Primary leukemia cells (Supplementary Table 1 ) were cultured on OP9 stroma cells in Alpha MEM without ribonucleotides and deoxyribonucleotides, supplemented with 20% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 IU/ml penicillin, and 100 μg/ml streptomycin. Human ALL cell lines were maintained in RPMI with GlutaMAX containing 20% FBS, 100 IU/ml penicillin, and 100 μg/ml streptomycin. Mouse BCR-ABL1-transformed ALL cells were maintained in IMDM with GlutaMAX containing 20% FBS, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 50 μM 2-mercaptoethanol. Cell cultures were kept at 37°C in a humidified incubator under a 5% CO2 atmosphere.
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Deoxyribonucleotides
Deoxyribonucleotides
Deoxyribonucleotides are the fundamental building blocks of DNA, the genetic material essential for all cellular life.
These molecules consist of a deoxyribose sugar, a phosphate group, and one of four nitrogenous bases: adenine, guanine, cytosine, or thymine.
Deoxyribonucleotides play a crucial role in DNA replication, transcription, and repair processes, enabling the storage, transmission, and expression of genetic information.
Understanding the properties and behaviors of deoxyribonucleotides is crucial for advancing research in fields such as molecular biology, genetics, and biotechnology.
PubCompare.ai's AI-powered comparisons can help identify the most reproducible and accurate methods for working with deoxyribonucleotides, allowing researchers to optimize their protocols and advance their investigations more efficiently.
These molecules consist of a deoxyribose sugar, a phosphate group, and one of four nitrogenous bases: adenine, guanine, cytosine, or thymine.
Deoxyribonucleotides play a crucial role in DNA replication, transcription, and repair processes, enabling the storage, transmission, and expression of genetic information.
Understanding the properties and behaviors of deoxyribonucleotides is crucial for advancing research in fields such as molecular biology, genetics, and biotechnology.
PubCompare.ai's AI-powered comparisons can help identify the most reproducible and accurate methods for working with deoxyribonucleotides, allowing researchers to optimize their protocols and advance their investigations more efficiently.
Most cited protocols related to «Deoxyribonucleotides»
2-Mercaptoethanol
alpha minimal essential medium
Atmosphere
Cell Culture Techniques
Cell Lines
Cells
Cultured Cells
Deoxyribonucleotides
Glutamine
Homo sapiens
Leukemia
Mus
Penicillins
Pyruvate
Ribonucleotides
Sodium
Streptomycin
2'-deoxycytidine 5'-triphosphate
Biotin
biotin-14-dCTP
Chromatin
Cytokinesis
Deoxyribonucleotides
DNA Library
DNA Polymerase I
Enzymes
Ligation
Technique, Dilution
All PCR experiments were performed on a Perkin-Elmer 480 thermal cycler. Restriction enzymes, DNA ligase and DNA polymerase were obtained from New England Biolabs. Deoxyribonucleotide triphosphates (dNTPs) were purchased from Promega and the primers were obtained from Sigma-Aldrich Pty. Ltd. The mutagenic primers (Table 1 ) differed from the wild-type sequence by containing substitutions to change one amino acid residue. Mini preparation of plasmid DNA was performed using the Promega Plasmid Preparation Kit.
Our new strategy has been applied to four different proteins but for illustration the strategy to construct a mutant of E. coli KARI will be described in detail. Isolating clones followed by DNA sequencing was used to assess the success of the strategy. For the other three enzyme constructs, the designed mutagenic primers contained altered restriction enzyme recognition sites to permit facile screening for the mutant (Table1 ). In one case; the target codon change was chosen so as to create a restriction enzyme recognition site, while in a second case an existing site was removed. A third mutagenic primer contained a second substitution that is silent with respect to the protein sequence but which introduced a restriction enzyme recognition site. The resulting mutants were screened using diagnostic restriction digestion.
The ilvC gene (1482 bp) encodes KARI and the gene product is a single polypeptide with a deduced molecular weight of 54 kDa [14 (link)]. We have described previously the cloning of the gene into the pET30a(+) vector [15 (link)]. The pET-C (6879 bp) plasmid was used as the template.
