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Genomics and Bionanotechnology
Published in Anil Kumar Anal, Bionanotechnology, 2018
The classical technique is convenient in the presence of well-defined restriction site in the sequence. On the other hand, this approach is not suitable in case of absence of restriction site and most of the eukaryotic genes are interrupted by intervening sequences (introns), which makes the gene of interest very large. As manipulation of the large genomic DNA is difficult due to size capacity of cloning vectors and multiple restriction endonucleases, alternative cloning approaches such as PCR-mediated TA cloning, ligation-independent cloning (LIC), recombinase-dependent cloning, and PCR-mediated cloning are being developed (Souii et al. 2013). In PCR-mediated TA cloning method, DNA fragment generated from PCR is directly stitched with vector without the use of any restriction enzymes. As DNA fragments with adenine (A) residue known as A-tailed DNA fragment and vectors with thymine (T) residue known as T-tailed are directly ligated, this approach is known as TA cloning.LIC is done by adding short sequence of DNA to the insert DNA, which is homologous to the vector. Complementary cohesive ends between the insert and vector are formed by the enzymes with 3′–5′ exonuclease activity and the resulting two molecules are annealed together such that plasmid will have four single-stranded DNA nicks.The seamless cloning method allows sequence-independent and scar-free, that is, free of restriction enzyme sites and unwanted sequence, insertion of DNA insert into vector. This method includes addition of homologous region at each end of insert to be cloned and with action of enzyme exonuclease, DNA polymerase and ligase-recombinant DNA are formed.Recombinatorial cloning technique utilizes site-specific DNA recombinase enzymes that can swap pieces of DNA between two molecules with appropriate recombination site. Initially, the recombination sites are added on either side of insert by PCR followed by recombination with vector to make an entry clone, which is further recombined with destination vector to develop final construct. Gateway cloning system is one of the widely used cloning techniques based on this approach (Bertero et al. 2017).
Biodegradation of polycyclic aromatic hydrocarbons (PAHs) by microbial consortium from paddy rice soil
Published in Journal of Environmental Science and Health, Part A, 2023
Hernando P. Bacosa, Genese Divine B. Cayabo, Chihiro Inoue
Nearly full-length fragment of the 16S rRNA gene was PCR amplified by primers 10 F and 1500 R using PCR Master Mix (Promega, USA).[20] The PCR amplicons were cloned into the pCR 2.1 TOPO vector following the TOPO TA Cloning Kit protocol (Invitrogen, CA, USA). Ligation products were transformed into competent TOP10 E. coli cells. The positive clones were picked, the plasmids were extracted using MagExtractor Plasmid Purification Kit in the MFX-2000 Nucleic Acid Purification System (Toyobo, Japan), and sequenced in 3130 Genetic Analyzer (Applied Biosystems Inc.) using BigDye Terminator v3.1 chemistry.[19,21,26]
Enhancement of heavy metal tolerance and accumulation efficiency by expressing Arabidopsis ATP sulfurylase gene in alfalfa
Published in International Journal of Phytoremediation, 2019
V. Kumar, S. AlMomin, A. Al-Shatti, H. Al-Aqeel, F. Al-Salameen, A. B. Shajan, S. M. Nair
The Arabidopsis seeds were incubated at 4 °C in the dark for 4 days. Following the incubation period, they were surface-sterilized and germinated in magenta boxes containing 40 ml of pre-sterilized, half-strength Murashige and Skoog (MS) medium (Murashige and Skoog 1962) of pH 5.8, prepared with 2% sucrose and solidified with 0.7% agar. The seeds were incubated in a plant growth chamber set at 22 °C and 16:8 h light regime. For the induction of heavy metal responsive genes, the seedlings were removed carefully from the solid medium and transferred to a medium containing 10 µM and 100 µM concentrations of lead nitrate or sodium vanadate and incubated for 24 h. Five seedlings from each treatment were pooled, and the total RNA was isolated using the total plant RNA kit (Sigma, St. Louis, MO, USA), according to the manufacturer's instructions. The isolated RNA samples were converted to cDNA using Taqman Reverse Transcriptase Reagents (Applied Biosystems-Grand Island, NY, USA), following the protocol provided by the manufacturer. The reaction consisted of 1 μl 10X RT buffer, 2.2 μl 25 mM MgCl2, 2 μl dNTPs mixture, 0.5 μl oligo dT, 0.2 μl RNase Inhibitor, 0.25 μl multiscribe reverse transcriptase, and 400 ng of total RNA for each sample, in 10 μl final volume. The reaction was performed with the following cycle profile: 25 °C for 10 min, 48 °C for 30 min, 95 °C for 5 min, and 4 °C for infinity. The cDNA obtained was subjected to PCR with primer pair APS2F and APS2R (Supporting Table S1). The PCR reactions consisted of ∼40 ng cDNA, 1 µl each of APS2 forward and reverseprimers, 0.5 µl DNA polymerase enzyme, 2 µl MgCl2, 2.5 µl dNTPs, 2.5 µl PCR buffer, and 14.5 µl water. The reaction was carried out at 50 °C annealing temperature for 35 cycles. The PCR products were run on agarose gel and documented using a gel documentation system. The PCR products were purified and the DNA samples were then ligated into a PCR2.1 vector following the protocol provided in the instruction manual of the TOPO TA Cloning Kit (Thermo Fisher Scientific, Waltham, MA USA). The ligated vectors were transfected into TOP10 chemically competent E. coli cells, using the CaCl2 mediated transformation method (Sambrook et al. 1989).