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Principles and Techniques for Deoxyribonucleic Acid (DNA) Manipulation
Published in Hajiya Mairo Inuwa, Ifeoma Maureen Ezeonu, Charles Oluwaseun Adetunji, Emmanuel Olufemi Ekundayo, Abubakar Gidado, Abdulrazak B. Ibrahim, Benjamin Ewa Ubi, Medical Biotechnology, Biopharmaceutics, Forensic Science and Bioinformatics, 2022
Nwadiuto (Diuto) Esiobu, Ifeoma M. Ezeonu, Francisca Nwaokorie
The process of expanding the new DNA strands seems continuous. However, it terminates in two ways; either when there is no more DNA template left to replicate especially at the end of the chromosome or when two replication forks meet at the terminus called terC. On completion of DNA synthesis, it is important to proofread and ascertain that newly synthesized strands are bound and stabilized. In the lagging strands, these activities are performed by two enzymes: RNAase H and DNA Ligase. The enzyme RNAase H catalyzes the removal of the RNA primers present at the beginning of each Okazaki fragment in lagging strands. The new strand is proofread to ensure that there are no mistakes in the new DNA sequence. The DNA polymerase I proofreads the newly formed daughter strands and replaces RNA Primer with sections of DNA. In addition to these enzymes, eukaryotic cells produce telomeres to prevent erosion of genes. On the other hand, DNA Ligase joins two fragments together to create one complete strand. This enzyme seals up the sequence of DNA into two continuous double strands. Thus, the result of DNA replication is two DNA molecules consisting of one new and one old chain of nucleotides. The daughter DNA molecules produced consist of one progeny and one parental strand. This concept defines why DNA replication is described as semi-conservative. That is, one half of the chain is part of the original DNA molecule, while the other half is wholly brand new. When DNA replication is successfully completed, the new DNA automatically winds up into a double helix to give the characteristic feature of DNA (Figure 1.2b).
Effects of glycerol and glucose on docosahexaenoic acid synthesis in Aurantiochyrium limacinum SFD-1502 by transcriptome analysis
Published in Preparative Biochemistry & Biotechnology, 2023
Huaqiu Zhang, Xiangying Zhao, Chen Zhao, Jiaxiang Zhang, Yang Liu, Mingjing Yao, Jianjun Liu
For library construction and sequencing, firstly, mRNA was purified from total RNA by poly-T oligo-attached magnetic beads, followed by a fragmentation step which was carried out by divalent cations under elevated temperature in Illumina proprietary fragmentation buffer. The first strand cDNA was synthesized by random oligonucleotides and Super Script II. Then, the second strand cDNA synthesis was subsequently performed by DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities, and the enzymes were also removed. After the 3′ends of the DNA fragments adenylation, Illumina PE adapter oligonucleotides were ligated to prepare for hybridization. To select cDNA fragments of the preferred 400–500 bp in length, the library fragments were purified by the AMPure XP system (Beckman Coulter, USA). DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 15cycle PCR reaction. Products were purified by AMPure XP system (Beckman Coulter, USA) and quantified by the Agilent high sensitivity DNA assay on a Bioanalyzer 2100 system (Agilent, USA). The sequencing library was sequenced on NovaSeq 6000 platform (Illumina).
Transcriptome analysis reveals that yeast extract inhibits synthesis of prodigiosin by Serratia marcescens SDSPY-136
Published in Preparative Biochemistry & Biotechnology, 2023
Junqing Wang, Tingting Zhang, Yang Liu, Shanshan Wang, Zerun Li, Ping Sun, Hui Xu
After 12 h of cell growth, samples were harvested for transcriptome analysis. Three duplicates were used for the experimental group grown in yeast extract (TR1, TR2, and TR3) and the control group grown in peptone (CK1, CK2, and CK3). TRIzol reagent (Invitrogen Life Technologies, USA) was used to isolate total RNA. To assess the quality and integrity, a NanoDrop spectrophotometer (Thermo Scientific, USA) and a Bioanalyzer 2100 system (Agilent, USA) were applied. The Zymo-Seq RiboFree Total RNA Library Kit (Zymo Research, USA) was used to remove ribosomal RNA (rRNA) from total RNA. The first strand of cDNA was constructed using random oligonucleotides and SuperScript III (Thermo Scientific). Following that, second-strand cDNA synthesis was performed with DNA polymerase I and RNase H (Thermo Scientific). Exonuclease/polymerase operations transformed the remaining overhangs into blunt ends and the enzymes were eliminated. As preparation for hybridization, Illumina PE adapter oligonucleotides were then ligated after the 3′ ends of the DNA fragments and adenylated. AMPure XP technology was employed to purify the library fragments and choose cDNA fragments of the preferred 400–500 bp length (Beckman Coulter, USA). Using the Illumina PCR Primer Cocktail, DNA fragments containing ligated adaptor molecules from both ends were preferentially selected for the 15-cycle PCR reaction. The products were separated using the AMPure XP system and measured using the high-sensitivity DNA assay on a Bioanalyzer 2100 system (Agilent). The sequencing library was then sequenced on NovaSeq 6000 platform (Illumina) by Shanghai Personal Biotechnology Cp. Ltd.