This new strategy consists of two rounds of PCR amplification. In the first round, the reaction was carried out in total volume of 50 μL and contained 2 U of Vent polymerase, 1X ThermoPol PCR buffer (10 mM KCl, 10 mM (NH4)2SO4, 20 mM Tris-HCl (pH 8.8 at 25°C), 2 mM MgSO4 and 0.1% Triton® X-100), 50–100 ng of pET-C DNA, 0.2 mM dNTP, 1.0 pmole of internal mutagenic forward primer (Table1 ) and 0.05 pmole of the reverse flanking primer (T7 terminator, GGTTATGCTAGTTATTGCTCAGCGGTGGC). The PCR was carried out under the following conditions: denaturation, 95°C for 1 minute; annealing, 50°C for 30 seconds; and extension, 72°C for 2 minutes. This cycle was repeated 5 times and followed by an additional extension step at 72°C for 35 minutes. In the second round of PCR, the forward flanking primer (T7 promoter, CGCGAAATTAATACGACTCACTATAGGGG) was added into the same tube. After an initial denaturation of 96°C for 1 minute, 25 PCR cycles were used as follows: denaturation, 96°C for 1 minute; annealing, 55°C for 1 minute; and extension, 72°C for 2 minutes. These cycles were followed by an additional extension step at 72°C for 10 minutes. The second PCR product (1837 bp) having desired mutation was purified and digested with BamHI and HindIII to give a mutated ilvC gene fragment (1482 bp). The resulting fragment was purified and cloned into the expression vector pPROEX™HTb (4779 bp) (Gibco BRL) after digesting vector with BamHI and HindIII. The resulting mutant plasmid (6192 bp) was used to transform E. coli strain CU505 (Purdue University, Indiana, USA) competent cells and 10% of the transformed culture was cultured on Luria-Bertani agar plates supplemented with 10 μg/ml ampicillin. The plasmid isolated from selected colonies was sequenced using BigDye™ Terminator chemistry at the Australian Genome Research Facility (Brisbane, Queensland, Australia).
Our new strategy has been applied to four different proteins but for illustration the strategy to construct a mutant of E. coli KARI will be described in detail. Isolating clones followed by DNA sequencing was used to assess the success of the strategy. For the other three enzyme constructs, the designed mutagenic primers contained altered restriction enzyme recognition sites to permit facile screening for the mutant (Table
The ilvC gene (1482 bp) encodes KARI and the gene product is a single polypeptide with a deduced molecular weight of 54 kDa [14 (link)]. We have described previously the cloning of the gene into the pET30a(+) vector [15 (link)]. The pET-C (6879 bp) plasmid was used as the template.
This new strategy consists of two rounds of PCR amplification. In the first round, the reaction was carried out in total volume of 50 μL and contained 2 U of Vent polymerase, 1X ThermoPol PCR buffer (10 mM KCl, 10 mM (NH4)2SO4, 20 mM Tris-HCl (pH 8.8 at 25°C), 2 mM MgSO4 and 0.1% Triton® X-100), 50–100 ng of pET-C DNA, 0.2 mM dNTP, 1.0 pmole of internal mutagenic forward primer (Table
Agar
Amino Acids
Amino Acid Sequence
Ampicillin
Buffers
Cells
Cloning Vectors
Codon
Deoxyribonucleotides
Diagnosis
Digestion
DNA-Directed DNA Polymerase
DNA Ligases
DNA Restriction Enzymes
Enzymes
Escherichia coli
Genes
Genome
Mutagenesis
Mutation
Oligonucleotide Primers
Plasmids
Polypeptides
Promega
Proteins
Strains
Sulfate, Magnesium
triphosphate
Triton X-100
Tromethamine
vent polymerase
The PALB2 genomic sequence was obtained from the National Center for Biotechnology Information (reference sequence number NG_007406.1). Primers (Geneworks, Hindmarsh, South Australia, Australia) (Additional file 1 ) were designed by using Primer3 software (Whitehead Institute and Howard Hughes Medical Institute, Cambridge, MA, USA). For optimal performance of the HRM curve analysis, primers were designed to amplify DNA products between 100 and 310 bp. A total of 35 fragments were designed to cover the coding and flanking intronic regions of PALB2. The primer sequences and annealing temperatures are listed in Additional file 2 . Initially, 96 DNA were Sanger-sequenced as described in Tischkowitz et al. [22 (link)]. These data and the corresponding DNA