For the genotyping of SNP using ARMS-PCR, the PCR reactions contained 1 μL of template DNA, each primer at 500 nM, 200 μM dNTP, 1.5 mM MgCl2, and 0.4 U Phusion DNA polymerase (Thermo Scientific, Waltham, USA) in a total volume of 20 μL with 1× Phusion HF buffer. The primers and PCR conditions are listed in Table
1 . The PCR products were resolved in 2% agarose gels and photographed using an imaging system. The PCR for the genotyping of SNP with RFLP was carried out with the same protocol, and the PCR products were digested with HpyCH4III (New England Biolabs, Ipswich, USA) following the manufacturer’s instructions. The digested DNA was resolved in 2% agarose gels and photographed using an imaging system.
Hpych4iii
Manufactured by New England Biolabs
Sourced in United States, United Kingdom
HpyCH4III is a type IIB restriction endonuclease that recognizes and cleaves the DNA sequence 5'-ACNGT-3' producing a 2-base 5' overhang. The enzyme is supplied with the appropriate reaction buffer.
Automatically generated - may contain errors
Lab products found in correlation
Phusion dna polymerase, by Thermo Fisher Scientific (1 mentions)
Hpy188i, by New England Biolabs (1 mentions)
Nlaiii, by New England Biolabs (1 mentions)
Chip seq sample prep kit, by Illumina (1 mentions)
Gel extraction kit, by Qiagen (1 mentions)
Minelute pcr purification kit, by Qiagen (1 mentions)
Hpy166ii, by New England Biolabs (1 mentions)
Cfx96 real time thermocycler, by Bio-Rad (1 mentions)
D100 screen tape, by Agilent Technologies (1 mentions)
Cac8i, by New England Biolabs (1 mentions)
6 protocols using hpych4iii
1
Genotyping SNPs using ARMS-PCR and RFLP
2
Efficient Single-Stranded DNA Production
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dsDNA was prepared with two-step PCR. The first-round PCR was performed using non-modified primers followed by treatment with DpnI and exonuclease I, as described above. The specific PCR product was gel purified and then used as a template for the second-round PCR with two short primers (about 25 nt), one of which contains five sequential PS bonds at the 5’ end. After column purification, the dsDNA was reacted with T7 exonuclease as described above. For the preparation of T7RE donors, HpyCH4III (0.025 U/µL), Hpy188I (0.1 U/µL), NlaIII (0.025 U/µL), and RsaI (0.05 U/µL) purchased from New England Biolabs were added directly to the T7 exonuclease reaction mix and incubated at 37 °C for 15 min. After the enzymatic reactions, ssDNA was column-purified using Buffer NTC (Macherey–Nagel) as a binding buffer. Typically, 4 to 5 µg of ssDNA was obtained from 15 µg of dsDNA, when elution with 15 µL of nuclease-free water was conducted twice.
3
Random-Primed PCR for Bisulfite-Converted DNA
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DNA is single-stranded following bisulfite conversion. To make DNA double-stranded and generate enough material for constructing a sequencing library, we performed a random-primed PCR amplification. Bisulfite converted DNA was subjected to a round of linear amplification using an adapter combined with a random 9-nucleotide (nt) primer [GTTTCCCAGTCACGGTC(N)9], purified, and PCR-amplified using primers against the adapter sequences. The PCR reaction was purified with MinElute PCR Purification Kit (Qiagen) and digested with HpyCH4III (NEB) to remove the adapter sequences. The digest was resolved on a 2% agarose gel; the smear in the range of 100–500 bp was excised, purified with the Gel Extraction Kit (Qiagen), and used for library construction with the ChIP-Seq Sample Prep Kit (Illumina) according to the manufacturer’s instructions. The library was sequenced on an Illumina GAII to collect paired-end reads of 36 nt each.
4
Genotyping NC_000005.10 c.*3+80T>G SNP
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The genomic region harboring the NC_000005.10 (NM_000344.3):c.*3+80T>G (rs143838139) single nucleotide polymorphism (also referred to as g.27134T>G in the literature) was coamplified along with a control to monitor digestion efficiency (F: 5′‐TGG GTT TTA TTT CCA GAC TTC A‐3′ and R: 5′‐TGC TTT GAT GAC GCT TCT GT‐3′) (Luo et al., 2014 (link)). Five microliters of the resulting PCR products were digested with 2.5 units of HpyCH4III (New England Biolabs, Ipswich, MA) for 1 hr at 37 °C and analyzed by capillary electrophoresis to size the digestion products (ABI 3130 DNA Analyzer). Alleles harboring the c.*3+80T>G SNP were sensitive to HpyCH4III digestion and resulted in a 136 bp product, whereas those that did not harbor the SNP were resistant to HpyCH4III digestion and remained intact (169 bp product).
5
Allele-Specific PCR-RFLP Genotyping
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We used allele-specific polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) to test the genotypes of NUDT15 R139C (rs116855232) and TPMT*3C (rs1142345). The sequences of the primers for NUDT15 R139C are: forward, 5-AGCTTACCCAAATAAACACCCT-3 and reverse, 5-TGGGGGATACAT TAAGAGACTGC-3, and those for TPMT*3C are: forward, 5-AAGTGTTGGGATTA CAGGTG-3 and reverse, 5-TCCTCAAAAACATGTCAGTGTG-3. PCR amplification started at 94 °C for 5 min, and continued with 30 cycles of 94 °C for 60 s, 59 °C for 30 s, and 72 °C for 30 s. Final extension was performed at 72 °C for 10 min. The PCR product was digested with restriction enzyme HpyCH4III (New England Biolabs, Hertfordshire, United Kingdom) for NUDT15 R139C testing and the enzyme AccI (New England Biolabs, Hertfordshire, United Kingdom) for TPMT*3C testing.
6
Quantitative LAMP Reaction Analysis
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The MATLAB script processes a .txt file with temperature-time data generated from the TE Tech Controller and a TIF stack containing 2-channel images of the LAMP and melt curve from the LEICA microscope. Partitions are identified using a custom iterative thresholding algorithm and labels are propagated throughout the TIF stack using a custom labeling algorithm. Average well intensity is tracked over time to generate LAMP curves and plotted against temperature to generate the melt curves. Complete details of the script are in the Supplementary Materials and Methods , ‘MATLAB script.’
Bulk LAMP reactions were conducted in 10 μl volumes within a well plate on a CFX96 Real-time Thermocycler (Bio-Rad) at buffer conditions and temperatures matching the dLAMP reactions.
Enzymatic digestions of bulk LAMP products were conducted using CAC8I (#R0579S), Hpy166II (#R0616S), ACCI (#R0161S), AciI (#SR0551S), MseI (#R0525S) and HpyCH4III (#R0618S) purchased from New England Biolabs and were conducted in 50 μl reaction volumes containing 1 μl enzyme, 1 μg DNA, in 1 × Cut Smart Buffer and incubated for 1 h at 37°C. Samples were inactivated for 1 h at 80°C and diluted to 1 ng/μl (∼1:300) to run on an Agilent 4200 TapeStation using High Sensitivity D5000 ScreenTape (#5067-5592) with ladder (#5190-7747), and D100 ScreenTape (#5067-5584) with High Sensitivity D1000 Reagents (#5067-5585).
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