U10 QUESTIONS
- DNA cloning is the technique in molecular biology which reproduces many identical pieces of DNA, e.g., genes. The process entails the insertion of the target gene into a circular piece of DNA known as the plasmid. The plasmid gets introduced into the bacteria’susing the transformation process. Thebacteria’s that carry the plasmid are then identified and categorized using antibiotics. Thebacteria that are identified to have the right amount of plasmid are then used to make more and more plasmid DNA and, in some instances, they are used to make proteins or to express the gene (Zhang et al. 55).
Theword clone means to make an exact copy of something genetically. Cloning doesn’t entail making the whole organism but copying some bits of something and not the whole thing. DNA cloning is the process of making multiple andsimilar copies of a specific piece of DNA. The first step in the procedure of DNA cloning is the insertion of the important human protein gene into the circular piece of DNA referred to as the plasmid (Zhang et al. 55). The gene is inserted into the plasmid using enzymes that replicate the DNA. The insertion triggers the production of Recombinant DNA molecules.
The recombinant plasmid is then introduced into the bacteria. Thebacteria that carry the plasmid are selected and grown. As these bacteriago on reproducing, they go forth to replicate the plasmid and later on pass on the plasmid to their offspring’s, hence making the copies of the DNA. The steps involved in DNA cloning include the cut and paste of DNA. The DNA of multiple sources may be joined together using two enzymes, namely the restriction enzymes and the DNA ligase enzymes. Restriction enzymes are DNA-cutting enzymes that recognize a certain target sequence and proceed to cut the DNA into two pieces (Zhang et al. 55). A lot of restriction enzymes produce cut ends that have short and single-stranded overhangs. In cases where we have two molecules with matching, overhangs may stick and join together. The union of the two molecules doesn’t stick together for long until the DNA ligase is used to seal the gaps located in the DNA backbone.
The main aim of cloning being the insertion of the target viable human gene into the plasmid. The plasmid is cut singly and the target gene that has a cut site near each ending. The fragments are combined by the DNA ligase, which links the two into forming recombinant plasmid, which harbors the gene. Step two is the bacteria transformation and bacteria selection. The plasmid is reintroduced into the bacteria as harmless organisms. The process of reintroducing the plasmids is known as transformation (Zhang et al. 55). During the transformation process, the bacterial cells are given a shock, which makes them ready to take on foreign DNA.
Step three is the production of protein, whereby upon finding a bacterial colony with the correct amount of plasmid, the scientist can grow larger cultures of plasmid harboringbacteria. The bacteria are then given signals through chemicals to instruct them to generate the target proteins. Thebacteria are like factories that churn out significant amounts of proteins. Upon the production of protein, the bacterial cells may be split open to release the proteins. The presence of many proteins in the bacteria the target protein needs purification through biochemical techniques. The protein that has been purified may now be used for experiments or injected into patients (Zhang et al. 55).
DNA cloning has various limitations, including increaseduncertainty as there is no way of knowing the effects this cloning has on people. There is also the issue of new diseases that may arise due to cell mutations that may cause critical genetic disorders to the patients. DNA cloning may lead to a decrease in diversity people will become weak as they are unable to adapt. This may leave them susceptible to new diseases. Inbreeding also limits the success of DNA cloning as all the people will bear the same genotypes, a thing that may lead humanity to extinction (Zhang et al. 55).
- Fluorescence in situ hybridization (FISH) initiated the new era of study of the chromosome structure and functions. FISH entails the use of virtual techniques, which are appealing as they can provide the immediate degree ofresolution between chromosomal investigations and DNA analysis. At the same time, maintain the information at the single-cell level. FISH has been used to support the significant mapping and sequencing efforts related to the human genome project (Bridger et al.). Fish proved to be accurate and versatile in biological and medical researches as it provided the researchers with a wide range of applications as well as the FISH diagnostic assays.
