Journal of Genetic Engineering and Biotechnology (2013) 11, 17–24
Academy of Scientific Research & Technology and National Research Center, Egypt
Journal of Genetic Engineering and Biotechnology www.elsevier.com/locate/jgeb
ARTICLE
Optimization of DNA isolation process and enhancement of RAPD PCR for low quality genomic DNA of Terminalia arjuna Pramod Sairkar, Shweta Chouhan *, Nitin Batav, Rajesh Sharma Centre for Excellence in Biotechnology, M.P. Council of Science & Technology, Vigyan Bhawan, Nehru Nagar, Bhopal, India Received 20 June 2012; revised 2 February 2013; accepted 6 February 2013 Available online 7 March 2013
KEYWORDS Terminalia arjuna (Roxburgh); RAPD PCR; FTA plant saver card; PCR enhancement; BSA; Formamide; DMSO; Glycerol
Abstract Owing to the presence of higher amount of polyphenolic and polysaccharide compounds, Terminalia arjuna (Roxburgh) is a significant medicinal plant in the Indian primeval medicine system. Polyphenolic and polysaccharide compounds also acts as inhibitors during Genomic DNA isolation from young leaves of T. arjuna, resulting in recovery of low quality genomic DNA, which affects downstream applications like PCR, restriction digestion’s, etc. In this study, nine different methods of genomic DNA isolation were used, out of which two methods were modified CTAB based methods, third one was HEPES based method and remaining six methods was FTA Plant Saver Card based. Out of the six FTA card based methods, in the first method, leaves were directly pressed inside the circle of FTA card. In the second and third methods, the leaves were homogenized with PBS and DNase RNase free water and the sample was applied on the FTA card. In the fourth and fifth methods: finally recovered DNA from two modified CTAB based methods was directly applied to the FTA card. In the sixth method, DNA precipitated after first phenol:chloroform:isoamyl alcohol precipitation of modified CTAB based methods dissolved in DNase RNase free water and applied to FTA Card. To optimize the PCR conditions, BSA (400 ng/ll), formamide (2.5%), DMSO (5% and 10%) and glycerol (5%, 10%, 15%, and 20%) was added into the PCR mix as enhancement agents for amplification of low quality genomic DNA (A260/A280 – 1.27 ± 0.090) of T. arjuna recovered using the HEPES Based method. It was found that the BSA was the best among them followed by 10% glycerol. In addition of BSA to the PCR mixture, it specifically enhances the amplification of the low quality DNA. It reduces the noise in-between the amplified bands and increases the intensity of PCR product. ª 2013 Academy of Scientific Research & Technology. Production and hosting by Elsevier B.V. All rights reserved.
* Corresponding author. Tel.: +91 9993136536. E-mail addresses:
[email protected] (P. Sairkar),
[email protected] (S. Chouhan),
[email protected] (N. Batav). Peer review under responsibility of National Research Center, Egypt.
