Molecular Techniques in Biosciences (D224P6 MY)

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15 APRIL 2013

DNA extraction and Polymerase Chain Reaction for Green Fluorescent Protein from transformed Sauropus androgynus culture
By student ID 009921
School of Biosciences, University of Nottingham Malaysia Campus, Jalan Broga, 43500, Semenyih, Selangor.

INTRODUCTION
The development of the molecular technique especially polymerase chain reaction (PCR) make the amplification of a small amount of DNA possible to create up to billions DNA copies number. This revolutionary molecular technique has been developed by Kary Mullis in 1980s in Cetus Coperation, California (Barlett et al., 2003) and has been used in many fields. PCR utilize the thermostable DNA polymerase an enzyme obtained from the thermophilic bacterium to amplify the target sequence. Taq DNA polymerase first discovered by Chien et al. (1976) in Thermophilus aquaticus isolated from hot springs and have been widely used DNA polymerase after it has been optimize for the PCR process by Saiki et al. (1988). PCR reaction requires the genomic DNA to be denatured in denaturation process at 95oC so that both forward and reverse primer sequences are able to bind to complementary DNA in annealing stage at 58oC. Then in extension phase the DNA polymerase play its important role to copy the target sequence that occur at 72oC. This cycle is repeated until the target DNA copy number is achieved. However, it was a tedious task to complete all the cycle when one need to monitor different temperature and time for different phases. But recent technology have save a lot of time and the tedious task by using thermocycler or PCR machine first developed by Weier et al. (1988) that can be automatically set the desired temperature and time. One crucial step one need to perform before PCR is the genomic DNA isolation where the DNA is extracted from target organism that carried target sequence. Plant DNA extraction is crucial to break the plants components that problematic to obtain pure genomic DNA. The components include are cell wall, natural occurring phenolic compound, proteins, lipids and many others. Proper extraction that utilized proper solution is the key component to break these barriers. This paper aimed to isolate the genomic DNA from both transformed and untransformed Sauropus androgynus tissue culture that previously developed (unpublished result) in lab though particle bombardment with green fluorescent protein (GFP) as gene of interest. The DNA of GFP obtained from the extraction then undergoes PCR and verified through gel electrophoresis. This paper also aimed to analyse the protocols used for both procedure and further optimization is also suggested in discussion part of this paper.

METHODOLOGY
1. DNA extraction from S. androgynus cultures Three leaf samples transformed and untransformed from S. androgynus cultures selected and freeze by liquid nitrogen to be grind into a fine powder using pastel and mortar and placed in different microcentrifuge tube. The transformed culture is chosen from previous study a week after particle bombardment (unpublished data). Buffer A, Buffer B and Sodium dodecyl sulphate (SDS) is added and vortex gently. The sample is incubated in 65oC waterbath for 10 minutes and centrifuged for 2 minutes. The tube is carefully removed to ensure the pellet is undisturbed. The supernatant is transferred using pipette into clean labelled microcentrifuge tube. Potassium acetate solution is added and mix gently by inversion and place in ice for 5 minutes. The mixture is then centrifuged and the clear supernatant is pipetted into clean microcetrifuged tube. Isopropanol is added and mix thoroughly by inversion before incubated at room temperature. The sample is centrifuged and the supernatant is discarded carefully. 70% ice cold ethanol is added to wash the DNA pellet and centrifuged again. At this stage the DNA pellet might be visible. The supernatant is discarded and let the pellet dried. For storage, TE buffer is added.

2. Polymerase chain reaction procedure 2 reaction tube is prepared for transformed and untransformed samples. PCR cocktail is prepared for three reaction by mixing the following solutions; polymerase buffer, magnesium chloride (MgCl2), dNTP, forward and reverse primer and sterile water. The mix is vortex and centrifuge. 24.5 l PCR cocktail then mixed with 0.5 l of extracted DNA template in clean PCR tube to be vortex and centrifuge. Taq polymerase then added into the tube and to be vortex and centrifuge again. The sample is run for PCR in thermal cycler with following conditions for approximately 2.5 hours; initial denaturation (4 min, 95oC, 1 cycle), denaturation (60 sec, 95oC, 30 cycle), annealing (60 sec, 58oC, 30 cycle), extension (60 sec, 72oC, 30 cycle), final extension denaturation (6 min, 72oC, 1 cycle) and holding temperature (10oC, 1 cycle). The product then undergoes gel electrophoresis in 1% agarose gel stained with SYBR safe.

