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Practical Approach to Molecular Biology in Hematopathology
Published in Harold R. Schumacher, William A. Rock, Sanford A. Stass, Handbook of Hematologic Pathology, 2019
Anwar Mikhael, Harold R. Schumacher
Typically, extracted DNA is digested with three different restriction enzymes (EcoRI, BamHI, and HindIII), followed by agarose gel electrophoresis, transfer to nylon filter, hybridization with 32P-labeled DNA probe, and subsequent autoradiography. The size of the germline bands have been determined based on the restriction enzyme map of the Ig and TCR loci (Fig. 2).
Optimal Detection of and Effect of Vitamin D3 on Extrachromosomal Oncogene Sequences
Published in Maryce M. Jacobs, Vitamins and Minerals in the Prevention and Treatment of Cancer, 2018
Daniel D. Von Hoff, Donald R. VanDevanter
4. DNA Electrophoresis. Horizontal 1.0% agarose (BRL UltraPure) electrophoretic gels were run in 45 mM Tris Borate, 1 mM EDTA, pH 8.3 (0.5X TBE) buffer with recirculation. Gels were stained after running with 1.0 μg/ml ethidium bromide and photographed with a red filter while transilluminated by a 300 nm UV source. Low voltage electrophoresis (LoVE) was performed at room temperature for 24 hours at a field strength of 1.7 V/Cm. All other gels were run at 14°C with a field strength of 5.6 V/cm. FIGE was performed with a forward to reverse pulse ratio of 3.0, with pulses ramped linearly from an initial forward pulse time of 3 seconds to a final forward pulse time of 60 seconds over 12 hours. Linear DNA molecular weights were determined with intact chromosomes of S. cerevisiae (Beckman) and coliphage lambda DNA digested with HINDIII restriction endonuclease (BRL).
HLA-DR and -DQ Typing by DNA-RFLP Analysis
Published in M. Kam, Jeffrey L. Bidwell, Handbook of HLA TYPING TECHNIQUES, 2020
According to the probe required (Table 1 and Figure 3), select the appropriate restriction endonuclease and proceed as follows: In a 1.5-ml microcentrifuge tube, set up the following restriction digest:0.1 ml plasmid DNA (equivalent to 150 μg)15 μl 10× Restriction endonuclease buffer (see suppliers’ recommendations)300 U Restriction endonuclease (usually 12 to 30 μl)Make up to 150 μl with ddH2OVortex and incubate for 3 h at the temperature recommended for the enzyme (37°C for PstI and AvaI). Vortex occasionally during the incubation. After 3 h, remove 2 μl and check that the digest is complete (follow instructions for minigel electrophoresis as described earlier). Use a HindIII digest of phage lambda as a molecular weight marker and check that the expected fragments (Table 1) are present. If the digest is incomplete, add more enzyme and reincubate, then check a further 2 μl aliquot.When digestion is complete, add 30 μl 6x gel loading buffer, vortex, and proceed to preparative electrophoresis.
Beta Globin Gene Cluster Haplotypes in Beta Thalassemia in the Kurdistan Region of Iraq
Published in Hemoglobin, 2023
Shaima Al-Zebari, Nasir AS Al-Allawi, Farida Nerweyi
All eligible enrollees had their DNA extracted by blood DNA extraction Kit (Qiagen, Germany). Their original molecular diagnoses were reported by a recent study [10]. In addition, an extra 12 patients diagnosed after conclusion of the latter study were included using Beta globin StripAssay (ViennaLAb Diagnostics GmBH, Austria) and sequencing as detailed elsewhere [10]. Haplotype analysis was done by using the technique of restriction fragment length polymorphism (RFLP) at seven restriction sites across the β-globin gene cluster and as detailed in an earlier study [8]. The RFLPs include: HindII 5′ε, HindIIIGγ, HindIII Aγ, Hind II 5′ψβ, Hind II 3′ψβ, AvaII β, and BamHI 3′β. Haplotypes were assigned as proposed by Orkin et al. (1982) and Kazazian et al. (1984) [4,11]. For the purposes of the current study, it was assumed that a heterozygous individual had one classic (as defined by Orkin et al. [4]) and one atypical haplotype, rather than two atypical ones [12].