were then used to establish the optimal conditions for HRM curve analysis. DNA extracted from peripheral blood samples provided by 1,473 case probands (695 from women diagnosed with breast cancer under the age of 40 years participating in the ABCFR and 778 from kConFab) were screened for germline PALB2 mutations using HRM curve analysis [23 (link),24 (link)]. DNA was then systematically screened using this established method. HRM reactions were carried out in 15-μL volumes and included 1.5 μL of 10 × polymerase chain reaction (PCR) buffer (Applied Biosystems, Victoria, Australia), a 3 mM final concentration of MgCl2 (Applied Biosystems), a 100 μM final concentration of deoxyribonucleotide triphosphate (dNTP) (Bioline, Alexandria, New South Wales, Australia), a 200 nM final concentration of each primer (Geneworks) (Additonal File 2 ), a 2.3 μM final concentration of Syto9 (Invitrogen, Victoria, Australia), 0.25 U of AmpliTaq Gold (Applied Biosystems) and 3 μL of Q solution (Qiagen, Victoria, Australia). Each reaction underwent a hold of 10 minutes at 95°C and 40 cycles of amplification of 30 s at 95°C and 1 minute at annealing temperature followed by melting to dissociate double-stranded DNA. The temperature range for melting was set at ± 10°C of the melting temperature of each amplicon with a rise in temperature of 0.05°C/s. HRM analysis was performed using Rotor-Gene 6000 Series Software 1.7 (Qiagen). Fragments displaying aberrant melt curves were sequenced to determine potential underlying genetic variations. For sequencing reactions, we utilized larger amplicons than those generated during HRM curve analysis (Additional file 1 ). Sequencing was carried out in 10-μL reactions, which included 1 μL of 10 × PCR buffer (Applied Biosystems), a 3 mM final concentration of MgCl2 (Applied Biosystems), a 100 μM final concentration of dNTP (Bioline), a 200 nM concentration of each primer (Geneworks), 0.25 U of AmpliTaq Gold (Applied Biosystems) and 3 μL of Q solution (Qiagen). PCR products were purified and analyzed on a 3130xl Genetic Analyser (Applied Biosystems) and the results were viewed using Chromas 1.45 (Technelusium, Tewantin, Queensland, Australia).
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BLOOD
Buffers
Deoxyribonucleotides
DNA, Double-Stranded
Genes
Genetic Diversity
Genome
Germ-Line Mutation
Gold
Introns
Magnesium Chloride
Malignant Neoplasm of Breast
Oligonucleotide Primers
PALB2 protein, human
Polymerase Chain Reaction
Reproduction
triphosphate
Woman
The prokaryotic 16S rRNA (V3-V4 region) [20 (link)] was amplified from directly frozen samples and from material after culture in thioglycollate broth using V3-V4 primers as presented in Table 1 . After amplification, the PCR products were purified individually using a clean-up kit (Gene JET PCR Purification kit, Thermo Scientific). Quality and concentration of amplified DNA were checked using gel electrophoresis. PCR products from individual samples were pooled by group and stored at −80°C until analysis.
Attempts to amplify the V3-V4 region directly from DNA from frozen liver were unsuccessful. However, three of the frozen samples yielded a band when amplified using Helicobacter genus-specific primers (C97 and C98). These PCR products, when re-amplified using the V3-V4 region primers yielded new bands (Fig 1 , S2 Fig ). Helicobacter pylori DNA (LMG 8775 DMST 20165 type strain) was obtained from the National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Thailand and was used as a positive control. PCR amplification was performed with a thermal cycler and an Expand High-Fidelity PCR system (Bio Rad C100™thermal cycler). Each reaction (20 μl) contained 1× Expand High-Fidelity buffer, 1U Platinum Taq DNA polymerase, a 5 μM concentration of primers, a 10 mM concentration of each deoxyribonucleotide triphosphate, and 50 mM MgCl2. Amplification conditions are shown in Table 1 .
Liver samples yielding a positive PCR result for the genus Helicobacter were further investigated for the presence of H. pylori using species-specific ureA gene primers following the protocol inTable 1 .