RESULTS AND FINDINGS

1. DNA extraction of S. androgynus culture From the nanodrop reading (Table 1.0), the DNA concentration is very low which only 136.69 ng/l. Based on 260/280, the sample have slightly protein contamination (pure DNA have reading approximately 1.8) and contains RNA contamination as the ratio close to 2.0. 260/230 value indicates the nucleic acid purity and commonly range between 2.0-2.2. The 260/230 value from the experiment is far from range (0.96) which indicates contaminants absorbed at 230nm.
Table 1.0 : Nandrop reading obtained from spectrophotometer. Absorbance at 260nm (A260) shows the DNA concentration and absorbance at 280nm (A280) indicates the protein contamination. A good quality DNA should have a ratio 260/280 about 1.7-2.0, larger value indicates the contamination of proteins and other molecules.

Sample ID W4

ng/l 136.69

A260 2.734

A280 1.392

260/280 1.96

260/230 0.96

Gel electrophoresis of the plant genomic DNA shows no DNA bands nor smearing present (Figure 1). To validate the result, gel electrophoresis is run again and still shows no sign of DNA bands or smearing (Figure 2).

23130bp

10Kbp 1Kbp

2322bp

125bp

Figure 1: Gel electrophoresis of the plant genomic DNA. L1; Lamda DNA/HindIII marker, W4; plant genomic DNA, L2; 1kb DNA ladder

23130bp 10Kbp 3Kbp 1Kbp 2322bp 1Kbp

Figure 2: Repeated result of gel electrophoresis of the plant genomic DNA. L1; 1kb DNA ladder, W4; plant genomic DNA, L2; Lamda DNA/HindIII marker

2. Polymerase chain reaction Gel electrophoresis shows no DNA band for GFP (at 700bp) for the transformed plant. This probably shows that the GFP gene did not integrate into the plant DNA genome. So the plant did not transformed. Untransformed sample act as control in this experiment. Some smearing can be observed near 100bp for both transformed and untransformed sample that might be an indication of primer dimer formation.

500bp 100bp

Figure 3: PCR for both transformed and untransformed sample for GFP gene detection at 700bp L; 100bp DNA Ladder, W4; ID of plant genomic DNA, UT; untransformed sample, T; transformed sample

DISCUSSION
1. DNA extraction of S. androgynus culture This study is unable to extract plant genomic DNA from S. androgynus culture - probably due to low concentration of DNA as indicated in nanodrop reading. This then further justified through gel electrophoresis twice where not a single band nor smearing present while the DNA ladder appears to be normal. The low amount of DNA content is might due to the high number of plant tissues per extraction buffer unit as reported by Moyo et al., (2008) and could be improve by increasing the extraction buffer volume or decreasing amount of the tissue number. Improper pipetting technique during the experiment need to be considered as the experiment is conducted by novice researchers. Dealing with micro-litre unit of concentration needs proper pipetting skill because inaccuracy might alter solutions volume thus affecting DNA concentration. It is suspected that the DNA sample contains contamination of protein complexes, lipids, carbohydrate and other nucleic acid (in this experiment, RNA) (Buckingham, 2007). It is crucial to understand that successful nucleic acid purification rely on several important key steps; effective disruption of cells or tissue, denaturation of nucleoprotein complexes and inactivation of nucleases (Doyle, 1996). Methodology of the experiment is using hexacetyltrimethyl ammonium bromide (CTAB) in DNA buffer to bind to polysaccharides and removed the contaminants from the DNA sample (Clarke, 2009) refer Table 2.0 for the list of solution used for DNA extraction buffer. Along with CTAB, NaCl is also used to remove polysaccharides (Moreira, 2011). The presence of polysaccharides in the DNA sample is problematic during PCR because it leads to inhibition of Taq polymerase (Fang et al., 1992). It is suggest to use higher concentration of NaCl up to 6M (Aljanabi et al., 1999) to improve the quality of DNA harvested free from carbohydrates. Once the cell disrupt, the DNA is protected by EDTA by deactivating metal-dependant enzyme (DNAses) from digesting the DNA. EDTA will chelate the metal ion that required by the enzymes (Auld, 1995). Phenolics and polyphenols compound is common in plant tissues. These compound could damage the DNA sample when oxidized which bind covalently to DNA and make it useless for further study (Katterman and Shattuck, 1983). In this experiment, the situation is countered using polyvinylpyrolidone (PVP). Higher concentration of PVP with low molecular weight (10,000) rather

