bp) Sequence (5 3 ) HBV 122-bp F CCTAGCAGCTTGTTTTGCTCGCAGCCG-3 HBV 122-bp R TCCAGTTGGCAGCACAGCCTAGCAGC-3 HBV 169-bp F GCACGGGACCATGCAGAACCTGCACGAT-3 HBV 169-bp R AGCCAGGAGAAACGGACTGAGGCCCAC-3 HBV 188-bp F GCGGACGACCCGTCTCGGGGCCGTTTG-3 HBV 188-bp R GACCTGGTGGGCGTTCACGGTGGTCTCCAT-3 HBV 222-bp F ACGTCCTTTGTCTACGTCCCGTCGGCGCTG-3 HBV 222-bp R CCTGGTGGGCGTTCACGGTGGTCTCCATGC-3 HCV 87-bp F CCGAAATTGCCAGGACGACCGGGTCC-3 HCV 87-bp R GCTAGCAGTCAGGCGGGGGCACGC-3 HCV 105-bp F CCGGAATTGCCAGGACGACCGGGTCC-3 HCV 105-bp R CGCGACCCAACACTATCTGGCTAGCAG-3 HIV 208-bp F CCGAGGGGACCCGACAGGCCCGAAGGAAT -3 HIV 208-bp R GGGCTTCCCACCCCCTGCGTCCCAGAAGTT-3 HIV 306-bp F ACGCAGGGGGTGGGAAGCCCTCAAATATT-3 HIV 306-bp R CCACCCCATCTGCTGCTGGCTCAGCTCGTC-3 HIV 397-bp F CTGGGACGATCTGCGGAGCCTGTGCCTCTT-3 HIV 397-bp R CCCACCCCATCTGCTGCTGGCTCAGCTCG-3 HIV 500-bp F CCAATCCCGAGGGGACCCGACAGGCCCGAA -3 HIV 500-bp R CCACCCCATCTGCTGCTGGCTCAGCTCGT-3, HIV 613-bp F AGCCAGCAGCAGATGGGGTGGGAGCAGTAT-3 HIV 613-bp R CCGCCCAGGCCACGCCTCCCTGGAAA-3 HIV 745-bp F GAGGCGAGGGGCGGCGACTGGTGAGTACG-3 HIV 745-bp R GGCCTGGTGCAATAGGCCCTGCATGCACT-3 HIV 816-bp F ACGCAGGGGGTGGGAAGCCCTCAAATATT -3 HIV 816-bp R CCGGATGCAGCTCTCGGGCCACGTGATG -3 HBV 199-bp F GGCCATAGGCCATCGGCGCATGCGT-3 HBV 199-bp R CCCGCGCAGGATCCAGTTGGCAGCACA-3 HBV 213-bp F CCTTTGTCTACGTCCCGTCGGCGCTG-3 HBV 213-bp R CTGGTGGGCGTTCACGGTGGTCTCCAT-3 The average length of primers designed for CCPCR is 28 nucleotides (ranging in 26 primers) 2

3 Supplementary Figure S2. The oil-sample interface temperature decreases with increasing sample volume when the dry bath is set at 95 C. Supplementary Table S2. The list of seven sets of primers with different melting temperatures for amplifying HBV amplicons. Name mer Tm ( C) 5 3 HBV set 1-F GCGGAACTCCTAGCAGCTTGTTTTGCTCGCAGCCG HBV set 1-R CGCAGGATCCAGTTGGCAGCACAGCCTAGCAGC HBV set 2-F CCTA GCAG CTTG TTTT GCTC GCAG CCG HBV set 2-R TCCA GTTG GCAG CACA GCCT AGCA GC HBV set 3-F GCAGCTTGTTTTGCTCGCAGCCG HBV set 3-R GTTGGCAGCACAGCCTAGCAGC HBV set 4-F GCTTGTTTTGCTCGCAGCCG HBV set 4-R GGCAGCACAGCCTAGCAGC HBV set 5-F TTGTTTTGCTCTCGCAGCCG HBV set 5-R CAGCACAGCCTAGCAGC HBV set 6-F TTTTGCTCGCAGCCG HBV set 6-R GCACAGCCTAGCAGC HBV set 7-F TTGCTCGCAGCCG HBV set 7-R CACAGCCTAGCAGC All primer sets were designed by software (LightCycler Probe Design Software 2.0, Roche) to minimize secondary structure formation and anneal in the same region of the HBV genome to maintain the consistency of PCR amplification. 3

