Research on the Expressed Sequence Tag of Olives Essay

The vital components and techniques of gene cloning are as follows, the DNA sequence that contains the desired gene (EZH2) is amplified by Polymerase chain reaction. PCR was established by Kary Mullis in 1985, popularly known to amplify target sequences of DNA (EZH2) to a billion fold in several hours using thermophilic polymerases (Taq) ,primers and other cofactors (Sambrook and Russell, 2001). Three crucial steps are involved which are Denaturation (at 95°), Annealing of the forward and reverse primers (55-65°) and lastly primer extension (at 72°). After amplification the desired sequence is integrated into the circular vector (pbluescript) forming the recombinant molecule. For the compatibility of the insert and vector, both were digested with (EcoR1) so the same cohesive ends are generated in both, making it easier to ligate. EcoR1 is a restriction enzyme that belongs to the type II endonuclease class which cuts within dsDNA at its recognition site “GAATTC” (Clark 2010; Sambrook and Russell, 2001).

This however, was unsuccessful, as the restriction digestion created fragments of many different lengths, resulting in smears at different lengths on the gel electrophoresis due to the restriction enzyme recognition sites being present in-between the telomeric repeat sequences, the analysis was thus

The Basic Local Alignment Search Tool (BLAST) was the final portion of this lab report. BLAST is provided courtesy of the National Center for Biotechnology Information (NCBI). After running the five chosen tests, students were given the two 16S rRNA sequences that were associated with their assigned culture. The BLAST results served to confirm or disprove the hypothesis of what the unknown may be. The BLAST program compared the given 16S rRNA sequence to a database of known sequences and searched for similarity. (10) This search tool is capable of comparing nucleotide or protein sequences to find statistical significance between the matches. A perfect match to the unknown sequence is indicated by a

2. McClean, Phillip. (1997). Cloning and molecular analysis of genes: Polymerase chain reaction (or PCR). Retrieved on December 6, 2014, from http://www.ndsu.edu/pubweb/~mcclean/plsc431/cloning/ clone9.htm. 3.

It is of great interest to see how far the scientific community has come across with identifying the genes and functions of each encoding protein. The science brought different interventions, as each gene has the possibility of interaction and the whole cellular environment has a type of role to engage in the complex day to day regulation of our eukaryotic cells.

350 of each sample are loaded on the gel. Based on Genomic Solutions, once fixing the changes of fresh 40% methanol and 10% glacial acetic acid for every 12 hour each, the gels were stained for 12 hours with Sypro Ruby solution in the dark. The gels were incubated in 10% methanol: 6% acetic acid for 4 hours for destaining procedure. The gels are the imaged by using ProPick Workstation. Triangulation and robotic excision are conducted for identification of protein spots. It was then submitted to tandem mass spectrometric analysis.

PCR was performed in 1 × PCR amplification buffer with 1 mM MgCl2 (Applied Biosystems, Tunis, Tunisia), 1 μM of each primer, 50 μM of each deoxynucleotide triphosphate (Promega, Tunis, Tunisia), 0.2 μg of DNA template, and 1.2 U of Taq DNA polymerase (Applied Biosystems, Tunis, Tunisia), in a final volume of 50 μl, using a GeneAmp PCR System 2400 (Perkin Elmer, Tunis, Tunisia). Cycling parameters were 95 °C for 3 min followed by 35 cycles of 94 °C for 1 min, 52 °C for 40 s, and 72 °C for 1 min, and then a final extension at 72 °C for 10 min. Control reactions lacking template DNA were performed simultaneously to rule out any putative contamination of PCR products. The ITS amplified fragments were visualized on 1% agarose gels stained with ethidium bromide, purified using a GeneClean kit (Q-BIOgene, Tunis, Tunisia), ligated into the pGEM-T Easy vector (Promega, Tunis, Tunisia), transformed into E. coli strain DH5α and plated on Luria Bertani agar medium containing ampicillin (50 μg ml-1) and X-Gal (5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside; 20 μg ml-1) (Sambrook et al., 1989). The presence of an insert encoding for ITS in the recombinants was confirmed by PCR amplification and sequenced using BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Tunis,

Several approaches can be envisaged to address the function of a gene. The techniques of molecular biology and biochemistry allowing for example to localize the expression of a gene or its product (Northern, western, in situ hybridization, immunofluorescence, etc.), to determine the structure of the protein (NMR, crystallography ...) or to find partners proteins (double hybrid, immunoprecipitation ...) give important information on the function of a gene (V Ecochard -2011).