than 40,000 (used in this study) is more preferable to address phenolic problems as recommended in one study (Couch and Fritz, 1990). High molecular weight of PVP tends to precipitate with nucleic acids (Zhang and Stewart, 2000) that aid the DNA extraction. Other component like mercaptoethanol act as strong reducing agent also used to remove polyphenols (Wicke, 2009) and ascorbic acid to inhibit the activity of polyphenol oxidase (Borse et al., 2011). Further improvement for the protocols to combat plant components could be used like repeated chloroform isoamylalcohol to remove chlorophyll and plant pigments (Sahu et al., 2012).
Table 2.0: The components of buffer used for DNA extraction Buffer A 2% Hexacetyltrimethyl ammonium bromide (CTAB) 100mM Tris-HCl (pH 8.0) 20mM EDTA 1.4M NaCl 0.4% polyvinylpyrolidone (PVP-40) 0.1 ascorbic acid 10mM -mercaptoethanol Buffer B 100mM Tris-HCl (pH 8.0) 50mM EDTA 100mM NaCl -mercaptoethanol

2. Polymerase chain reaction Analysis of gel electrophoresis deduce the target plant did not transformed because no DNA band that represent GFP gene is observed when compared to the control (untransformed plant, act as indicator for comparison of the target gene present). This is justified from plant genomic DNA results where no DNA yield is obtained. The smearing present in both transformed and untransformed sample is perhaps due to primer dimer (PD) formation - primers that hybridized each other. 3 end of the primers rich in guanine (G) and cytosine (C) is used to complement to the DNA template but at the same time reduced the specificity that responsible to PD formation (Mitsuhashi, 1996). PD formation is problematic because it reduced the efficiency of PCR amplification (Mitsuhashi et al., 1996) and

waste the PCR resources. Study showed that formation of PD not only reduce the primers concentration but also form nonspecific DNA (Das et al., 1999) that possibly lead to inaccuracy of gel electrophoresis. Approaches have been taken in this study to reduce this PD complication by using hot start (D'Aquila et al., 1991) and high annealing temperature (Wu et al., 1991) but this paper findings shows differently. Other possible solutions need to be are to redesign the primer which used genome specific sequence at the 3-end (Brownie et al., 1997) and optimize the recipe by using low concentration of primers with higher concentration of polymerase (Brownie et al., 1997). PD formation is not the only cause of smearing as there are many variables need to be taken into account. Other possible cause is the present of DNAse activity (Application Hints and Troubleshooting, 2008). This verified by the visible smearing near 100bp below than expected band size, 700bp and probably the other reason why no DNA band observed (Dube, n.d.). Nonoptimal use of concentration of the PCR cocktails buffer like MgCl2, primers, enzymes and buffer concentration also lead to smearing (Why do I get smeared PCR products?, 2013).

CONCLUSION
Unsuccessful attempt to obtain the plant genomic DNA from S. androgynus culture leads to unsuccessful PCR where GFP gene is absent. Failure is mainly due to contaminations due to lack of laboratory skills and protocols limitation during the DNA extraction. Further study is strongly suggest addressing the problems encountered in this experiment including especially of DNA extraction protocol and PD formation in PCR. Optimization of the concentration and volume of the different components of PCR needs to be done to ensure the high quality PCR reaction can be achieved.

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