Dna & Pcr Extraction From Raw Soybean (gmo)

Genetic Engineering

PRACTICAL DNA Extraction from Raw Soybean (GMO) Objectives 1. Extract DNA of raw soybean by using CTAB method. 2. To familiarize student on how to do aseptic lab work. Introduction Since GMO’s are widely used and entered the worldwide market especially in food industry, there is a need and reliable sensitive methods to provide the detection of GMOs in food matrices. The methods are DNA based on PCR method. In mid 1990s, when the first GM crop was on the market, several identification methods have been developed. The official was classified focused on the detection of the GM Roundup-Ready (RR) soybean. The extraction of genomic were used conventional method which from Swiss and Wizard German method. The CTAB method were developed and modified by Lipp et al. 1999 and were followed for extraction and purification of DNA. Materials and Methodology Extraction of Soybean Genomic 0.1g of raw soybean sample was weighted. ↓ 1.5mL CTAB buffer lysis was added and mixed well until homogenized. It was incubated in water bath at 65°C for 1 hour. ↓ 700µL of supernatant was taken into the new eppendorf tube. 700µL of chloroform isoamylalcohol (24:1) was added. It was mixed well and centrifuged at 12000rpm for 10 minutes. ↓ 400µL of upper layer was transferred and was added with 400µL CTAB precipitate. It was mixed gently and was left at room temperature for 30 minutes. 400µL of chloroform isoamylalcohol (24:1) was added, mixed gently, and centrifuged at 12000rpm for 10 minutes. ↓ 400µL of upper layer was transferred and was added with 200µL of 3M NaAc. It was mixed gently. Then 400µL of cold isopropanol was added, mixed gently, and centrifuged at 6000rpm for 6 minutes. Supernatant was discarded carefully. ↓ 500µL of cold ethanol was added, and then was centrifuged at 10000rpm for 5 minutes. Supernatant was discarded carefully. ↓ 500µL of cold ethanol was added, and then was centrifuged at 10000rpm for 5 minutes. Supernatant was discarded carefully. ↓ The pellet was dried at room temperature for 30 minutes. ↓ 40µL of sterile distilled water was added. ↓ 0.8% agarose gel was run.

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Genetic Engineering

Question 1. What are the differences between genomic extraction bacteria and plant? For bacteria, the cell pellet was resuspended in TE buffer, but for plant cell/tissue, it will be resuspended in CTAB. PCI solution was not used in plant genomic extraction. 2. What is the full name of CTAB? Explain why we used it. CTAB is cetyl trimethylammonium bromide or cetrimonium bromide. Its uses include providing a buffer solution for the extraction of DNA DNA extraction from plant tissue can vary depending on the material used. Essentially any mechanical means of breaking down the cell wall and membranes to allow access to nuclear material, without its degradation is required. For this, usually an initial grinding stage with liquid nitrogen is employed to break down cell wall material and allow access to DNA while harmful cellular enzymes and chemicals remain inactivated. Once the tissue has been sufficiently ground, it can then be resuspended in a suitable buffer, such as CTAB 3. Is there any different step with the bacterial step? And in which step? Yes. Step of resuspending the sample (see question 1) Conclusion Through this experiment, I become familiar on how to extract DNA of raw soybean by using CTAB method. References http://en.wikipedia.org/wiki/Cetyl_trimethylammonium_bromide (211008) http://www.cilr.uq.edu.au/UserImages/File/Plant%20Genomic%20DNA%20Extraction%20by %20CTAB%20_2__Fiona.pdf. (211008)

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Genetic Engineering

PRACTICAL PCR Detection of Lectin Gene in Soybean (GMO) Objectives 3. To familiarize student with PCR machine. 4. To familiarize student on how to do aseptic lab work. Introduction Roundup is a non-selective herbicide, effective against the majority of annual and perennial grasses and broad-leaved weeds. Glyphossate (N-phosphonomethyl-glycine) is an active ingredient in Roundup. Earlier research has indicated that glyphosate binds to and inhibits the action of 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase, EPSPS). EPSPS is present in all plants, bacteria, and fungi, but not in animals; animals do not make their own aromatic amino acids, but receive them from plants. EPSPS is an important enzyme in the aromatic amino acid biosynthetic pathway, catalyzing the reaction of S3P and PEP to form EPSP and phosphate. Therefore, by inhibiting EPSPS, glyphosate disrupts the aromatic amino acid synthesis in plants – many proteins cannot be made, resulting in death or severe growth reduction. Genetically altered Roundup Ready Soybean contains the roundup ready sequence to tolerate Roundup. Polymerase chain reaction (PCR) is widely used in many fields of analysis to detect even small amounts of DNA very specifically. Multiplex PCR enable the detection of the lectine gene (housekeeping gene) and the Roundup gene simultaneously. PCR target for the GMO and plant species

Promoter

Interest gene

Terminator

<-------------------------Inserted gene--------------------> Materials and Methodology DNA of Soybean Extraction 1. DNA was extracted using an alkaline-lysis method. 2. Purity and concentration of DNA was determined by gel electrophoresis, measured by spectrophotometer and stored at -20°C. 3. DNA was used as a DNA template in the PCR amplification. Polymerase Chain Reaction (PCR) PCR mixture 1. All the microcentrifuge tubes and PCR thin wall tubes was labelled accordingly on both on top of the cap and sides. 2. Master Mix was labelled. Reaction component was added in order into sterile 1.5mL centrifuge tube. 3. Contents in tube were vortex and spin-down. Reaction was kept in ice. 4. Master Mix was mixed gently and spin-down quick spin. 5. 23µL of the Master Mix was transferred into 4 new 0.2mL thin wall PCR tubes. 6. 2µL of appropriate DNA was added directly into each PCR tubes respectively with the appropriate labelled. 23µL Master Mix + 2µL DNA = 25µL total reaction volume 7. For PCR tube with label N, 2µL of sdH2O was added, replacing the DNA as a negative control.

