The coding sequences of the three gene models, Glyma.04G143300, Glyma.04G143400 and Glyma.04G143500, were analyzed for H-e3 and ZE. The coding sequences were amplified from the cDNAs by using primers listed in Supplementary Table S1 . The amplified fragments were cloned into a pGEM-T Easy vector (Promega) and sequenced with a BigDye Terminator v3.1 Cycle Sequencing kit and an ABI PRISM 3100 Avant Genetic Analyzer (both from Applied Biosystems, Japan) according to the manufacturer’s instructions. A derived cleaved amplified polymorphic sequence (dCAPS) marker targeting a single-base deletion observed in ZE was developed to discriminate the functional E1Lb allele of H-e3 from the loss-of-function e1lb allele of ZE. The 275-bp DNA fragment amplified from ZE by PCR with the forward primer 5′-GTGTAAACACTCAAAGTCCTT-3′ and the reverse primer 5′-CGTCTTCTTGATCTTCCAACG-3′ was digested with HpyCH4IV (New England Biolabs Japan) into two fragments, 254 bp and 21 bp, but the 276-bp fragment amplified from H-e3 was resistant to HpyCH4IV digestion. The PCR products were treated with HpyCH4IV for 1 h and then separated by electrophoresis in 2.5% NuSieve 3:1 gel (Lonza), stained with ethidium bromide, and visualized under UV light.
Hpych4iv
Manufactured by New England Biolabs
Sourced in United States, Japan
HpyCH4IV is a type II restriction endonuclease that cleaves DNA at the sequence 5'-ACGT-3'. It recognizes and cuts the palindromic DNA sequence ACGT.
Automatically generated - may contain errors
Lab products found in correlation
T4 dna ligase, by Thermo Fisher Scientific (4 mentions)
Hpaii, by New England Biolabs (3 mentions)
Biotaq hs dna polymerase, by Meridian Bioscience (2 mentions)
Ez dna methylation gold kit, by Zymo Research (2 mentions)
Pgem t easy vector, by Promega (2 mentions)
Multina, by Shimadzu (2 mentions)
Exonuclease 1, by Thermo Fisher Scientific (1 mentions)
Ultra gelred, by Vazyme (1 mentions)
Evagreen, by Biotium (1 mentions)
Ecori, by New England Biolabs (1 mentions)
Epitect bisulfite kit, by Qiagen (1 mentions)
Hinfi restriction enzyme, by New England Biolabs (1 mentions)
Gotaq master mix, by Promega (1 mentions)
Dna clean and concentrator 5 kit, by Zymo Research (1 mentions)
Fla 7000 phosphorimager, by Fujifilm (1 mentions)
Hybond n membrane, by GE Healthcare (1 mentions)
Pjet vector, by Thermo Fisher Scientific (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Mytaq dna polymerase kit, by Meridian Bioscience (1 mentions)
Gelred, by Biotium (1 mentions)
31 protocols using hpych4iv
1
Genotyping Soybean Maturity Genes
2
Oligonucleotide Protocols for Z-DNA Research
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T4 DNA ligase and Exonuclease I were obtained from Thermo Scientific (Pittsburgh, PA, USA). EcoRI, MboI, Hpych4Iv and SphI were purchased from New England Biolabs Inc. (Ipswich, MA, USA). The fluorescent dye of EvaGreen was from Biotium (Fremont, CA, USA), and Ultra GelRed (a dye staining both dsDNA and ssDNA) was purchased from Vazyme (Nanjing, China). Z-DNA-specific antibody (Z22) was from Absolute Antibody Ltd. (Oxford, UK), and ZBP1 (recombinant Z-DNA-binding protein) was from Wuhan USCN Business Co., Ltd. (Wuhan, China). All other chemicals were from Sigma-Aldrich (St. Louis, MO, USA).
All oligonucleotides used in this study (seeSupplemental Table S1 for sequences) were purchased from GENEWIZ (Suzhou, China). Sequence were designated as follows. ls: linear ssDNA strand; cs: circular ssDNA strand; Sp: direct the head-tail ligation of a linear oligonucleotide to a circular one; lc: hybridization of a linear strand (α) and a circular one (β); cl: hybridization of a circular strand (α) and a linear one (β); cc: hybridization of two circular strands. 740: the duplex containing a continuous APP sequence; 741 and 743 are duplexes containing non-APP sequences; 741–20 and 741–35 are duplexes containing 20 bp mismatches and 35 bp mismatches, respectively. cc-742 is a duplex formed with one strand of poly(purine) and the other strand of poly(pyrimidine).
