Molecular Cloning – Selected Applications in Medicine and Biology. Edited by Gregory G. Brown. Published by InTech. Janeza Trdine 9, Rijeka, Croatia. Recombinant DNA technology depends on the ability to produce large numbers of identical DNA molecules (clones). • Clones are typically generated by placing . PDF | Molecular cloning is the collection of experimental procedures required to isolate and expand a specific fragment of DNA into a host.

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Molecular Cloning Pdf

Molecular Cloning, A Laboratory Manual, 4th Edition, Cold Spring Harbor Protocols, This is a free sample of. Molecular cloning is a basic technique used in a molecular biology labs. In this manual, we include a protocol for isolating the luciferase gene from. DNA using. Gene Cloning Technology. Also known as: Genetic engineering or Genetic manipulation (GM) technology. – implies precision engineering being applied.

Over the last decades, molecular cloning has transformed biological sciences. Having profoundly impacted various areas such as basic science, clinical, pharmaceutical, and environmental fields, the use of recombinant DNA has successfully started to enter the field of cellular engineering. Here, the polymerase chain reaction PCR represents one of the most essential tools. Due to the emergence of novel and efficient PCR reagents, cloning kits, and software, there is a need for a concise and comprehensive protocol that explains all steps of PCR cloning starting from the primer design, performing PCR, sequencing PCR products, analysis of the sequencing data, and finally the assessment of gene expression. It is the aim of this methodology paper to provide a comprehensive protocol with a viable example for applying PCR in gene cloning. Exemplarily the sequence of the tdTomato fluorescent gene was amplified with PCR primers wherein proper restriction enzyme sites were embedded. Practical criteria for the selection of restriction enzymes and the design of PCR primers are explained. Efficient cloning of PCR products into a plasmid for sequencing and free web-based software for the consecutive analysis of sequencing data is introduced. Finally, confirmation of successful cloning is explained using a fluorescent gene of interest and murine target cells. Using a practical example, comprehensive PCR-based protocol with important tips was introduced. This methodology paper can serve as a roadmap for researchers who want to quickly exploit the power of PCR-cloning but have their main focus on functional in vitro and in vivo aspects of cellular engineering. The online version of this article doi: Various techniques were introduced for assembling new DNA sequences [ 1 — 3 ], yet the use of restriction endonuclease enzymes is the most widely used technique in molecular cloning. Whenever compatible restriction enzyme sites are available on both, insert and vector DNA sequences, cloning is straightforward; however, if restriction sites are incompatible or if there is even no restriction site available in the vicinity of the insert cassette, cloning might become more complex. The use of PCR primers, in which compatible restriction enzyme sites are embedded, can effectively solve this problem and facilitate multistep cloning procedures.

Kinetics Parameters Kinetic studies of Amy were determined under standard conditions using different concentrations of soluble starch 0. As obtained from the Lineweaver-Burk plot, the and values were 2.

SH3 2. In Figure 7 , Amy displayed the highest specificity towards amylose Amy displayed the highest rate of hydrolysis towards pea starch Amy showed different hydrolysis abilities towards various starches, due to the difference in particle size and shape, the ratio of amylose and amylopectin, and structure of the amylose and amylopectin molecules [ 40 ].

The result of oligosaccharides hydrolysis showed that the enzyme could not degrade other oligosaccharides except maltooligosaccharides and no glucose was observed even after incubation for 48 h.

In addition, the enzyme was not able to hydrolyze pNPG. Figure 7: Substrate specificity of purified Amy The hydrolysis products of soluble starch analyzed by TLC for various periods were shown in Figure 8 a. At the early stage of hydrolysis, soluble starch was hydrolyzed into maltose G2 , maltotriose G3 , maltotetraose G4 , and a small amount of higher molecular weight oligosaccharides.

As the reaction proceeded, glucose G1 gradually increased; however, G2 was still the main hydrolysis product after incubation for 48 h. Figure 8: TLC analysis of product formation during degradation of soluble starch and maltooligosaccharides.