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Genetic Engineering

8. Caps of the 0.2mL thin wall PCR tubes were closed firmly and were quick spin. 9. All the labelled 0.2mL thin wall PCR tubes were placed into the thermal cycler. 10. PCR machine was started. Master Mix Volume reaction (µL) 2.5 1.5 1.0 1.0 1.0 1.0 15.0 23.0

PCR Reagents 5X PCR buffer MgCl2 (25mM) 1.25mM dNTP (4mM) 0.025mM Lf (sense)(Lectin) Lr (antisense)(Lectin) Taq DNA polymerase (5 U/µL) Sterile distilled water Total volume

Volume (µL) 2.5 2.5 2.0 0.5 0.5 0.3 16.7 25.0

PCR amplification (a) Primers for lectin gene Primers Primer Lectin 01 forward Primer Lectin 02 reverse

Product (bp)

Targeted Gene

164bp

Lectin gene in soybean

(b) Required parameter as given below was set in the PCR thermal cycler Step 1 Step 2 Step 3 Step 4 Step 5 Step 6

Initial denaturation Denaturation Annealing Extension Final extension Hold

95°C 95°C 72°C 72°C 72°C 4°C Use heated lead: YES

12 minutes 1 minutes 30 seconds 30 seconds 10 minutes forever

1 cycle 40 cycles 1 cycle -

1. The reaction tubes were placed in the slots and the lid was closed carefully and tightly. 2. Programme started. 3. The tubes was taken out and placed in ice when the reaction was over. 4. The product was analyzed by separation on 2% agarose gel. 5. Agarose gel was run using 10µL each of the reaction mixture with loading blue dye. 6. 70 volt was run for 40 minutes. 7. Results are analyzed using normal ethidium bromide staining and visualized under UV transilluminator or visualization under gel documentation.

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Genetic Engineering

Question 4. Explain the PCR process. PCR (polymerase chain reaction) is a process used to make many copies of select regions of DNA. The process involves three repeated steps. • The template (original) DNA is first denatured or broken apart. The double stranded helix is separated so that single stranded DNA exists. This is done by heating the DNA to 94oC. • The temperature is then reduced in order to allow the primer to bind to the single stranded template (original) DNA. • Then the temperature is raised to the temperature where optimum extension (or replication) of the DNA takes place. In order for the process to take place a mix of the original/template DNA, primers, individual nucleotides (dNTPs - A [adenine], T [thymine], G [guanine], C [cytosine]), MgCl2, a buffer solution, and Taq DNA polymerase (the enzyme responsible for replicating the DNA, originally extracted from bacteria discovered in hot springs Thermus aquaticus) must be made up. Primers are simply small segments of DNA that are synthesized to be complimentary to a specific region(s) of the template DNA. It is characterized by having A's matched with T's (or T's with A's) and G's matched with C's (or C's matched with G's). PCR is used for many genetic analyses ranging from paternity testing and criminal forensic analyses to speciation and phylogenetic tests to examinations of variation between species' subpopulations. Depending upon the specificity of the primers that are used the number of locations within the genome that are copied and amplified varies. This means that for very specific primers either one or two sizes of DNA fragments will be produced while for other less specific primers many different sizes of DNA fragments are produced. The various sizes of DNA originate from multiple sites within the genome where the primers can bind and Taq DNA polymerase can replicate the fragments. 5. What is primer? Why there are forward and reverse primers in this experiment? A primer is a strand of nucleic acid that serves as a starting point for DNA replication. They are required because the enzymes that catalyze replication, DNA polymerases, can only add new nucleotides to an existing strand of DNA. The polymerase starts replication at the 3'-end of the primer, and copies the opposite strand. Primers in PCR are usually short, chemically synthesized oligonucleotides, with a length of about twenty bases. They are hybridized to a target DNA, which is then copied by the polymerase. Forward and reverse primers were used to increase the chances of getting the desired product. It is also important for the sequence-reading preferences of enzymes. 6. How do you know that you get the PCR product? When the result gives the correct band, which is the sequence was not completely unexpected and nonsensical. PCR primers will not give a completely blank lane or give two simultaneous and superimposed (and thus unreadable) sequences.

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Genetic Engineering

Conclusion Through this experiment, I become familiar with the use and application of PCR machine. References http://genamics.com/expression/primer.htm (251008) http://en.wikipedia.org/wiki/RNA_primer (251008) http://seqcore.brcf.med.umich.edu/doc/dnaseq/pcr.html (251008)

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Genetic Engineering

Result

7