Molecular analyses were carried out on genomic DNA extracted from iPSCs using the Flexigen 250 kit (Qiagen, Hilden, Germany). Beta S (βS) globin gene cluster haplotypes were investigated using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method [37 (link)]. Briefly, PCR amplification was performed using five primer pairs (3/4, 5/6, 6/7, 8/9, and 10/11). The reaction mixture contained genomic DNA, primer (25 pmol/μL), dNTPs (2 mM) (Thermo Fisher Scientific), Taq polymerase (5U/μL), MgCl2 (50 mM), buffer 10X (Taq DNA Polymerase Kit, Thermo Fisher Scientific), and a sufficient volume of free DNase H2O for 50 μL. PCR was performed at 94 °C for 10 min for initial denaturation, followed by 35 cycles of denaturation at 94 °C for 45 s, annealing at a specific temperature (Table 1 ) for 45 s and an extension at 72 °C for 90 s, and a final extension at 72 °C for 10 min. Amplicon (20 μL) was digested with the restriction enzymes XmnI, HincII, HindIII, or HinfI (Thermo Fisher Scientific). Buffer 10X and a suitable amount of PCR H2O were added to this mixture. Regarding the reaction containing XmnI, bovine serum albumin was added. Amplicons and digested DNA fragments were electrophoresed on 1% and 2% agarose gels (Thermo Fisher Scientific) for 1–2 h, respectively, and visualized under ultraviolet light (Table 1 ).
Hincii
Manufactured by Thermo Fisher Scientific
Sourced in United States
HincII is a type II restriction endonuclease enzyme that recognizes and cleaves the palindromic DNA sequence 'G*ANTC' where 'N' can be any nucleotide. This enzyme is commonly used in molecular biology applications such as DNA cloning, mapping, and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Fastdigest green buffer, by Thermo Fisher Scientific (2 mentions)
Sexai, by Thermo Fisher Scientific (2 mentions)
Gel doc xr imaging system, by Bio-Rad (2 mentions)
Ecori, by Thermo Fisher Scientific (2 mentions)
Dntps, by Thermo Fisher Scientific (2 mentions)
Taq polymerase, by Thermo Fisher Scientific (1 mentions)
Hinfi, by Thermo Fisher Scientific (1 mentions)
Flexigen 250 kit, by Qiagen (1 mentions)
Taq dna polymerase kit, by Thermo Fisher Scientific (1 mentions)
Hindiii, by Thermo Fisher Scientific (1 mentions)
T4 polynucleotide kinase, by Thermo Fisher Scientific (1 mentions)
Generuler 1 kb dna ladder, by Thermo Fisher Scientific (1 mentions)
Generuler 1 kb plus dna ladder, by Thermo Fisher Scientific (1 mentions)
6 protocols using hincii
1
Molecular Characterization of Beta Globin Haplotypes
2
Mitochondrial DNA Southern Blot Analysis
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2D-AGE and subsequent Southern blot were performed as previously described (24 (link)). HincII (ThermoScientific) was used for mtDNA fragmentation. A PCR probe spanning nucleotides 35–611 (corresponding to the mtDNA non-coding region) random-prime labelled with [α-32P]dCTP was used for nucleic acid hybridization.
For the analysis of sheared DNA samples for ChIP protocol, reverse cross-linked DNA was precipitated and purified using standard phenol-chloroform extraction. 2.5 μg of non-sheared and sheared DNA was separated over a 1% agarose gel in 1× TBE buffer. Southern blotting was performed by using standard procedures. An oligonucleotide mapping to the ND5 region of the mtDNA (Supplementary Table S5 ) was labelled at the 5′-end with [γ-32P]ATP using T4 Polynucleotide kinase (ThermoScientific) according to the manufacturer's instructions. Probe hybridization was performed using standard procedures (24 (link)). The membrane was exposed to a storage phosphor screen and scanned with the Typhoon Scanner 9400 (Amersham Biosciences).