Lane M, maltooligosaccharide standards G1 glucose, G2 maltose, G3 maltotriose, G4 maltotetraose, and G5 maltopentaose. Lanes the end products of G2 for 1 h, 24 h, and 48 h; Lanes the end products of G3 for 1 h, 24 h, and 48 h; Lanes the end products of G4 for 1 h, 24 h, and 48 h, respectively. To determine whether Amy could perform transglycosylation, the hydrolysis products of maltooligosaccharides were analyzed by TLC Figure 8 b.

Amy released a small amount of glucose from maltose at 1 h. When maltotriose and maltotetraose were degraded, Amy also produced oligosaccharides that were one-glucose unit smaller than the substrates. It is noteworthy that the oligosaccharides that were larger than the original substrates were produced by Amy, especially when maltotriose was hydrolyzed for 1 h.

The results suggested that Amy possessed transglycosylation activity. Detergency Characteristics To confirm the application of Amy in detergent formulations, its stability was investigated in presence of various commercial detergents Figure 9.

The result revealed that Amy exhibited extreme stability with all the tested commercial laundry detergents and more than Figure 9: Detergent compatibility study of the purified Amy Although the detergent alone showed fainted washing effect on chocolate and tomato sauce spots starch rich , addition of Amy with This result indicated that Amy could be employed as potential laundry detergent additive.

Figure Wash performance analysis of Amy, the amylase from Pseudoalteromonas sp. Rows: T: clothes stained with tomato sauce; Lane C: clothes stained with chocolate.

M was cloned, expressed, and characterized.

Amy had seven highly conserved regions and the putative catalytic triad. Furthermore, the mammalian amino acid transporter also contained the oligo-1,6-glucosidase-type of QPDLN [ 12 ].

Its coverage value 0.

BioMed Research International

However, its sequence identity with Amy was only On the other hand, this trehalose synthase was described as a homotetramer, while the result of native-PAGE indicated that Amy should be a monomer. The result that Amy degraded the soluble starch to several maltooligosaccharides suggests that Amy mainly hydrolyze starch internally Figure 8 a.

Moreover, Amy possessed transglycosylation activity. Furthermore, Amy could still keep Cold-adapted enzymes can carry out their functions at very low temperature because of their flexible structures [ 22 ].

Arginine is famous as a stabilizing residue and can reduce the flexibility by forming hydrogen bands and salt bridges with the guanidinium group [ 42 , 43 ]. Another noticeable characteristic of Amy was its salt-tolerance. Amy exhibited the activity in a wide range of M NaCl with the highest activity in the presence of 1 M NaCl EMB8 [ 30 ] and Halorubrum xinjiangense [ 31 ].

Some researches showed that the hydrophobic interactions of enzyme core structures were possibly enhanced by salting-out effect under high salinity and made enzymes more compact and stable [ 26 ], which might be helpful to enhance the stability of these enzymes. An abundance of acidic amino acids produces a negative surface potential, promoting the formation of the hydrated salt ions network that reduces the tendency of aggregation and keeps the enzyme activity and stability under high salinity [ 48 — 50 ].

The proportion of acidic amino acid excess of Amy is 5. In addition, Amy was predicted to be an extracellular enzyme with N-terminal signal peptide of 23 amino acids. Qin et al.

Additionally, effects of various metal ions and chemical reagents on enzyme activity were studied Tables 2 and 3. These metal ions may inhibit the enzyme activity by either binding to catalytic residues or replacing the required metal ions [ 53 ]. The enzyme resistance towards SDS is a good characteristic, particularly in detergent industry, and SDS-stable amylases have been rarely reported [ 59 ]. Furthermore, Amy exhibited the tolerance to other chemical reagents and could keep more than Arikan reported that some alkaline amylases were unaffected by chelator EDTA [ 63 ].

It is probable that some metal ions can activate Amy, but they are not essential for the catalytic reaction process. Amy demonstrated not only good tolerance towards some chemical reagents, but also excellent stability against all the tested commercial detergents. Therefore, the residual amylase activity is the result of combining effects of different ingredients in the detergents. Furthermore, the addition of Amy led to better stain removal from cotton fabrics than that of detergent and water alone.

