Light cycler 480 high resolution melting master kit

Detection of small nucleotide alterations within the BRCA1 coding region was performed by”high resolution melting“(HRM) analysis as previously described [31 (link)] using a Lightcycler 480 instrument and the Lightcycler 480 high resolution melting master kit (Roche, Mannheim, Germany). The reaction volume of 20 μl contained 50 ng tumor DNA, 4 mM MgCl₂ and 10 μl HRM melting master solution. M13 tagged-PCR primer pairs [31 (link)] in a final concentration of 250 nM were used. Data analysis was performed with the Gene Scanning module and normalized melting curves were visualized as Difference Plots. Samples indicating differences in melting were subsequently subjected to sequencing analysis on an ABI 3100 capillary sequencer (Applied Biosystems, Darmstadt, Germany). Only clear pathogenic frameshift, nonsense or splice site aberrations were classified as BRCA1 mutations. International databases such as the BIC database (Breast Cancer Information core: [http://www.research.nhgri.nih.gov]) were searched for these aberrations. BRCA1 copy number variations in mutation carriers were analysed by the MLPA-based P002-C1 test (MRC-Holland, Amsterdam, The Netherlands) as described previously [32 (link)].

Genomic DNA was prepared using QIAamp DNA Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer instruction. Real-time PCR cycling and conditions and primers for HRM analysis of all the examined DNA repair SNPs are summarized in Table 3. CR amplification was performed with support of a Light Cycler^® 480 High Resolution Melting Master Kit (Roche, Mannheim, Germany), according to the manufacturer's recommendations. The HRM technique was carried out in a LightCycler^® 96 (Roche, Mannheim, Germany) Thermocycler. A non-template control contained water, instead of genomic DNA, as a negative control. Additionally, positive controls (DNA samples with known genotype) were employed in each run of HRM analysis. All the control DNA samples were employed in each run of HRM analysis. The collected data were analyzed, using the LightCycler^® 96 software version SW 1.1 (Roche, Mannheim, Germany). SNPs in DNA repair genes were selected using the public domain of the National Center for Biotechnology Information at http://www.ncbi.nlm.nih.gov/snp (Bethesda, MD, USA). Primer3 software (http://frodo.wi.mit.edu/, Tartu, Estonia) was used for primers design.

Transformants were screened for DNA sequence mutations in the target region of the CpSRP54 gene (Supplementary Fig. 3) by performing high-resolution melting (HRM) based PCR assays on the region spanning the target site of the Cas9-sgRNA complex. A small amount of cells derived from each transformant were lysed7 (link). An amplicon with a size of approx. 500 bp surrounding the target site were amplified by PCR using the lysate as template and ExTaq DNA polymerase and Buffer system (TaKaRa). The PCR products were purified using Wizard^® SV Gel and PCR Clean-Up kit (Promega). The concentration of the purified PCR products were adjusted to 5 ng/μl, diluted 1:10⁶ and used as DNA template in following HRM analyses. An amplicon of approx. 100 bp surrounding the target site was amplified using the LightCycler 480 High Resolution Melting Master kit (Roche) following the manufacturer’s instructions. The PCR reactions and HRM curve analyses were performed on a LightCycler 96 instrument (Roche). Mutations were confirmed by Sanger sequencing. The 500 bp PCR product was cloned into pCR™4-TOPO^® TA vector (Invitrogen), and re-sequenced to get an overview of the different indels present within a colony. Primers used are presented in Supplementary Table 3.

DNA samples were isolated from G. parasuis clinical isolates using the TIANamp Bacteria DNA Kit (Tiangen, China) according to the manufacturer’s instructions. The HRM primers (P1, P2) were designed to amplify the sequences (approximately 100 bp) surrounding the target site. The annealing temperatures of the HRM primers were optimized to approximately 60°C and purified by HPLC.
A sample of 20 ng of the genomic DNA of a G. parasuis strain was used as a template in a 20 μl PCR reaction using a LightCycler® 480 High Resolution Melting Master kit (Roche), with 1× LightCycler® 480 High Resolution Melting Master Mix, and final concentrations of 200 nM of forward and reverse primers and 3 mM MgCl₂. A total of 108 bp PCR products surrounding the target site were amplified in the LightCycler® 96 instrument (Roche), using the following cycling conditions: pre-incubation 10 min 95°C, 45 cycles 3-step amplification (95°C for 10 s, 57°C for 10 s, 72°C for 20 s) followed by one cycle of high resolution melting [95°C for 60 s, 40°C for 60 s, 65°C for 1 s, and finally using a ramp rate 0.07°C/s and an acquisition mode setting of 15 readings/s to reach the target temperature 95°C (1 s)]. Raw data were analyzed using the LightCycler® 96 software 1.1 (Roche), which sorted the samples into groups based on their normalized melting curves. Each sample included 2–3 technical replicates.

Apple leaves from 56 apple cultivars or lines were collected for DNA extraction. DNA extraction was performed with a plant genomic DNA kit (Tiangen Biochemical Technology Co., Ltd, Beijing, China), following the manuals provided by manufacturer. The extracted DNA was used for PCR, KASP, and high-resolution melting analysis (HRM). The HRM was conducted with a LightCycler^®480 High Resolution Melting Master Kit on a LightCycler^® 480 System (Roche, Switzerland). The reaction included 1 μL of genomic DNA (10 ng μL⁻¹), 7.5 μL of 2 × Master Mix, 1.5 μL MgCl₂ (2 mmol L⁻¹), 0.5 μL of each primer (0.2 μmol L⁻¹), and 4 μL of water. The program for PCR amplification was as follows: predenature at 95 °C for 10 min followed by 45 cycles of 95 °C for 15 s, 55 °C for 15 s, and 72 °C for 10 s. The HRM detection program for the amplified products was as follows: 95 °C for 1 min, 40 °C for 1 min, 65 °C for 1 s. The fluorescence information was collected at a frequency of 25 times °C⁻¹ during the process of increasing 65 °C to 95 °C, and finally cooling down to 40 °C using the gene scanning software for HRM analysis of the LightCycler^®480 system.