All oligonucleotides used in this study (see
3
Quantitative DNA Methylation Analysis
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DNA methylation levels were evaluated by COBRA as we previously reported15 (link),16 (link). In brief, sodium bisulfite treatment was performed using an EpiTect Bisulfite kit (Qiagen) according to the conditions as follows: 95 °C for 5 min, 65 °C for 85 min, 95 °C for 5 min and 65 °C for 175 min. After sodium bisulfite treatment, PCR was performed using one unit of Biotaq HS DNA polymerase (Bioline, London, UK) and the primer sets shown in Supplementary Table S11 online under the thermocycling conditions (35 to 38 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s, with an initial step of 95 °C for 10 min and a final step of 72 °C for 7 min). A part of the PCR product was digested with the restriction enzyme TaqI (Takara, Tokyo, Japan) or HpyCH4IV (New England Biolabs, Ipswich, MA). The treated PCR product was electrophoresed by 3% agarose gel. PCR products from methylated DNA and unmethylated DNA are digested and undigested by the treatment with the restriction enzyme. The intensity of the signals of the digested and undigested PCR products was measured by densitometry. Methylation levels (%) were calculated as the ratio of the digested PCR product in the total PCR product (digested + undigested products).
4
Bisulfite-based DNA Methylation Analysis
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cDNA derived from the bisulfite treated RNA was used to generate PCR products from tRNA Asp(GTC) and helix 70 of 25S nuclear rRNA using the primers forward Asp tRNA_At_FWD and reverse Asp tRNA_At_REV and primers forward 25S_rRNA_F and reverse 25S_rRNA_R, respectively. The 25S_rRNA_F dCAPS primer contains a G mismatch at position four from the 3′ end to generate a HinfI restriction site. PCR products were digested with HpyCH4IV or HinfI restriction enzymes (New England Biolabs), respectively. The tRNAAsp(GTC) 72bp PCR product is digested by HpyCH4IV resulting in two digestion products of 35bp and 37bp if C38 is methylated, and is undigested if C38 is non-methylated and thus converted to T38 by bisulfite treatment, causing the HpyCH4IV restriction enzyme site to be lost. The 25S rRNA PCR product is 155bp in length. When C2268 is methylated, the restriction enzyme HinfI cleaves the PCR product into two fragments of 29bp and 126bp. Non-methylated 25S rRNA has a T at position 2268 after bisulfite treatment, and this eliminates the HinfI restriction enzyme site, leaving the product at 155bp and undigested. Undigested PCR products were used as loading controls. Primer sequences used are provided (Additional file 1 : Table S6).
5
Detection of MYH3 Gene Variants via PCR
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A set of primers was designed for the detection of the FSVs in the
MYH3 gene using available sequence information
(XM_013981330.2). PCR was performed using a Maxime PCR Premix kit, and reactions
included 20 µL of reaction mixture including 100 ng of DNA, 0.5 nmol of
MYH3 promoter-specific primers (MYH3_1_F,
5’-TGGTCTTTCCTAATTGGTGACAT-3’, MYH3_1_R,
5’-AGTTTTGAGCAAGGCTTTTGTT-3’) and distilled water (iNtRON,
Seongnam, Korea). PCR conditions were as follows: initial heating was at
95°C for 5 min, followed by 35 cycles of 30 s for denaturation at
94°C, 30 s for annealing at 65°C, and 30 s for an extension at
72°C, followed by a final extension at 72°C for 10 min in a Nexus
PCR machine (Eppendorf, Hamburg, Germany). And the amplicons were digested with
the restriction enzyme, HpyCH4IV (NEB, Ipswich, MA, USA). The
PCR products were separated on 2.5% agarose gels (Lonza, Basel, Switzerland) and
visualized by UV illumination with a BioFACT 100 bp plus DNA ladder marker
(BioFACT, Daejeon, Korea) [2 (link)].
MYH3 gene using available sequence information
(XM_013981330.2). PCR was performed using a Maxime PCR Premix kit, and reactions
included 20 µL of reaction mixture including 100 ng of DNA, 0.5 nmol of
MYH3 promoter-specific primers (MYH3_1_F,
5’-TGGTCTTTCCTAATTGGTGACAT-3’, MYH3_1_R,
5’-AGTTTTGAGCAAGGCTTTTGTT-3’) and distilled water (iNtRON,
Seongnam, Korea). PCR conditions were as follows: initial heating was at
95°C for 5 min, followed by 35 cycles of 30 s for denaturation at
94°C, 30 s for annealing at 65°C, and 30 s for an extension at
72°C, followed by a final extension at 72°C for 10 min in a Nexus
PCR machine (Eppendorf, Hamburg, Germany). And the amplicons were digested with
the restriction enzyme, HpyCH4IV (NEB, Ipswich, MA, USA). The
PCR products were separated on 2.5% agarose gels (Lonza, Basel, Switzerland) and
visualized by UV illumination with a BioFACT 100 bp plus DNA ladder marker
(BioFACT, Daejeon, Korea) [2 (link)].
6
Generating Methylated Filler DNA
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To generate filler DNA, enterobacteria phage λ DNA was polymerase chain reaction (PCR) amplified with GoTaq Master Mix (Promega, Madison, WI, USA). The primer sequences were as follows: forward primer 5′- CGATGGGTTAATTCGCTCGTTGTGG-3′, reverse primer 5′-GCACAACGGAAAGAGCACTG-3′. The 274-bp amplicons were treated with CpG methyltransferase (M.SssI, Thermo Fisher Scientific, Waltham, MA, USA) to methylate amplicons. Methylated amplicons were purified using a DNA Clean and Concentrator-5 Kit (ZYMO Research, Irvine, CA, USA) and quantified using a Qubit Fluorometer. CpG methylation-sensitive restriction enzyme HpyCH4IV (New England BioLabs, Ipswitch, MA, USA) digestion, followed by agarose gel electrophoresis, was performed to ensure the complete methylation of filler DNA.
