Please, some vocabulary no need to change like (Purification, synthesis, amplification, digestion ) because no synonym for it should use

it.

1. Abstract: Genome sequencing has been one of the most notable achievements in modern science. Aided with the expanse of faster and faster sequencing machines, researchers have tremendously accelerated the pace of DNA sequencing, which shows much promising research. Brachyury plays a major role in regulating the normal migration of cells during early vertebrate embryogenesis. Recently, Brachyury has been implicated in several cancers. It is marker of chordoma, and duplication of the Brachyury locus confers susceptibility to familial chordoma.DNA was isolated from human cells by using the Qiagen RNeasy Plus Micro Kit. In this study, Brachyury gene was amplified by polymerase chain reaction (PCR) and DNA was cut by restriction enzymes, such as, EcoRI. It was inserted into plasmid in E.coli and it was cultured into LB agar, which included X-gal and IPTG. They were detected colonies by colour. This process is called blue\white selection screening which is determined the recombinant pNEB193::Brachyury plasmids. We show in this report a successful result where the desired protein is correctly expressed and verified by a functional assay.

1- Introduction The significant destructive and intractable diseases are caused by genetic defects. Errors or variations in single genes (monogenic diseases) or in multiple genes (polygenic diseases) can predispose to the disease process(Varmus 1984). The contribution of the Human Genome Project to our knowledge of gene sequences and structure has been immense. However, the availability of this tremendous knowledge base generates new challenges: to elucidate the function and regulation of genes, to establish how genetic errors cause disease, and most importantly, to translate these advances into innovative and effective therapies(Abate-Shen, Shen 2000). The Human Molecular Genetics Program is a center of translational genetic medicine, with innovative basic science generating new routes to therapy for inherited diseases. Central research themes include the regulation of gene expression, chromatin
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structure and modification in differentiated cells and stem cells, novel therapeutic approaches to neuromuscular disease and signaling mechanisms in cancer(Bishop 1987). Cloning is a vital laboratory process and is used to obtain new generations that are identical to the original in function and structure. Modern techniques, such as PCR, help to provide genetic information within a short time frame(Roselli, Fernando et al. 2012). Another technique is inserting genes into organisms (e.g. E. coli), which causes the organism to biologically clone the gene. The organism then produces proteins or other molecules that protect the organism from attack by other organisms or produces antibiotics. Some organisms have cloning vectors that can be used by viruses to amplify a viruss genome(Sarkar, Shields et al. 2012). These processes are essential in the discovery and development of new drugs and antibiotics. The eponymous T mutation, or Brachyury, was originally identified in the 1920s but the gene underlying the mutation was not cloned until about 60 years later(Herrmann, Labeit et al. 1990). The existence of other genes similar to T was recognized when a region of homology was identified between T and a Drosophila gene, omb(Pflugfelder, Roth et al. 1992). The homologous region was a DNA binding domain, styled the T-box, which was used to identify a family of T-box genes in the mouse(Georgia 1994). Homologues of existing genes, and new family members, have been identified in most organisms examined so far(Papaioannou 2001). Alignment of T-box sequences shows that the family is an ancient one, and has arisen due to gene duplication and cluster dispersion(Wattler, Russ et al. 1998). Brachyury is shown by various research groups to be highly expressed in a variety of cancers, including breast, colon, lung and prostate(Palena, Polev et al. 2007). This made Brachyury an interesting candidate for clinical trial.Furthermore, understanding the role of Brachyury is also crucial to study this gene as a stem cell marker(Sarkar, Shields et al. 2012). Brachyury has been shown to be a potential marker for colorectal cancer and also as a colon cancer stem cell marker. A possible link between the expressions of Brachyury with the regulation of the pluripotency gene Nanog, has been shown in human colon cancer cell line(Sarkar, Shields et al. 2012). The oncoprotein -catenin, which is itself a modulator of stem signaling pathways, is known to influence the levels of Brachyury(Sarkar, Shields et al. 2012). This would suggest that, the latter might be an important factor in transducing the -catenin
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signaling pathway in the maintenance of cells with a CSC-like phenotype. Brachyury regulates expression of the pluripotency gene Nanog, by binding to Brachyury with upstream regulatory elements in the Nanog promoter in mesenchymal-like cancer cells. Brachyury moved from basic science to clinical trial significantly fast, many answers are yet unknown about its fundamental role in human body. Completion of the clinical trial will definitely tell a lot more in this field of study, but continuation of Brachyury to be studied in basic science is still needed to answer many questions. Cloning involves four essential steps. The first is fragmentation whereby the DNA strand is broken up, followed by ligation where the DNA fragments are glued together in a sequence following the complementbase pairing rule. The third step is transformation, in which the new fragments enter cells that are replicating. The final step involves screening and selection of the cells, which have successfully taken up the transformed DNA, and these are selected and grown. Sometimes, when the gene of interest is extracted, it needs to be amplified or increased in numbers, so a polymerase chain reaction (PCR) is used.PCR is a method used to magnify DNA molecules and to detect nucleic acid sequencesand quantify them. Compared to other methods, the major advantages of PCR are it ishigh throughput, sensitivity, accuracy and versatility (Edwards, et al., 1991). It is the seven most important way to determine the number of DNA copies; any problem with the accuracy of this figure will have an effect on PCR applications such as pathogen detection, food regulation and scientific research (Lin, et al., 2011). Finally, this practicalwill demonstrate the efficiency of high fidelity PCR by amplify of the Brachyury gene from SW480 and cloning it into the plasmid pNEB193 using the high fidelity restriction enzymes of EcoR1. After that, rejoining them together by ligation. The blue/white colonies screening technique is used to select the recombinants and non-recombinants plasmid and then it will confirm if the Brachyury cloned or not by use the restriction analysis on purified plasmids.