Production and hosting by Elsevier 1687-157X ª 2013 Academy of Scientific Research & Technology. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jgeb.2013.02.001
18 1. Introduction Terminalia arjuna (Roxburgh) Wight and Arnott is a large 20– 30 m high tree, belong to family Combretaceae comprising of nearly 200 species distributed around the world. Nearly 24 species of Terminalia have been reported from various parts of India [46]. It is an economically important medicinal plant due to the thick white-to-pinkish-grey bark, which has been used in both Ayurvedic and Unani system of medicine. According to Ayurvedic medicine system, T. arjuna is an important cardiotonic plant [48], its bark is useful in the treatment of coronary artery diseases, heart failure, hypercholestrolemia [25,16] with the antioxidant activity of the chloroform extract in diabetic rats [23]. It is also useful in the treatment of Cancer [32], fractures, ulcers, heart diseases, biliousness, urinary discharges, asthma, tumours, leucoderma, anemia, etc. In Unani System, it is used in problems related to urinary discharges [41]. This plant has an excellent antimicrobial [38] property like antiviral activity against HSV-2 [4] antifungal [18] and antibacterial activity [47] against Escherichia coli, Klebsiella aerogenes, Proteus vulgaris, Pseudomonas aeruginosa [31], multi-drug resistant Salmonella typhi [37] with great potency of antifertility [44], antimutagenic activity [43] and anti-human immunodeficiency virus [19] induced diseases. T. arjuna has reported many therapeutic active constituents which include tannins, cardenolide, triterpenoid saponins (arjunic acid, arjunolic acid, arjungenin, arjunglycosides), flavonoids (arjunone, arjunolone, luteolin), gallic acid, ellagic acid, oligomeric proanthocyanidins (OPCs), phytosterols, Calcium, Magnesium, Zinc and Copper [3,15]. Presence of Polyphenolic contents in T. arjuna were reported [2], which acts as inhibitors in isolation of DNA through precipitated along with the DNA, thus deteriorating the quality and yield of the DNA. The HEPES based DNA isolation method [5] claimed for recovering adequate yields with high quality genomic DNA from fresh as well as dry leaves of T. arjuna and other similar species of family Combretaceae e.g. Terminalia bellerica, Terminalia chebula and Terminalia tomentosa. The polymerase chain reaction (PCR) is one of the most rapid and versatile methods of producing large quantities of DNA for molecular analysis [26]. When performing the polymerase chain reaction (PCR) for amplification of specific targeted site, three criteria are critical to its efficacy, specificity, efficiency, i.e. a sufficient product yield; and fidelity, i.e. the lack of sequence errors in the final product [12]. In the case of RAPD PCR, the randomness of primer is essential, which create noise between bands. The good quality of DNA is required for any type of PCR. Quantitative DNA analysis is performed by measuring the optical density (OD) at a wavelength of 260 and 280 nm using UV–Vis Spectrophotometer. The ratio of A260/A280 should be 1.8; smaller ratios indicate protein contamination, whereas lower values may be indicative of significant protein contamination. However, the A260/A280 ratio is not always an accurate representation of DNA purity. Certain samples may be very difficult to evaluate due to interference from aromatic organic compounds such as phenol, added enzymes, protein stabilizers or crosslinked proteins in samples derived from fixed tissue [39,10]. In this study diverse methods were applied for the isolating DNA from young leaves of T. arjuna, mainly CTAB based