Akkermansia muciniphila upregulates genes involved in maintaining the intestinal barrier function via ADP-heptose-dependent activation of the ALPK1/TIFA pathway
Published in Gut Microbes, 2022
Camille Martin-Gallausiaux, Diego Garcia-Weber, Amandine Lashermes, Pierre Larraufie, Ludovica Marinelli, Veronica Teixeira, Alice Rolland, Fabienne Béguet-Crespel, Vincent Brochard, Timothé Quatremare, Alexandre Jamet, Joël Doré, Scott D. Gray-Owen, Hervé M. Blottière, Cécile Arrieumerlou, Nicolas Lapaque
E. coli K-12 WT and ΔHldE E. coli (JW3024) are from the Keio collection49 (Dharmacon). hldE from A. muciniphila was amplified using the following primers: (i) AhldEfor_NheI (5’-GGGGGCTAGCAGGAGGTAAATAATGAACCGGCTGCATACAT-3’) creating a new NheI restriction site (GCTAGC) and adding a Shine-Dalgarno sequence (AGGAGG) located 6 bases upstream of the start codon ATG and (ii) AhldErev_HindIII (5’-GGGGAAGCTTTCATTCCGGGCTGCTTTTC-3’) creating a new HindIII restriction site (AAGCTT). The 1523 pb fragment was amplified using Phusion High-Fidelity DNA polymerase, A-tail with GoTaq polymerase and cloned in pGEM®-T Easy vector (Promega Corporation) to generate pGEM-T-AhldE. After NheI HindIII restriction of the pGEM-T-AhldE, the AhldE fragment was cloned into NheI-HindIII-restricted pBAD24 generated pBAD24-AhldE vector (amp). pBAD24-AhldE and the empty vector pBAD24 were purified and used to electroporate E. coli ΔHldE. Overnight bacterial cultured were washed in PBS and resuspended at an OD = 1, boiled for 30 min and stored at -20°C until use.
Dual functions of discoidinolysin, a cholesterol-dependent cytolysin with N-terminal discoidin domain produced from Streptococcus mitis strain Nm-76
Published in Journal of Oral Microbiology, 2022
Atsushi Tabata, Airi Matsumoto, Ai Fujimoto, Kazuto Ohkura, Takuya Ikeda, Hiroki Oda, Shuto Yokohata, Miho Kobayashi, Toshifumi Tomoyasu, Ayuko Takao, Hisashi Ohkuni, Hideaki Nagamune
The recombinant protein expression system was constructed according to a previously reported method [50]. Briefly, to construct recombinant DLY (rDLY), the fragment encoding DLY without a predicted signal sequence consisting of 44 amino acids, as underlined in Figure 1a, was amplified using PrimeSTAR HS DNA polymerase (TaKaRa Bio Inc.) with primers 3 and 4 (Table S1) and genomic DNA purified from S. mitis Nm-76 as the template. The amplified fragment was digested by both BamHI and HindIII and purified and cloned into pQE-9 (Qiagen, Hilden, Germany) using DNA Ligation Kit <Mighty Mix> (TaKaRa Bio Inc.). Subsequently, E. coli JM109 competent cells were transformed with the vector. The target clones were selected after PCR using GoTaq DNA polymerase (Promega Corp.) with primers 15 and 16 (Table S1), and isolated colonies were used as templates. Sequencing of the purified plasmids from PCR-positive clones was outsourced (BEX Co., Ltd.). The expression systems of the DD and DD-deleted DLY (ΔDD) were also constructed according to the method described above using a primer sets: 3 and 17 and 18 and 4, respectively (Table S1).