For the analysis of sheared DNA samples for ChIP protocol, reverse cross-linked DNA was precipitated and purified using standard phenol-chloroform extraction. 2.5 μg of non-sheared and sheared DNA was separated over a 1% agarose gel in 1× TBE buffer. Southern blotting was performed by using standard procedures. An oligonucleotide mapping to the ND5 region of the mtDNA (
3
PCR-RFLP Analysis of CDH1 Polymorphisms
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The polymerase chain reaction (PCR)-restriction fragment length polymorphism method was applied to analyze -160C > A and -347GA > G polymorphisms separately. A 447 bp PCR product containing both -160C > A and -347GA > G polymorphisms was achieved from CDH1 gene promoter amplification. Two primers: 5'-GCCCCGACTTGTCTCTCTAC-3' (forward) and 5'-TACCGCTGATTGGCTGAGGG-3' (reverse) were applied. The PCR amplification was carried out in a 25 μl reaction mixture containing 2 μl template DNA, 12 μl ready ×2 PCR Master Mix (Amplicon, Denmark), 10 μl distilled water, and 0.5 μl of each forward and reverse primer. The amplification was performed under the following conditions: 1 cycle of 95°C for 2 min; 35 cycles of 95°C for 30 s, 65°C for 30 s, and 72°C for 30 s; and a final cycle of 72°C for 5 min. The PCR product was digested with HincII and BanII (Thermo Fisher Scientific) at 37°C overnight in two separate tubes, respectively [Table 3 ].
To detect the –160C > A and –347GA > G polymorphisms, PCR products were digested with HincII restriction enzymes for –160C > A (rs16260) and BanII restriction enzyme (Thermo Fisher Scientific) for -347GA > G (rs5030625) polymorphisms, respectively [Table 3 ]. After digestion, the products were separated by 3% agarose gel electrophoresis and stained with Green Viewer™ DNA Staining Dye [Figure 1 ].
To detect the –160C > A and –347GA > G polymorphisms, PCR products were digested with HincII restriction enzymes for –160C > A (rs16260) and BanII restriction enzyme (Thermo Fisher Scientific) for -347GA > G (rs5030625) polymorphisms, respectively [
4
Restriction Digestion Protocol
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Restriction digestions were carried out using the restriction enzymes EcoRI, HincII, NsiI, PstI, SalI, ScaI, SexAI, and SpeI (Thermo Fischer Scientific). These enzymes gave several well separated bands when in silico digested by the NEBcutter (http://nc2.neb.com/NEBcutter2/ ). The digestions were carried out in a final volume of 10 µL, containing DNA (ca. 300 ng), 0.5 µL of enzyme and 1 µL of Fast digest green buffer (10×, Thermo Fisher Scientific). After 2–16 h incubation at 37 °C the restriction fragments along with undigested DNA and GeneRuler 1 kb DNA Ladder (Thermo Fisher Scientific) were loaded on 1% (w/v) agarose gel including 0.005% (w/v) Midori green. After the electrophoresis the fragments were visualized using UV transillumination and images recorded using the BioRad GelDoc XR+ imaging system.
5
Restriction Enzyme Digestion Analysis
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Restriction digestions were carried out using the restriction enzymes EcoRI, HincII, NsiI, PstI, SalI, ScaI, SexAI, and SpeI (Thermo Fischer Scientific). These enzymes gave several well separated bands when in silico digested by the NEBcutter (http://nc2.neb.com/NEBcutter2/ (accessed on 8 May 2021)). The digestions were carried out in a final volume of 10 µL, containing DNA (ca. 300 ng), 0.5 µL of enzyme, and 1 µL of Fast digest green buffer (10×, Thermo Fisher Scientific). After 2–16 h incubation at 37 °C, the restriction fragments along with undigested DNA and GeneRuler 1 kb plus DNA Ladder (Thermo Fisher Scientific) were separated on 1% (w/v) agarose gel and detected with ethidium bromide staining using UV transillumination. Images were recorded using the BioRad GelDoc XR+ imaging system.
6
SNP Genotyping by PCR-RFLP Analysis
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Restriction digestion of the PCR product will determine the presence or absence of the desired SNPs at the restriction site. RFLP assay confirms the frequencies of desired SNP within a specified sample cohort. After getting the specific PCR products, they were digested with restriction endonucleases. PCR products for CDH1 gene and Exo1 gene were digested with 2 units of HincII (Thermo Fisher Scientific, USA) at 37°C for overnight with Msel (Tru1l) (Thermo Fisher Scientific, USA) at 65°C for 16 h, respectively, following the manufacturers' standard protocol. The digested products of both genes were then evaluated by electrophoresis through 3% agarose gel (Figures 2(b) and 3(b)). Homozygous A/A genotype of CDH1 was cut by HincII yielding two fragments with 368 and 80 bp, C/C genotype produced a single 448 bp band because of absence of cutting site, whereas heterozygous genotype resulted all three bands. Similarly, on MseI digestion of Exo1 PCR products, homozygous A/A allele yielded 196 and 110 bp fragments, homozygous G/G genotype yielded single 306 bp, whereas heterozygosity yielded all three fragments. We checked our initial results by regenotyping randomly selected 20 samples (10% of our total samples) by the previously followed PCR-RFLP method.
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