7
Quantifying Genomic Satellite DNA
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Southern blot on genomic DNA was performed as described in Thijssen et al.63 (link). For major satellite analysis 200 ng of genomic DNA were digested with HpyCH4IV (New England Biolabs) for 1h at 37 °C, while for minor satellites 500ng of gDNA were digested with HpaII (New England Biolabs) and 300 ng with MspI (New England Biolabs), both O/N at 37 °C. Digested samples were separated for 5 h on 1% agarose gel. Gels were then denaturated in a 1.5 M NaCl and 0.5 M NaOH solution for 20 min and neutralized with 0.5 M Tris-HCl pH 7.5 and 1.5 M NaCl for 40 min. Transfer was performed O/N on Hybond-N+ membranes (GE Healthcare) in SSC 20X. After ultraviolet crosslinking, membranes were pre-hybridized in SSC 6X, Denhardt 5X and 0.1% SDS for 1 h at 42 °C and hybridized with 32P-labelled probes for 2 h at 42 °C. After membrane washing, signals were detected using FLA 7000 phosphorimager (Fuji). Images were then analyzed with ImageJ (imagej.nih.gov/ij) to perform linescan for major satellites and intensity ratio HpaII/MspI for the lower six bands of each lane for minor satellites. Probe used: Major satellites 5′-CAC GTC CTA CAG TGG ACA TTT CTA AAT TTT CCA CCT TTT TCA GTT-3′ and minor satellites 5′-ACA TTC GTT GGA AAC GGG ATT TGT AGA ACA GTG TAT ATC AAT GAG TTA CAA TGA GAA ACA T-3′.
8
Bisulfite Conversion and Sequencing of DNA
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Genomic DNA was extracted by solution D from normal fibroblasts, fibroblasts transfected with vector control and fibroblasts that overexpressed Peblr20. DNA bisulfite (BS) conversion was performed using the EZ DNA Methylation-Gold kit (Zymo Research, Orange, CA) according to manufacturer's protocol. PCR was performed using BS-treated DNA samples as template, and BS-specific primers were shown in Table S1
9
Genetic Variant Analysis via PCR and Sequencing
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Analysis of rs147546939, rs529135220 and rs187887338 was performed by PCR analysis followed by Sanger sequencing using BigDye terminator v3.2 and analysis on an ABI3100XL platform. Primers used for PCR were as follows: rs147546939: Rs147546939-FH acc cac tgc tta ctg gct tat cAG GGC AGA AAA CTT CAC CAA and Rs147546939-RH gag ggg caa aca aca gat ggc TCA ACA TGG TTC TTT AAT GTC CAC; rs529135220: TYR-c1184-17845-FH acc cac tgc tta ctg gct tat cGG CGG ATT ATG AGG TCA GGA and TYR-c1184-7845-RH gag ggg caa aca aca gat ggc TTT GGC CCA CTG TTT GTT CC; rs187887338: TYR-s187887338-FH acc cac tgc tta ctg gct tat cTC TCG CTC ATA GGT GGG AAC and TYR-s187887338-RH gag ggg caa aca aca gat ggc CCA TGA AAT CTT TGG GGC ACA. Bases in lower case letters were used for sequencing and are not gene specific. Analysis of rs145409367 was performed by PCR using primers TYR-s145409367-FH acc cac tgc tta ctg gct tat cTG GAT TTG GCA AAG GTG TTT T and TYR-s145409367-RH gag ggg caa aca aca gat ggc TGA ATC CTA CTA ATC CAA TGG CT followed by restrictionenzyme analysis with HpyCH4IV (New England Biolabs, Ipswich, MA, USA) which only cuts the C allele (major allele).
10
BDNF rs6265 Genotyping Protocol
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The BDNF rs6265 genotyping was performed using a polymerase chain reaction (PCR) combined with restriction enzyme digestion. An 84 base pairs (bp) fragment was amplified from 20 ng of genomic DNA (reaction volume of 20 μL), using a MyTaq DNA polymerase kit (Bioline, Taunton, MA, USA), and 10 mM of each primer (forward BD-F and reverse BD-R) (Santoro et al., 2016). The amplification conditions were as follows: 2 min at 95 °C, initial denaturation; 15 s at 95 °C, 30 s at 60 °C and 30 s at 72 °C for 35 cycles; 7 min at 72 °C, final extension. The PCR products, digested with 3 units of HpyCH4IV (cleavage sequence 5′-ACGT-3′) (New England Biolabs, Beverly, MA, USA) restriction enzyme, were resolved by electrophoresis on 2% agarose gels stained with Gel Red (Biotium, Fremont, CA, USA).
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