The increased diversification of the initial FISH protocol into the amazing number of procedures that are done nowadays are owed to the followingfactors; Advancement in the field of fluorescence microscopy and the field of digital imaging, the growth, and availability of the genomic bioinformatic resources and advancement in sensitivity, specificity, and resolution. FISH is a straightforward technique that entails hybridizing DNA probes to their complementary sequences on chromosomal preparations, which were previously fixed on slides (Bridger et al.). The probes may be labeled in two ways: directly through incorporation fluorescent nucleotides and indirectly through the introduction of reporter molecules that are detected by the fluorescent antibodies.
The probes and the targets are then visualized in situ suing microscopy analysis. The FISHtechnique was famous for its support for large-scale mapping and its accuracy and adaptability, which saw its significanceincrease asit began being used in biological and medical research. The wide range of FISH applications has been developed to incorporate other fields of investigation, ranging from neuroscience, clinicalgenetics, toxicology, microbial ecology, and evolutionary biology, just to mention a few. Factors like the specificity and resolution of FISH have become handy in understanding mora about chemical and physical components of nucleic acids and chromatin (Bridger et al.).
ACM-FISH is a multiple colored FISH assay responsible for detecting structural and numerical chromosolabnormalities in the sperm cells.ACM stands for concurrent hybridization of DNA probes for the alpha, classical and midi satellites of chromosomes, each with a different function.ACM technique has its origin from the integration of technical aspects and biological results from the prior studies on FISH and chromosomal rearrangements in the sperm and lymphocytes(Bridger et al.). The ACM has been responsible for significant discoveries in sperm cell abnormalities in healthy men. This discovery points out that structural defects are more frequent than numerical defects. The discoveries also found that chromosomal breaks are always existence in the human sperms long before fertilization. This technique has been used to analyze the sperms of infertile men, which resulted in more findings of the increased levels of chromosomal defects.
Arm Fish is a FISH variant made up of 42 colors. It is responsible for detecting chromosomal anomalies’ technique. It entails the combinationof commerciallyavailable MFISH kits with chromosome arm-specific paintingprobes. The advantages of this technique are the increased resolution of chromosome arms’ level and the resulting ability to recognize pericentric inversions (Bridger et al.). The assay has been previously used to expose the increased chromosomal instability in the glioma cell lines.
Fiber FISH is a technique that facilitates high-resolution mapping of genes and the permitting physical ordering of DNA probes to a resolution of 1000 bp. Fiber FISH also allows the assessment of gaps and overlaps in contigs. Fiber fish is applied during the release of chromatin, which is done through extraction and stretching and fixing them on a microscope slide before they are hybridized. The technique was advanced to allow for DNA stretching uniformity and reproducibility, made possible by the implementation of molecular combining protocol. The FISH techniques have been adapted to incorporate later changes that may come (Bridger et al.). The technique allows for improvements hence making it able to accommodate all changes that need to be affected.
3.In Vitro Translation is the tool used by a molecular biologist in the synthesis ofproteins. In Vitro Translation has many applications, whichinclude; protein folding studies, incorporation of modified amino acids for functional studies, and rapid identification of gene products. The main reason why scientists recommend the use of In Vitro Translation because of its advantages over In Vivo. In the case of overexpression of toxic products to the host cells, when the product forms inclusion bodies or is insoluble. In vitrotranslation can produce proteins even from dead cells. This technique ensures that the synthesis of proteins is not constrained by cell walls conditions necessary for maintaining cell viability (Capece 299). The fact that it is not constrained by cell walls or homeostasis makes it the best option for many scientists.
Cell-free protein synthesis ensures direct access and the control of the translation environment, giving this technique more advantages over the other bacterial expression systems. The In Vitro translation is advantageous as it takes a shorter period in preparation of the extract. The in Vivo systems used to take two weeks to prepare the extract, but the In vitro systems prepare this extract in several two days maximum. CFPSis an open reaction compared to In Vivo, which requires a lot to obtain accurateresults (Capece 300). In Vitro systems being open reactions makes it easierto take samples, monitorconcentrations easily, and monitor the reaction.
CFPS doesn’t care about the toxicity of the proteins. The lack of concern for toxicity makes it superior to the In Vivo systems (Capece 298). The toxicity isn’t a concern as dead cells are used; hence no significant concern is required. In vitro systems are useful in the insertion of the modified tRNAs as they provide the ideal conditions required for the reaction.