All DNA tests were changed in accordance with 100 ng/μl. A 1000 ng (1 μl) aliquot of every example's DNA was utilized for a 50 μl PCR response. The 16S all inclusive eubac-terial preliminaries 530F (5'- GTG CCA GCM GCN GCG G) and 1100R (5'- GGG TTN CGN TCG TTG) were utilized for increasing the 600 bp area of 16S rRNA qualities. HotStar Taq Plus Master Mix Kit (QIAGEN Inc.) was utilized for PCR under the accompanying condi-tions: 94°C for 3 min took after by 32 cycles of 94°C for 30 sec; 60°C for 40 sec and 72°C for 1 min; and a last prolongation venture at 72°C for 5 min. A second-ary PCR was performed for FLX (Roche, Nutley, NJ) amplicon sequencing under the same condition by using designed special fusion primers with differ-ent tag sequences: LinkerA-Tags-530F and LinkerB-1100R. The resultant individual sample after parsing the tags into individual FASTA files was assembled using CAP3. The ace files generated by CAP3 were then processed to generate a secondary FASTA file containing the tentative consensus (TC) sequences of the assembly along with the number of reads in-tegrated into each consensus. The TC was required to have at least a 3-fold coverage. The resulting TC FASTA for each sample was then evaluated using BLASTn (Altschul et al., 1990) against a custom data-base derived from the RDP-II database (Cole et al., 2005) and GenBank website (http://www.ncbi.nlm. nih.gov/). The sequences contained within the

Many work has been done to perform an integrated analysis using genetic and gene expression data.

We performed gel electrophoresis on DNA that had been cut by a restiction enzyme, in order to separate the strands based on length. It is a routine procedure that allows the separation analyzis, and purification of samples of DNA.4

The purpose of the Bioinformatics/Molecular Evolution lab is to explore a particular protein given to us by its amino acid sequence. By entering this protein into the Entrez Protein Database in the National Center for Biotechnology Information, and using tools within this database such as Jpred, SMART, and Protoparm, the given protein could be fully analyzed. This experiment was very successful as our particular protein could be identified, and the tools within the NCBI database allowed us to fully analyze both chemical and physical aspects of this protein.

4. Gallego-giraldo L, Ubeda-tomás, Gisbert C, García-martínez, Moritz T, López-díaz. Gibberellin homeostasis in tobacco is regulated by gibberellin metabolism genes with different gibberellin sensitivity. Plant Cell Physiol 2008 05;49(5):679-90.results have not been consistent.

The Ramy3D promoter and 5′ UTR were amplified using PCR reaction from rice genomic DNA. As shown in Figure 1, a 995 bp fragment was obtained from PCR reaction. The product sizes were consistent with our expected length. The amplified fragment was ligated into the pTG19-T vector to obtain pTG19-RamyPro recombinant vector. The pTG19-RamyPro vector was digested with BamH I restriction enzyme to further confirm the cloning of the desired fragment. The digestion reaction showed the correct insertion of the desired fragment into pTG19-T vector (Figure 2).

Various data was collected from this experiment taken from four different sites on exons targeted for cleavage. This was to see if a CRISPR/Cas 9 construct can be used for initiating mutagenesis in grape leaves (6). The results indicated that there was some successful CRISPR/Cas 9 induced mutagenesis, but at low levels in the data collected from grape calli. The physical characteristics of the wild type grape plants were normal, but transgenic plants differed in the physical appearance of their leaves. The study included an image (figure 1) that showed that there were noticeable white and pale green spots on the plants leaves. This physical difference demonstrates that, in the leaves, there was a larger number of mutations. A trend found when analyzing these leaves were newer leaves measured lower for mutations than older leaves. (9). The number of successful mutation ranged greatly from plant to plant type. The plant altered by CRISPR/Cas 9 did show that the expression of Cas 9 has some effect on the level of mutations measured using western blot analysis (12).