Lightcycler 480 high resolution melting master

Primers for the screening of BDNF rs6265, rs1048218, rs1048220 were designed using Primer3Plus (Table 1) [29 ]. High resolution melting was carried out using LightCycler 480 (Roche, Switzerland) in a volume of 10μl consisting of 1X LightCycler 480 High Resolution Melting Master (Roche, Switzerland), 15ng of genomic DNA and 0.35μmol forward and reverse primers, 2mM MgCl₂ and PCR-grade water. The conditions for the thermal cycling were as follows: initial denaturation at 95°C for 10 min followed by 45 cycles of amplification at 95°C for 10s and annealing at 60°C for 15s and a final extension at 72°C for 10s. Subsequently, amplicons were subjected to HRM analysis followed by melting analysis in the same machine using a temperature range of 65°C to 95°C with 25 acquisitions per every 1°C increment. To obtain the best discrimination, samples with different melting profiles denoting the different genotypes were randomly selected from the first run and were validated by sequencing and were included in each run to serve as references. The HRM data were analysed using the Light Cycler release 1.5.1.62SP3 software. The normalized melting curves and the temperature shifted differential plots were obtained from the gene scanning module of the software to determine genotype for each sample based on the profiles of samples validated by sequencing.

All DNA oligonucleotides (Supplementary Table S1) were synthesized and purified with HPLC by Sangon Biotech Company, Ltd (Shanghai, China). T4 polynucleotide kinase (PNK), T4 DNA ligase, phi29 DNA polymerase, EcoRV, adenosine 5′-triphosphates (ATP) and deoxyribonucleoside 5′-triphosphates (dNTPs) were purchased from New England Biolabs (NEB, Beijing, China). γ-[³²P]-ATP was acquired from Perkin Elmer (Woodbridge, ON, Canada). Human α-thrombin was purchased from Haematologic Technologies. The process of protein immobilization on magnetic beads (M-270, carboxylic acid functionalized, Life Technologies) was performed according to the manufacturer's protocol for two-step coupling using ethyl (dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (Sigma). LightCycler^® 480 high resolution melting master was purchased from Roche. Human serum was purchased from XinFan Biotech (Shanghai, China). Water was purified with a Milli-Q Synthesis from a Millipore system. All other chemicals were purchased from Sigma-Aldrich and used without further purification.

The her9 target sites and single strand DNA oligonucleotides used to generate the guide RNAs were selected using the ZiFit online tool (https://zifit.partners.org/ZiFiT/). The target sites for CRISPR/Cas9 genome editing were selected within the first and third exons of the her9 gene. The first target site is 54 bp 3′ of the translation start site and 46 bp upstream of the beginning of the bHLH domain, while the second target site is immediately after the region corresponding to the Orange domain (sequences listed in Supplemental Table 2).
The pT3TS-nls-zCas9-nls (Addgene: 46757) expression vector was used to produce Cas9 mRNA. This vector contains a zebrafish codon-optimized Cas9 coding sequence flanked by the nuclear localization sequence. The plasmid was linearized using XbaI (NEB: R0145S) and purified by phenol:chloroform extraction and ethanol precipitation. Cas9 mRNA was generated using Ambion mMESSAGE mMACHINE T3 Transcription Kit (Life Technologies: AM1304) and purified by phenol:chloroform extraction and ethanol precipitation.
Screening for mutations in her9 was performed by high resolution melting analysis (HRMA). Genomic DNA was collected from 24 hpf embryos as described below. HRMA was performed using the LightCycler 480 High-Resolution Melting Master (Roche) kit according to the manufacturer's instructions on a LightCycler 96 Real-Time PCR System (Roche).

The samples were genotyped using CADMA-based genotyping as previously published with slight modifications to the PCR mastermix (30 (link)). In brief, the final reaction mix consisted of 2 µl template DNA (10 ng/µl), 5 µl LightCycler^® 480 High Resolution Melting Master (Roche Diagnostics, Ropkreuz, Switzerland), 1.2 µl MgCl2 (25 µM, Roche Diagnostics, Ropkreuz, Switzerland), 0.2 µl wild-type primer 5′-TATGCATCCGTCCACCTGAGTTCTTC-3′ (10 µM), 0.2 µl mutation primer 5′-TATGCATCTATCCACATGATTTCTTT-3′ (10 µM), 0.2 µl common reverse primer 5′-GAGGTTTTTCTCTGCAAATGCAG-3′ (10 µM), and finally adding 1.2 µl ddH₂O to a final volume of 10 µl.

To generate HRM data for the development of our data analysis approach, we used four EpiMelt assays, targeting the APC, BRCA1, H19, and MGMT genes (provided by MethylDetect ApS, Aalborg, Denmark). A reference range of controls with different methylation levels was generated. The provided assays included a methylated and non-methylated control. They were mixed to gain a dilution range of 0, 0.1, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% of the methylated control in a non-methylated background. PCR amplification was performed using LightCycler® 480 High Resolution Melting Master (catalogue number: 04909631001) and Roche LightCycler480 platform (Roche Applied Science, Laval, PQ, Canada). Briefly, the PCR amplification mix contained: 3 mM MgCl_2, 1 x LightCycler® High Resolution Melting Master (Roche), 500 nM of the primer mix, and 6 μL of the provided control mix in a total volume of 20 μL. All the reactions were run in triplicates.