Attempts to amplify the V3-V4 region directly from DNA from frozen liver were unsuccessful. However, three of the frozen samples yielded a band when amplified using Helicobacter genus-specific primers (C97 and C98). These PCR products, when re-amplified using the V3-V4 region primers yielded new bands (
Liver samples yielding a positive PCR result for the genus Helicobacter were further investigated for the presence of H. pylori using species-specific ureA gene primers following the protocol in
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Buffers
Deoxyribonucleotides
Electrophoresis
Freezing
Genes
Helicobacter
Helicobacter pylori
Liver
Magnesium Chloride
Oligonucleotide Primers
Platinum
Prokaryotic Cells
RNA, Ribosomal, 16S
Strains
Taq Polymerase
Thioglycolates
triphosphate
Urea
Most recents protocols related to «Deoxyribonucleotides»
In this study, after morphological identification of the field-collected D. nuttalli, we randomly selected five females and males to extract DNA for molecular identification of ticks. And for the first-laboratory generation, we also randomly selected five males and five females for molecular identification. DNA from the field-collected and first-laboratory generation adult males and females was extracted using the QIAamp DNA Mini Kit (QIAGEN, Germany) according to the manufacturer's manual. The DNA concentration was determined using a NanoDrop 2000 (Thermo Fisher Scientific, USA), and the DNA was stored at−80°C until further use. To further confirm the tick species, PCRs based on the 12S ribosomal RNA (12S rRNA), 16S ribosomal RNA (16S rRNA), cytochrome c oxidase subunit I (COI), and internal transcribed spacer 2 (ITS2) genes were conducted, respectively. All primers are listed in Table 1 . The PCR assay volume was 10 μl including 2 μl of DNA, 0.5 μl of each forward and reverse primer (100 μM), 0.2 μl of deoxyribonucleotide triphosphate (200 μM, New England BioLab, USA), 1 μl of 10× ThermoPol Reaction Buffer (New England BioLab, USA), 0.1 μl of Taq polymerase (0.5 U, New England BioLab, USA) and enough double-distilled water to reach a final volume of 10 μl. Double-distilled water was used as a negative control and D. nuttalli DNA stored in our laboratory was used as a positive control.
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Adult
Biological Assay
Buffers
Deoxyribonucleotides
Females
Genes
Males
Oligonucleotide Primers
Oxidase, Cytochrome-c
Protein Subunits
RNA, ribosomal, 12S
RNA, Ribosomal, 16S
Taq Polymerase
Ticks
triphosphate
Cells were cultured in 6-well plates to ~85% confluence and washed with 2 mL ice cold 1X Phosphate-Buffered Saline (PBS). The cells were then harvested in 300 µL freezing 80% acetonitrile (v/v) into 1.5 mL tubes and lysed by Bullet Blender (Next Advance) at 4 °C followed by centrifugation at 21,000 × g for 5 min at 4 °C. The supernatant was dried by speedvac and reconstituted in 7.5 µL of 66% acetonitrile and 2 µL was separated by a ZIC‐HILIC column (150 × 2.1 mm, EMD Millipore) coupled with a Q Exactive HF Orbitrap MS (Thermo Fisher) in negative detection mode. Metabolites were eluted within a 45 min gradient (buffer A: 10 mM ammonium acetate in 90% acetonitrile, pH = 8; buffer B: 10 mM ammonium acetate in 100% H2O, pH = 8). The MS was operated by a full scan method followed by targeted selected ion monitoring and data-dependent MS/MS (tSIM/dd-MS2). MS settings included full scan (120,000 resolution, 350–550 m/z, 3 × 106 AGC and 50 ms maximal ion time), tSIM scan (120,000 resolution, 1 × 105 AGC, 4 m/z isolation window and 50 ms maximal ion time) and data-dependent MS2 scan (30,000 resolution, 2 × 105 AGC, ~50 ms maximal ion time, HCD, Stepped NCE (50, 100, 150), and 10 s dynamic exclusion). Data were quantified using Xcalibur software (Thermo Fisher Scientific) and normalized by cell numbers. Ribonucleotide and deoxyribonucleotides were validated by authentic standards.
