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DNA sequencing refers to the determination of the exact order of nucleotide bases in a molecule of DNA. DNA sequence is crucial for any biological research. Sequences of nucleotides can be determined by the locations of the labelled nuceleotides. The DNA fragments are then separated by gel electrophoresis which are then analysed using different methods of sequence analysis. Such sequence analysis methods include, Sanger dideoxy chain termination, Maxam-Gilbert method. Sanger dideoxy chain termination sequence is the most common method to determine the exact nucleotide sequence. The DNA strand is used as a template which is a strand of DNA which sets the genetic sequence of new strands. DNA polymerase is also used to generate complimentary strands using primers. Primers are strand of nucleic acid that provides as a starting point for DNA replication. Four different PCR reaction mixtures are prepared. Each will contain a percentage of dideoxynucleoside triphosphate (ddNTP) analogs which are building blocks of DNA, to one of the four nucleotides which are ATP, CTP, GTP or TTP. Radio lableld primer is added. ddNTP do not contain 3'-OH group, this prevents the formation of a phosphodiester bond with the nucleotide at 3'end. DNA strands are produced until one of the analogs is included by which the strand is cut. Different lengths of DNA strands will be contained in PCR reactions. The PCR reaction will end with nucleotide which was dideoxylabeled for that specific reaction. Gel electrophoresis is then used to separate the fragments. The sequence of the original template is then determined based on what lengths of strands end with what nucleotide. Presence of fragments can be visualised by using autoradiography.
Sequences of an amino acid of a protein which is encoded by a cloned gene is compared to proteins of known function. With such comparison similarity in sequences can be determined which will then lead to knowing the function of the encoded protein. Amino acid sequence can be found by using automated DNA sequencing. Automated DNA sequencing is a modification of the dideoxy chain termination method. It is carried out by the chain termination procedure. Automated DNA sequencing fluorescent dyes for the detection of the electrophoretically resolved DNA fragments (Rosenblum, Lee, et al., 1997). Fluorescent dyes are used to label DNA fragments which fluoresce when a laser hits them. Fluorescently labelled chain terminators are used such as ddTTP, ddATP, ddGTP and ddCTP. These fluorescent labels are attached to the dideoxynuclotide. Each chain terminated molecule carries a single label at its 3'. The fluorophore has four different colours which are used to label each of the four bases. The fluorescent emissions are collected and the wavelength of the specific colour is determined. Random dye-terminator incorporation makes certain of complete sequencing. The use of thermal cycling and a vector base primer makes this quick and efficient. The strands are separated by PAGE. Fluorescene detectors record each band as it migrates through the gel. Fluorescent signals are detected using an imaging system. The products are loaded into a single well of a polyacrylamide gel which is then runned past the fluorescence detector. The fluorescence detector identifies the fluorescent signal emitted by each band and saves the information on the computer. Advantages of automated sequencing is that fluorescent dye is not a hazardous chemical and is used instead of radioactive isotope. It presents more reliable results, fast and efficient. Moreover, sequence data is directly fed and stored into a computer, etc.
NCBI is a powerful tool for genetic analysis which provides biomedical information, bioinformatic tools and molecular and genomic databases. To know if two genes are homologous computer analysis should be made which is done by computer software programs. BLAST program is widely used to search for homologous sequences in nucleotide and protein databases (Tatusova and Madden, 1999). After the analysis and the statistical significance of the matches if the two genes are homologous it means that they are evolved from a common ancestor. This program can help deduce evolutionary and functional relationships between sequences and also identify members of gene families.
Nicotiana tabacum or cultivated tobacco is a perennial herbaceous plant. Osmotin is a basic 24 kDa protein that was originally identified in Nicotiana tabacum (Aslam, Singh, et al., 2009). It accumulates in the cells adapted to low water potentials (LaRosa, Singh, 1989). In response to osmotic stress, osmotin in high concentration is produced. The aim of this experiment was to sequence a subcloned promoter fragment (pOS2) of the osmotin gene of N. tabacum. Automated sequence analysis was employed to recognize homologous sequences of the promoter fragment and to find sequence motifs connected with regulation of osmotin gene expression.
MATERIALS AND METHODS :
The reagents used in this practical were : GTE buffer, 10 mM EDTA (pH 8.0), 0.2 N NaOH/1% SDS%, 3 M Potassium acetate (pH4.8), 10mg/ml RNase A, 1xTE, pellet paint, 3 M sodium acetate, 4 M NaCl, 13% PEG8000, 100% isopropanol, phenol/choloroform and chloroform, 70% and 100% ethanol and sterile double distilled water.