The amylases can help to enhance wash performance by effectively breaking down starch rich stains, protect the environment due to the biodegradability of enzymes, and make laundry detergent more sustainable [ 20 ]. All above results suggested that Amy could be added to laundry detergent formulations, for enhancing the ability of detergents to clean clothes in cold water. M, was expressed and purified. With the global trend of using low temperature processing to save energy, cold-active amylases such as Amy would be a promising enzyme candidate to be used for industries such as food, detergent, and textile.

Data Availability The data used to support the findings of this study are available from the corresponding author upon request. Conflicts of Interest The authors declare that there are no conflicts of interest regarding this study. Cuijuan Shi and Qiuju xie contributed to the statistical analysis. Guangfeng Kan and Xiulian Ren participated in the study design.

Geng Yu and Michael Betenbaugh helped to draft the manuscript. All authors read and approved the final manuscript. References R. Gupta, P. Gigras, H. Mohapatra, V. Goswami, and B. Rajagopalan and C. Reddy, A. Nimmagadda, and K. View at Google Scholar A.

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Charlton, H. Ertan, S.

DNA Cloning with Plasmid Vectors - Molecular Cell Biology - NCBI Bookshelf

Omar, K. Siddiqui, and T. Santiago, C. Zamora, and L.

New York: W. Freeman ; Search term Section 7. In the case of DNA , this is feasible for relatively short molecules such as the genomes of small viruses. But genomes of even the simplest cells are much too large to directly analyze in detail at the molecular level.

The problem is compounded for complex organisms. Cleavage of human DNA with restriction enzymes that produce about one cut for every base pairs yields some 2 million fragments, far too many to separate from each other directly. With these methods virtually any gene can be purified, its sequence determined, and the functional regions of the sequence explored by altering it in planned ways and reintroducing the DNA into cells and into whole organisms.

Molecular cloning using polymerase chain reaction, an educational guide for cellular engineering

When a single recombinant DNA molecule, composed of a vector plus an inserted DNA fragment, is introduced into a host cell, the inserted DNA is reproduced along with the vector, producing large numbers of recombinant DNA molecules that include the fragment of DNA originally linked to the vector.

Two types of vectors are most commonly used: E. In this section, the general procedure for cloning DNA fragments in E. These extrachromosomal DNAs, which occur naturally in bacteria, yeast, and some higher eukaryotic cells, exist in a parasitic or symbiotic relationship with their host cell.

Plasmids range in size from a few thousand base pairs to more than kilobases kb. During cell division, at least one copy of the plasmid DNA is segregated to each daughter cell, assuring continued propagation of the plasmid through successive generations of the host cell. For example, some bacterial plasmids encode enzymes that inactivate antibiotics.

Such drug-resistance plasmids have become a major problem in the treatment of a number of common bacterial pathogens. As antibiotic use became widespread, plasmids containing several drug-resistance genes evolved, making their host cells resistant to a variety of different antibiotics simultaneously.

Such transfer can result in the rapid spread of drug-resistance plasmids, expanding the number of antibiotic-resistant bacteria in an environment such as a hospital. Coping with the spread of drug-resistance plasmids is an important challenge for modern medicine. Generally, these plasmids have been engineered to optimize their use as vectors in DNA cloning. Most plasmid vectors contain little more than the essential nucleotide sequences required for their use in DNA cloning: a replication origin , a drug-resistance gene , and a region in which exogenous DNA fragments can be inserted Figure Diagram of a simple cloning vector derived from a plasmid, a circular, double-stranded DNA molecule that can replicate within an E.

Host-cell enzymes bind to ORI, initiating replication of the circular plasmid. Plasmid DNA replication. The parental strands are shown in blue, and newly synthesized daughter strands are shown in red. Once DNA replication more Selection of Transformed Cells In , O.

Avery, C.

Molecular cloning using polymerase chain reaction, an educational guide for cellular engineering

Macleod, and M. This process involved the genetic alteration of a bacterial cell by the uptake of DNA isolated from a genetically different bacterium and its recombination with the host-cell genome.

Their experiments provided the first evidence that DNA is the genetic material. Later studies showed that such genetic alteration of a recipient cell can result from the uptake of exogenous extrachromosomal DNA e.

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