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1-(trimethylsilyl)-1H-imidazole
acetonitrile
ammonium acetate
Buffers
Cells
Centrifugation
Cold Temperature
Deoxyribonucleotides
isolation
Phosphates
Radionuclide Imaging
Ribonucleotides
Saline Solution
Tandem Mass Spectrometry
Strains of Sis/pSeSD and Sis/pSeSD-aCcr1 were cultured in ATV medium under the conditions as described above. For transcriptomic analysis, culture was inoculated with an initial OD600 of 0.05. The cells were pelleted at 6000 g for 10 min after 12 h of cultivation when the OD600 reached approximately 0.2. The pellet was resuspended in 1 ml PBS buffer. The cells were pelleted again and stored at −80°C. Total RNA was extracted using the Trizol reagent (Ambion, Austin, TX, USA). Total amounts and the integrity of RNA were assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Transcriptomic analysis was performed by Novogene (Beijing, China). About 3 μg of high-quality RNA per sample was used for the construction of RNA-Seq libraries. Firstly, mRNA was purified from the total RNA by depleting the rRNA using the biotin-labelled probes against rRNA. First strand cDNA was synthesized using random hexamer primer and the M-MuLV reverse transcriptase. Then, RNaseH was used to degrade the template RNA. For the second strand of cDNA synthesis by DNA polymerase I, dUTP was used to replace the dTTP in the dNTP mixture of deoxyribonucleotides. The remaining overhangs were converted into blunt ends via the exonuclease/polymerase activities. After adenylation of 3′ ends of DNA fragments, adaptors with hairpin loop structures were ligated for hybridization. Then, the USER enzyme was used to degrade the second dU-containing strand of cDNA. In order to select cDNA fragments of preferentially 370–420 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). After PCR amplification, the product was purified by AMPure XP beads to obtain the libraries, which were sequenced using the Illumina NovaSeq 6000. Clean reads were aligned to the reference genome sequence of S. islandicus REY15A (31 (link)). The resulting data were then analysed by Fragments Per Kilobase of transcript sequence per Million base pairs sequenced (FPKM) analysis to reveal expression levels of all genes in the S. islandicus genome. Differential genome expression analysis (over-expression of aCcr1 versus empty vector) was performed using the DEGSeq R package. The resulting P-values were adjusted using the Benjamini and Hochberg's approach for controlling the false discovery rate padj < 0.05 and |log2(foldchange)| > 1 were set as the threshold for significantly differential expression. The ranscriptome experiments were performed in three biological repeats.
Anabolism
austin
Biological Assay
Biopharmaceuticals
Biotin
Buffers
cDNA Library
Cells
Cloning Vectors
Crossbreeding
Culture Media
Deoxyribonucleotides
deoxyuridine triphosphate
DNA, Complementary
DNA Polymerase I
Enzymes
Exonuclease
Gene Expression
Gene Expression Profiling
Genome
Moloney Leukemia Virus
Oligonucleotide Primers
Ribosomal RNA
RNA, Messenger
RNA-Directed DNA Polymerase
RNA-Seq
Strains
thymidine 5'-triphosphate
trizol
Total RNA from control or PPRH-transfected cells for 24 h and 48 h was extracted using Trizol Reagent (Life Technologies, Madrid, Spain), following the instructions of the manufacturer. Complementary DNA was synthesized in a 20 µL reaction mixture from 1 µg of total RNA, 0.5 mM of each deoxyribonucleotide triphosphate (dNTP, Epicentre, Madison, USA), 250 ng of random hexamers (Roche, Barcelona, Spain), 10 mM dithiothreitol, 200 units of a Moloney murine leukemia virus reverse transcriptase (RT), 20 units of RNase inhibitor, and 4 µL of buffer (5×) (all three from Lucigen, Middleton, WI, USA). The reaction was incubated at 42 °C for 1 h.
The BIRC5 mRNA TaqMan probe (Hs04194392_s1; ThermoFisher Scientific, Madrid, Spain) was used to determine survivin mRNA levels and the Cyclophilin (PP1A) mRNA TaqMan probe (Hs04194521_s1, ThermoFisher Scientific, Madrid, Spain) was used as the endogenous control. The reaction was conducted in 20 μL containing 1xTaqMan Universal PCR Mastermix (Applied Biosystems, Madrid, Spain), 0.5xTaqMan probe, and 3 μL of cDNA. PCR cycling conditions were 10 min denaturation at 95 °C, followed by 40 cycles of 15 s at 95 °C, and 1 min at 60 °C using a QuantStudio 3 Real-Time PCR System (Applied Biosystems, Barcelona, Spain). The quantification was performed using the ΔΔCt method, where Ct is the threshold cycle that corresponds to the cycle when the amount of amplified mRNA reaches the fluorescence threshold.