Preparation of plasmid DNA template for automated sequencing (Week 1 and week 2) :
Week 1 : DNA which is subcloned into a plasmid from pOS2 promoter of Nicotiana tabacum osmotin gene was prepared and sequenced. A 10 ml overnight E. coli cell culture was provided which was centrifuged at 5500 rpm for 10 minutes. By aspiration/pipette the supernatant was removed and in 2 mL of GTE buffer the bacterial pellet was resuspended by swirling the tube or passaging carefully with a P1000. Followed by addition of 3 ml of 0.2N NaOH/1% SDS the contents of the tube was mixed by rapidly inverting it 2-3 times. The tube was incubated on ice for 5 minutes, then neutralized with 3 ml of 3M postassium acetate (pH 4.8), followed by 5 minute incubation on ice after inverting the tube. The cellular debris was removed by centrifugation at 5500 rpm for 30 minutes at room temperature. the volume of supernatant was recorded as 8 ml. 0.6 volumes, ie, 4.8 ml of isopropanol was added and mixed by inversion and spun for 30 minutes at 3,500 rpm.The supernatant was discarded and then 2 ml of 70% ethanol was added and then tipped off and the pellet was allowed to dry. The pellet was then dissolved in 500 µl of 1xTE buffer. It was then transferred to a clean eppy tube and 2 ml of 10mg/ml RNase A agent was added and incubated at 37°C for 30 minutes. With 500 µl of phenol/chloroform followed by 500 µl chloroform the solution is extracted in the fumehood. 2 µl of pellet paint and 0.1 volume of sodium acetate was added and mixed well. By adding equal volume of 100% isopropanol and mixed by inversion DNA was precipitated. It was then placed in a freezer box. The tube was centrifuged at 13000 rpm for 10 minutes at room temperature and stored at -20°C for one week.
Week 2 : The second week, the supernatant from the tube was removed and the pink DNA pellet was washed with 500 µl of 70% ethanol. The tube was re-centrifuged for 2 minutes. 500 µl of 100% ethanol was added and then removed. Then ethanol was allowed to evaporate and pellet to dry by placing the tube on a 37°C heating block. The pellet was dissolved in 32 µl of 1xTE by flicking. 8 µl of 4M NacCL and then 40 µl of autoclaved 13% PEG8000 was added for the plasmid to precipitate. After mixing, the eppy tube was incubated on dry ethanol/ice for 20 minutes and then centrifuged for 20 minutes at 4°C. Formed supernatant was removed and the resulting pellet was rinsed with 500 µl of ice-cold 70% ethanol. The ethanol was removed and washed with 500 µl of 100% ethanol was added then again removed and then allowed to dry. In 15 µl of deionised H2O the DNA pellet was resuspended.
Quantification of nucleic acids using UV spectrometry :
Using the spectrophotometer (DNA samples) :
The DNA isolated was quantified via ultraviolet spectrometry.
In a microcentrifuge tube 99 µl water and 1 µl of DNA sample was added. In another microcentrifuge tube, 98 µl of water and 2 µl of DNA was added. The tubes were inverted to mix. The absorbance of the DNA solutions was measured at 260 nm, 280 nm, 230 nm and 320 nm. Using the absorbance readings, the concentration of nucleic acids in the sample was calculated.
Cycle sequencing :
Terminator premix was provided and in a 0.6 ml microcentrifuge 4.5 µl of the dye/buffer mix, 4 µl of the primer solution, µl of ds DNA template and µl of water were mixed and was then stored in ice. To prepare the sequencing reaction. In the thermal cycler, the dsDNA template was denatured at 96°C for 30 seconds. Then the primer was annealed to the plasmid DNA at 50°C for 15 seconds. The primer was then extended at 60°C for 4 minutes. The sequencing cycle was repeated 25 time and the reaction was held at 4°C until the next week.
Processing DNA from cycle sequencing reactions :
In this step, the residual dye-terminators by ethanol precipitation was removed. This was done by adding to a microcentrifuge tube 2 µl of 3M sodium acetate (pH4.6) and 50 µl of 95% ethanol. 20 µl of the extension product from week 2 was added to the microcentrifuge tube. The tube was briefly vortexed and placed on ice for 15 minutes and centrifuged at maximum speed for 20 minutes. The pellet was rinsed with 250 µl of 70% ethanol and centrifuged for 10 minutes. Ethanol was removed and the pellet was dried in a 90°C heating block for 1 minute. The DNA was resuspended in formamide/EDTA and loaded onto an automated sequencer. The sequence data was then collated and analysed by the computer in week 4.
Mapping of osmotin promoter fragment :
Genomic fragment from the genomic clone was found to hybridize by southern analysis and washing at high stringency to cDNA sequence. The cDNA clone of the osmotin gene was subcloned into the SalI restriction site of a pUC cloning vector.
Cloning sites in the polylinker of a pUC vector :
The pUC1 18, 3162 bp in length. The given subcloned DNA was digested with restriction enzymes and run on a gel. The length of the digested fragments was determined by gel electrophoresis. A graph was drawn to plot distance migrated (cm) versus Log of length (bp). The results were used to determine the restriction map of the cloned insert.