4 Supplementary Figure S3. Computational Fluid Dynamics (C.F.D.) results for the steady-state temperature and velocity corresponding to h/d = 7.7 and Ra = Computational solutions were obtained for the thermal flow in a capillary tube using the same boundary conditions as the PIV measurements. The solid model of the computational domain and mesh system was generated with the HEXA module of ANSYS ICEMCFD software. The computational domains were composed of the outer part of the solid glass tube with thickness of 0.45 mm and inner part of the curved liquid layer of DNA solution with height of 18 mm. The inner diameter of glass capillary tube is 2.3 mm. The total number of computational cells was ~630,000. The governing equations were the steady-state incompressible laminar Reynolds-averaged Navier Stokes equations, including one continuity equation, three directional momentum equations, and one energy equation for the fluid domain. For the solid domain (glass tube part), the axial and radial temperature gradients between the inner fluid column and outside air environment were modeled with the Fourier conduction heat transfer equations. Flow properties such as kinematic viscosity, thermal capacity and thermal conductivity were regarded as temperature-independent variables in the simulations. To treat the buoyancy effect, a Boussinesq approximation for the density of the fluid was applied in the momentum equation as a source term. The thermal-flow variables in the computational domain were solved with FLUENT version software based on the control volume method. The staggered pressurebased solver was selected because of the thermal-flow field that was assumed as incompressible steady flow. The convective flux and diffusive flux terms in the governing equations were evaluated using the third-order accuracy QUICK scheme and the second-order accuracy central difference scheme, respectively. The coupling between the pressure and the velocity in the momentum equations was achieved by means of the SIMPLEC algorithm. An algebraic multi-grid scheme was used to accelerate the convergence of scalar variables at each iteration. Nonslip velocity boundary conditions were assumed for all walls. The upper curved surface of the liquid column was also modeled as a wall boundary condition where the Marangoni stress due to the temperature gradient is applied. The thermal condition at the interface between the fluid domain and the solid domain was set as a coupled type, based on the energy flux balance. The outer tube wall was specified as a convective heat transfer boundary condition with constant heat transfer coefficient. The convergence criteria for the steady-state solution were judged by the reduction in the mass residual by a factor of six. Simulations were performed with a laptop computer with a Pentium D 3.0 GHz CPU and 2048 MB of DDR2 RAM. The number of iterations to finish a single run was ~5000. Supplementary Table S3. The information of four HBV amplicons (phbv-48, GenBank accession no. NC003977). Amplicon size (bp) Location T d T d * C C C C C C C C The denaturation temperature (T d ) of each amplicon was measured by melting curve analysis with SYBR Green dye. T d *, PCR mixture containing 5% DMSO. 4

5 Supplementary Figure S4. Capillary convective PCR with duplicate reactions in different surrounding air temperatures. Initial DNA copy number was 1000 copies. Primer set: HBV set2f/2r; 122 bp. P.C., positive control using traditional PCR. Supplementary Figure S5. Hot water bath and a portable heater for scent-based mosquito repellent driven by three AAA batteries are two possible ways to perform CCPCR. Initial DNA copy number is 1000 copies. First lane of gel picture is positive control amplified by traditional PCR. Second lane is CCPCR by hot water bath or portable heater. (red asterisk indicates the heating area for each device) 5