The BIRC5 mRNA TaqMan probe (Hs04194392_s1; ThermoFisher Scientific, Madrid, Spain) was used to determine survivin mRNA levels and the Cyclophilin (PP1A) mRNA TaqMan probe (Hs04194521_s1, ThermoFisher Scientific, Madrid, Spain) was used as the endogenous control. The reaction was conducted in 20 μL containing 1xTaqMan Universal PCR Mastermix (Applied Biosystems, Madrid, Spain), 0.5xTaqMan probe, and 3 μL of cDNA. PCR cycling conditions were 10 min denaturation at 95 °C, followed by 40 cycles of 15 s at 95 °C, and 1 min at 60 °C using a QuantStudio 3 Real-Time PCR System (Applied Biosystems, Barcelona, Spain). The quantification was performed using the ΔΔCt method, where Ct is the threshold cycle that corresponds to the cycle when the amount of amplified mRNA reaches the fluorescence threshold.
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BIRC5 protein, human
Buffers
Cells
Deoxyribonucleotides
Dithiothreitol
DNA, Complementary
Endoribonucleases
Fluorescence
Moloney Leukemia Virus
Peptidylprolyl Isomerase
RNA, Messenger
RNA-Directed DNA Polymerase
Survivin
triphosphate
trizol
All samples were analyzed in order to determine DNA content and to sequence two mtDNA markers: the control region (CR) and the cytochrome c oxidase subunit I (COI).
Genomic DNA was extracted both from muscle and hepatopancreas tissues using the Wizard® Genomic DNA Purification Kit (Promega Corporation, Madison, WI, USA) and DNeasy® Tissue Kit di Qiagen (Hilden, Germany, EU) and a good quality of DNA was obtained from both.
The primers used for mtDNA control-region amplification were previously published [34 (link)], and the reaction conditions were as follows: initial denaturation at 95 °C for 2 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 1 min and elongation at 72 °C for 1 min, followed by final extension at 72 °C for 10 min. Besides, a fragment of the mitochondrial gene coding for the cytochrome c oxidase subunit I was amplified using the primers published by Folmer et al., 1994 [40 (link)].
The PCR reactions contained 1X Buffer GoTaq, 2.5 mM of each deoxyribonucleotide triphosphate (dNTP), 0.3 µM of each primer, 0.03 U/µL of GoTaq DNA polymerase (Promega Corporation; Madison, WI, USA), 30 ng of genomic DNA and H2O to a final volume of 25 µL. The PCR amplification was carried out as follows: 94 °C for 2 min, followed by 35 cycles of 95 °C for 30 s, 55 °C for 1 min, 72 °C for 1 min and then 72 °C for 10 min. The PCR fragments were purified using exonuclease I and alkaline phosphatase (ExoSAP-IT enzymatic system-USB Corporation, Cleveland, OH, USA) and subsequently Sanger-sequenced with the following primers: LCO1490 and HCO2198 [40 (link)] for COI sequencing and a specifically designed primer (5′-CTTCTAAAAATGTTCCCCCC-3′) for CR region, by using Primer3 software. Sequences were aligned to the sequence considered as reference mitogenome (JN991197, [41 (link)]) for the haplotype (HT) annotation through the Sequencher software (www.genecodes.com (accessed on 12 August 2022)). In order to ensure the uniformity of all sequences, they were trimmed at the same range, from the nucleotide position (np) 4717 to np 5434 for the mtDNA CR marker, and from np 78 to np 651 for the COI region.
The COI region was investigated in 53 samples, selecting at least 2 samples for each CR HT from each basin.
Mitochondrial DNA sequence variation parameters were estimated by using DnaSP 5.1 software (www.ub.edu/dnasp/index_v5.html (accessed on 12 August 2022)). Haplotype number codes were assigned to each sample as previously classified in Dörr et al., 2021 [35 (link)] and with increasing numbering only for our convenience.
The evolutionary relationships among the haplotypes here identified were evaluated for each single mtDNA marker through median-joining trees built using Network software v.10.2.
In order to graphically display and summarize the mitogenetic relationships among the analyzed populations, a Principal Component Analysis (PCA) based on concatenated mtDNA (COI and CR) haplotypes was performed using the Excel software implemented by XLSTAT. Haplotype frequencies were used as input data.
Finally, to construct a Bayesian phylogeny for the two combined mtDNA regions, we used BEAST.v2.7.3 and the analyses were run for 100,000,000 generations [42 (link)].