2- Discussion: Part1:

We have shown that Brachyury can be cloned and amplified, ligated and transformed, translated and expressed. The results from the experiments above can be used in many interesting ways, not only to clone known genes, but also to disclose the limitless amount of undiscovered genes that have potential in medicine and gene therapy. Part2: Overall, the research was a success. The gene was cloned, amplified and made into functional protein. The implications of this research are limitless. Protein was expressed by E. coli that was originally in Brachyuryand was completely functional. The future for the gene has just begun. Future research could include transforming the expression vector in other competent cells or even cancerous cells and see if the protein remains stable. With the methods above, comparison is easier and more defendable. These methods could then be applied to many different proteins and used in future research for analysis of even more genes.

3-Results: The aspiration of this practical was to try to clone an expressed gene from a human cancer cell line (the Brachyury transcription factor from SW480 cells) and insert the gene into pNEB193 plasmid using the methods and materials that have been given. The goal was obtained and it is result will now be presented. Part1: The outcome of this protocol is extraction of total RNA from human cell lines to obtain optimal RNA yield and purity. The RNA was extraction from human cell lines by using the Qiagen RNeasy Plus Micro Kit and Measurements were performed with the Qubit fluorometer. The steps of this protocol only allow the purification of RNA molecules longer than 200 nucleotides. Part2: The purpose of this experiment is to use denaturing agarose gel electrophoresis to visualize the quality of the 28S and 18S RNA molecules. The quality of these 2 RNA-

species is used as an assessment as to the potential quality of the mRNA contained in preparations. Part3: The purpose of this experiment is to amplify mRNA transcripts into single stranded cDNA using Oligo (dT)20 primers. This protocol takes place in 3-stages. In stage 1 the Oligo (dT)20 primers are hybridised to the mRNA poly-A tails. In stage 2 the reverse transcriptase (SuperScript III RT) will hybridise to the Oligo (dT)20 primers/poly A duplex, read the mRNA transcript in a 3'-5' direction and synthesis a cDNA/mRNA duplex.In stage 3 the mRNA component of this duplex will be degraded using RNAseH to leave us with pure single stranded cDNA, which we will use to amplify Brachyury (and Actin). Part4: The purpose of this experiment is to test the suitability of the cDNA that created for PCR amplification. We use a pair of primers to amplify the human -actin gene to produce a 353-bp PCR product. As a result these primers can only produce a PCR product from full length cDNA and will not be able to amplify genomic DNA. Part5: The purpose of this experiment is to PCR amplify Brachyury transcripts from the cDNA that we created. In this experiment we use a pair of specific primers to attempt to amplify the predicted full length Brachyury transcript. Part6: The aim this experiment is to purify the DNA in the completed PCR reaction by removal of remaining reaction, such as excess primers, nucleotides, DNA polymerase, oil and salts. This step can achieve by using the Sigma GenElute PCR clean up kit NA1020 to purify DNA fragments from a PCR reaction mixture. Part7: The purpose of this experiment is to use the EcoRI restriction enzyme to digest purified Brachyury PCR product and the pNEB193 plasmid in readiness to perform a ligation reaction, also alkaline phosphatase to treat the digested pNEB193 plasmid.
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Part8: We using gel purification to specifically purify the linear cut phosphatase treated pNEB193 plasmid DNA molecules from any uncut plasmid molecules. The cut molecules will be used in the ligation reaction. Part9: The aim of this experiment is to analyze aliquots of digested, gel purified pNEB193 plasmid and digested T-6862 CDS, PCR fragment and determine the efficiency of the digestion reaction and 'estimate the Molar ratios' of your digested PCR and digested plasmid samples, in preparation for their ligation reaction. Part10: In this experiment you will ligate (stick) together gel purified plasmid and digested PCR fragments together to form a new 'recombinant' plasmid, also analysis of efficiency of plasmid gel purification and estimation of molar ratios Part11: After add aliquot of ligation reaction to chemically competent E.coli cells, the transformation process the E.coli cells to take upintact ligated-plasmids. Cells that have an intact plasmid will be growing on agar selection plates. Part12: The objective of this experiment is to use colony PCR to check to see if any of the white colonies on our transformation plates contain recombinant pNEB193: Brachyury plasmids. The colony PCR is a very useful way to quickly screening multiple bacterial colonies to see if they may contain recombinant plasmids with the approximately the required DNA sequence size.