Premix ex taq hs

The PCR reagent of the SARS-CoV-2 was composed of 1× Premix Ex Taq HS (TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China), 1× EvaGreen Dye (31000, Biotium, CA, USA), 500 nM of forward and reverse primers, and 104 to 106 copies per µL SARS-CoV-2 CDC positive plasmid (Sangon Biotech, Shanghai, China). Each test requires 20 µL of reagent and 15 µL mineral seal above that.
The PCR reagent of the Escherichia Coli was composed of 1× Premix Ex Taq HS, 250 nM of forward and reverse primers, and 1 × 105/mm³ of the suspension of Escherichia coli bacteria. Each test requires 20 µL of reagent and 15 µL mineral seal above that. The primer sequences were as Table 1.
Agarose powder (V900510, 2%, Sigma-Aldrich, St. Louis, MO, USA), DL2000 DNA marker (Jialan, Beijing, China), 0.5× TBE buffer (PH1755, Phygene Life Sciences Co., Ltd., Fuzhou, China), and Nucleic Acid GelStain (Gel-Green, KeyGEN Biotechnology, Nanjing, China) were used for agarose gel electrophoresis, which was detected at 302 nm with UV illuminator (ZF1-IIN, JIAPENG Co., Shanghai, China).

Genomic DNA was extracted with QIAamp Blood Maxi kit (Qiagen, cat. # 51192) in accordance with the manufacturer’s protocol. Using a nested PCR program, we generated barcoded amplicons containing the integrated gRNA sequences. Briefly, 10 separate 100-μL redundant reactions were performed, each containing 5 μg of DNA, Premix Ex Taq HS (TaKaRa, cat. # RR030A), and 6 μL of a 10 μM solution of each primer (F1 and R1) (Additional file 2). The first round of the PCR amplification program was as follows: step 1, 95 °C for 1 min; step 2, 95 °C for 30 s; step 3, 55 °C for 30 s; and step 4, 72 °C for 30 s; with steps 2–4 being repeated 15 times. Then, 5 μL of the PCR product was used to seed the second round of PCR, along with Premix Ex Taq HS, 6 μL of the R2 primer, and 6 μL of a 10 μM solution of the F2 primer in a staggered mixture that contained the Illumina adapters and a barcode to identify the sample after sequencing analysis (Additional file 2). The second-round PCR program was as follows: step 1, 95 °C for 1 min; step 2, 95 °C for 30 s; step 3, 63 °C for 30 s; and step 4, 72 °C for 30 s; with steps 2–4 being repeated 17 times.

The genomic DNA (gDNA) was extracted from amniocytes or peripheral blood using a Qiagen DNA Blood Midi/Mini kit (Qiagen), following the manufacturer's protocol. Variant screening of the TYR gene in 12 families (family 1‐10, 19, and 20) was performed with direct Sanger sequencing. Polymerase chain reaction (PCR) primers were designed by Primer Premier version 5.0 and contained the entire coding regions and the flanking introns of the TYR gene (Table S1). The 20 μL PCR reaction mixture contained 10‐50 ng template DNA, 10 μL Premix EX Taq HS (Takara), and 1 μL of each primer. Touchdown PCR was performed as follows: 95°C for 15 minutes; 11 cycles of 95°C for 45 seconds, 60°C‐0.5°C for 45 seconds, 72°C for 45 seconds; 24 cycles of 95°C for 45 s, 54°C for 45 seconds, 72°C for 45 seconds; and 72°C for 7 minutes. The PCR products were sequenced using an ABI 3130 automated DNA sequencer (Applied Biosystems). DNASTAR Lasergene SeqMan software was used for DNA sequence assembly, and sequences were compared with a wild‐type reference sequence.