Genomic DNA was extracted both from muscle and hepatopancreas tissues using the Wizard® Genomic DNA Purification Kit (Promega Corporation, Madison, WI, USA) and DNeasy® Tissue Kit di Qiagen (Hilden, Germany, EU) and a good quality of DNA was obtained from both.
The primers used for mtDNA control-region amplification were previously published [34 (link)], and the reaction conditions were as follows: initial denaturation at 95 °C for 2 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 1 min and elongation at 72 °C for 1 min, followed by final extension at 72 °C for 10 min. Besides, a fragment of the mitochondrial gene coding for the cytochrome c oxidase subunit I was amplified using the primers published by Folmer et al., 1994 [40 (link)].
The PCR reactions contained 1X Buffer GoTaq, 2.5 mM of each deoxyribonucleotide triphosphate (dNTP), 0.3 µM of each primer, 0.03 U/µL of GoTaq DNA polymerase (Promega Corporation; Madison, WI, USA), 30 ng of genomic DNA and H2O to a final volume of 25 µL. The PCR amplification was carried out as follows: 94 °C for 2 min, followed by 35 cycles of 95 °C for 30 s, 55 °C for 1 min, 72 °C for 1 min and then 72 °C for 10 min. The PCR fragments were purified using exonuclease I and alkaline phosphatase (ExoSAP-IT enzymatic system-USB Corporation, Cleveland, OH, USA) and subsequently Sanger-sequenced with the following primers: LCO1490 and HCO2198 [40 (link)] for COI sequencing and a specifically designed primer (5′-CTTCTAAAAATGTTCCCCCC-3′) for CR region, by using Primer3 software. Sequences were aligned to the sequence considered as reference mitogenome (JN991197, [41 (link)]) for the haplotype (HT) annotation through the Sequencher software (
The COI region was investigated in 53 samples, selecting at least 2 samples for each CR HT from each basin.
Mitochondrial DNA sequence variation parameters were estimated by using DnaSP 5.1 software (
The evolutionary relationships among the haplotypes here identified were evaluated for each single mtDNA marker through median-joining trees built using Network software v.10.2.
In order to graphically display and summarize the mitogenetic relationships among the analyzed populations, a Principal Component Analysis (PCA) based on concatenated mtDNA (COI and CR) haplotypes was performed using the Excel software implemented by XLSTAT. Haplotype frequencies were used as input data.
Finally, to construct a Bayesian phylogeny for the two combined mtDNA regions, we used BEAST.v2.7.3 and the analyses were run for 100,000,000 generations [42 (link)].
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Adjustment Disorders
Alkaline Phosphatase
Biological Evolution
Buffers
Deoxyribonucleotides
DNA, A-Form
DNA, Mitochondrial
DNA-Directed DNA Polymerase
Enzymes
EXO1 protein, human
Genes, Mitochondrial
Genetic Diversity
Genome
Haplotypes
Hepatopancreas
Mitogens
Muscle Tissue
Nucleotides
Oligonucleotide Primers
Oxidase, Cytochrome-c
Population Group
Promega
Protein Subunits
Tissues
Trees
triphosphate
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TRIzol reagent is a monophasic solution of phenol, guanidine isothiocyanate, and other proprietary components designed for the isolation of total RNA, DNA, and proteins from a variety of biological samples. The reagent maintains the integrity of the RNA while disrupting cells and dissolving cell components.
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Taq DNA polymerase is a thermostable enzyme used for DNA amplification in Polymerase Chain Reaction (PCR) applications. It is isolated from the thermophilic bacterium Thermus aquaticus, and its core function is to catalyze the synthesis of new DNA strands complementary to a template DNA sequence.
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TRIzol is a monophasic solution of phenol and guanidine isothiocyanate that is used for the isolation of total RNA from various biological samples. It is a reagent designed to facilitate the disruption of cells and the subsequent isolation of RNA.
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The In Situ Cell Death Detection Kit is a laboratory product designed for the detection of programmed cell death, or apoptosis, in cell samples. The kit utilizes a terminal deoxynucleotidyl transferase (TdT) to label DNA strand breaks, allowing for the visualization and quantification of cell death. The core function of this product is to provide researchers with a tool to study and analyze cell death processes.