A cell-free protein synthesis is an essential tool for molecular biologists in applied sciences. Thissynthesis has been used on several occasions through genotypesand proteomics compared to the protein expression in live cells. Cell-free protein synthesis is crucial during the generation of protein arrays and the engineering of enzymes using display technologies. The cell-free protein synthesis is a speedy way of correlating phenotype to genotypes. The synthesis may be performed in a few hours when mRNA templates or DNA templates are used. The cell-free protein expression of toxic proteins is said to undergo rigorous proteolytic degradation (Capece 301).
When choosing the best cell-free protein expression over cells, there are many available options to choosefrom. The choice, however, depends on the following; the origin of that template (RNA or DNA), proteinyield, orwhether the protein must be modified to ascertainway. A researcher should choose cell-free protein synthesis as it uses non-living cells. The use of non-living cells plays a significant role as there will be no need for being concerned about the toxic proteins (Capece 299).
4.RT-PCR is used to mean Reverse Transcription PCR; the starting genetic element in the PCR reaction is RNA. The RNA is transcribed in reverse to its DNA complements by the reverse transcriptaseenzyme. The new DNA that contains the reversed transcription is then amplified using traditional PCR. If one continues to read the results, they will find the presence or absence of the amplified gene product using agarose gel electrophoresis. On the other hand, PCR consists of a reaction between the PCR machine with a fluorescent dye. These two componentsbind with the amplified gene product to give CT values, which are later used to determine the number of RNAsand DNA in the reaction based on the standards used (Musso 53).
The polymerase Chain reaction is a simple and popularly used molecular biology technique used to amplify and detect DNA and RNA sequences. Compared to the traditional DNA cloning methods, PCR is superior as it takes a few hours to amplify and detect the sequences.PCR is very intensive and doesn’t require a maximum template to detect and amplify the specific sequences (Musso 55).
Quantitative PCR (qPCR) is used to detect, characterize, and quantify nucleic acids for different applications. While in RT-qPCR, the RNA transcripts are quantified using reverse transcribing. Thetranscripts are first reversed into cDNA, and then the qPCR is then done. In PCR, the DNA is amplified into three repetitive steps: denaturation, annealing, and elongation. On the other hand, in PCR, thefluorescent labeling allows for data collection as the PCR process is ongoing. Thedye-basedqPCR allows the quantification of the amplified DNA molecules by employing the use of ads DNA binding eye. Thefluorescent is measured each time, and it is worth noting that the fluorescence signal keeps on rising proportionally with the amount of DNA replicated (Musso 55).
Reverse transcription PCR is responsible for the use of the RNA template. Additional steps enhance the detection and amplification of RNA. The quality and purity of the RNA template are crucial for the success of the Reverse transcription PCR, the efficiency of the first strand reaction may affect the amplification process.RNA is very hard to work with as they are unstable and also single-stranded.
When there is a variation in the procedural reaction of PCR, many things may go wrong. If the reverse transcriptase enzyme is reduced or increased in the reverse stage of the RNA, then the error rate may be increased, leading to chemical reactions. Reversetranscriptaselacks a reverse capability; therefore, it may be hard to reduce the viral reverse transcriptase’s error rates, which provide a selective advantage to the host system. In the PCR reaction failing to denature the enzymes in different steps may cause the results of the reaction to be inaccurate(Musso 53). If the temperature is not raised during the denaturation stage would make the hydrogen bonds in between the cDNA strands not to break.
Works cited list
Bridger, Joanna M., and Emanuela V. Volpi, eds. Fluorescence in situ hybridization (FISH):
protocols, and applications. Totowa: Humana Press, 2010.
Capece, Mark C., et al. “A simple real-time assay for in vitro translation.” Rna 21.2 (2015): 296-
305.
Zhang, Yongwei, Uwe Werling, and Winfried Edelmann. “SLiCE: a novel bacterial cell extract-
based DNA cloning method.” Nucleic acids research 40.8 (2012): e55-e55.