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GoTaq DNA polymerase is a thermostable DNA polymerase enzyme used for DNA amplification. It catalyzes the polymerization of nucleotides into DNA strands in the presence of a DNA template and primers.
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The RNeasy Mini Kit is a laboratory equipment designed for the purification of total RNA from a variety of sample types, including animal cells, tissues, and other biological materials. The kit utilizes a silica-based membrane technology to selectively bind and isolate RNA molecules, allowing for efficient extraction and recovery of high-quality RNA.
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Deoxyribonucleotide triphosphate is a fundamental building block for DNA synthesis. It consists of a deoxyribose sugar, a phosphate group, and one of four nitrogenous bases: adenine, guanine, cytosine, or thymine. These molecules serve as the primary substrates for DNA polymerase enzymes, enabling the replication and repair of genetic material.
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Moloney murine leukemia virus reverse transcriptase is an enzyme that catalyzes the conversion of single-stranded RNA into double-stranded DNA.
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The G-25 spin columns are a lab equipment product designed for the purification and separation of biomolecules, such as proteins, nucleic acids, and small molecules. The columns are filled with a gel-filtration matrix that allows for the rapid removal of unwanted salts, buffers, and other small molecules from the sample. The sample is loaded onto the column, and the centrifugation process separates the biomolecules of interest from the unwanted components.
More about "Deoxyribonucleotides"
Deoxyribonucleotides, also known as dNTPs, are the fundamental building blocks of DNA, the essential genetic material for all cellular life.
These molecules consist of a deoxyribose sugar, a phosphate group, and one of four nitrogenous bases: adenine (A), guanine (G), cytosine (C), or thymine (T).
Deoxyribonucleotides play a crucial role in DNA replication, transcription, and repair processes, enabling the storage, transmission, and expression of genetic information.
Understanding the properties and behaviors of deoxyribonucleotides is essential for advancing research in fields such as molecular biology, genetics, and biotechnology.
Techniques like PCR (Polymerase Chain Reaction) and RT-PCR (Reverse Transcription-PCR) utilize dNTPs and enzymes like Taq DNA polymerase and Moloney murine leukemia virus reverse transcriptase to amplify and analyze DNA and RNA sequences.
Sample preparation methods, such as using TRIzol reagent and the RNeasy Mini Kit, can be optimized to ensure the quality and integrity of nucleic acids, including deoxyribonucleotides.
Moreover, instruments like the Agilent 2100 Bioanalyzer can provide detailed analysis of deoxyribonucleotide-related samples, aiding in the development of more reproducible and accurate research protocols.
PubCompare.ai's AI-powered comparisons can help identify the most reproducible and accurate methods for working with deoxyribonucleotides, allowing researchers to optimize their protocols and advance their investigations more efficiently.
By leveraging insights from published literature, pre-prints, and patents, PubCompare.ai can assist in locating the best products and procedures, such as the use of G-25 spin columns, to enhance deoxyribonucleotide-related research.
These molecules consist of a deoxyribose sugar, a phosphate group, and one of four nitrogenous bases: adenine (A), guanine (G), cytosine (C), or thymine (T).
Deoxyribonucleotides play a crucial role in DNA replication, transcription, and repair processes, enabling the storage, transmission, and expression of genetic information.
Understanding the properties and behaviors of deoxyribonucleotides is essential for advancing research in fields such as molecular biology, genetics, and biotechnology.
Techniques like PCR (Polymerase Chain Reaction) and RT-PCR (Reverse Transcription-PCR) utilize dNTPs and enzymes like Taq DNA polymerase and Moloney murine leukemia virus reverse transcriptase to amplify and analyze DNA and RNA sequences.
Sample preparation methods, such as using TRIzol reagent and the RNeasy Mini Kit, can be optimized to ensure the quality and integrity of nucleic acids, including deoxyribonucleotides.
Moreover, instruments like the Agilent 2100 Bioanalyzer can provide detailed analysis of deoxyribonucleotide-related samples, aiding in the development of more reproducible and accurate research protocols.
PubCompare.ai's AI-powered comparisons can help identify the most reproducible and accurate methods for working with deoxyribonucleotides, allowing researchers to optimize their protocols and advance their investigations more efficiently.
By leveraging insights from published literature, pre-prints, and patents, PubCompare.ai can assist in locating the best products and procedures, such as the use of G-25 spin columns, to enhance deoxyribonucleotide-related research.