P. Sairkar et al. method [6] modified CTAB based method [11,50], HEPES Based method [5]. We have also used FTA Plant Saver Card for isolating DNA using different ways of applying sample. After isolation of genomic DNA of T. arjuna by HEPES based method, ratios of recovered DNA was A260/A280 1.2–1.3 indicate protein contamination. In this study we have used various PCR enhancing agents for amplifying DNA (A260/A280 1.2– 1.3) especially for RAPD PCR. 2. Materials and methods Twenty-eight accessions of T. arjuna were collected and conserved at Human Herbal Health Care Garden (HHHCG), MPCST Bhopal, Madhya Pradesh, India from 6 different locations of 5 places in Madhya Pradesh; Gwalior, Sagar, Chitrakoot, Jabalpur and Bhopal namely (Table 1). Young leaves are used for isolation of genomic DNA from various described modified CTAB based methods and HEPES Based Method. The yield of DNA was measured using Nano Drop UV-Spectrophotometer (ND-1000) at 260 nm using DNase RNase free water as a blank. DNA isolation using FTA Card is very easy method and is used to preserve the DNA for a long time. As per the literature provided by the principle company (Whattman), leaves are pressed on FTA cards inside the marked circle. In the second and third methods, 10 mg of leaves were homogenized with PBS and DNase, RNase free water in a ratio of 1:5, by a mortar pestle and the homogenate is applied in the FTA card matrix using a micro pipette. Cards were allowed to air dry for 2 h at room temperature. In the fourth and fifth methods finally recovered DNA from modified CTAB method was dissolved in DNase RNase free water and applied directly on the FTA card, allowed to air dry. In the sixth method, DNA precipitated after first phenol:chloroform:isoamyl alcohol precipitation of modified CTAB based methods [50] were dissolved in DNase RNase free water and applied on FTA Card. In this method young leaf tissue (100 mg) was crushed in pre-chilled mortar and pestle after surface sterilization and immediately transferred to a 2 ml micro centrifuge tube containing 1 ml modified CTAB extraction buffer (100 mM Tris HCl (pH 8.0), 25 mM EDTA, 1.5 M NaCl, 2.5% CTAB, 0.2% 2 mercapto-ethanol (v/v), and 1% PVP (w/v), mixed well to form a slurry and incubated at 70 C for 30 min. The mixture was cooled at room temperature and an equal volume of the mixture of phenol:chloroform:isoamyl alcohol (25:24:1) centrifugation at 10,000 rpm for 10 min. Upper phase was collected in a new 1.5 ml micro centrifuge tube, in this aqueous phase equal volumes of ice-cold Isopropanol and 0.6 volumes 1.5 M NaCl was added and incubated at 20 C for 30 min followed by centrifugation at 10,000 rpm for 10 min at 4 C. The supernatant was decanted carefully and the pellet was dried at room temperature followed by dissolving in DNase RNase free water. 2.1. FTA sample purification After air dry of applied sample, FTA Plant Saver Card was placed on the FTA Sample Mat. A 2.0 mm disc from the center of the dried sample area was removed using the Harris Micro Punch tool and transferred into 0.2 ml PCR amplification tube. The disc was washed two times using 200 ll FTA purifi-
Optimization of DNA isolation process and enhancement of RAPD PCR for low quality genomic DNA Table 1
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Sample collection location and sample code.
SL
Location
District
No. of samples
Sample code
1. 2. 3. 4. 5. 6.
Van Parisar, Central Nursery Herbal Garden, MPCST Tropical Forestry Research Institute Jawahar Lal Nehru Krishi Vishvavidalaya Arogyadham, Chitrakoot Private Nursery
Gwalior Bhopal Jabalpur Jabalpur Satna Sagar
5 4 5 5 3 6
GTA-01 to GTA-05 BTA-01 to BTA-04 JTTA-01 to JTTA-05 JJTA-01 to JJTA-05 CTA-01 to CTA-03 STA-1 to STA-06
Table 2
Quantification of isolated DNA from 28 accessions of T. Arjuna.
SL
Sample code
DNA conc. (ng/ll)
OD at A260
OD at A280
A260/A280
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. Average ± SD
GTA-1 GTA-2 GTA-3 GTA-4 GTA-5* JJTA-1 JJTA-2* JJTA-3 JJTA-4 JJTA-5 JTTA-1 JTTA-2 JTTA-3 JTTA-4 JTTA-5 CTA-1 CTA-2 CTA-3 BTA-1 BTA-2 BTA-3 BTA-4*
997.15 900.55 407.12 264.63 817.17 260.30 135.86 20.49 447.91 793.01 446.50 306.34 397.33 982.59 260.26 173.66 771.53 609.63 231.35 263.33 421.24 385.90 467.90 ± 82.873
19.943 18.011 8.142 5.293 16.343 5.206 2.217 0.410 8.958 15.860 8.930 6.127 7.947 19.652 5.205 3.473 15.431 12.193 4.627 5.267 8.425 7.718 9.34 ± 1.657
15.411 14.126 6.287 4.176 14.423 4.213 2.060 0.328 6.987 12.478 7.302 4.682 6.615 15.971 4.106 2.800 12.328 9.344 3.837 4.023 6.470 5.287 7.42 ± 1.224
1.29 1.28 1.30 1.27 1.13 1.24 1.32 1.25 1.28 1.27 1.22 1.31 1.20 1.23 1.27 1.24 1.25 1.30 1.21 1.31 1.30 1.46 1.27 ± 0.090
*
Selected sample for PCR enhancement studies.
cation reagent after incubation of 4–5 min at room temperature (RT). Then two times wash the disc by 200 ll Isopropanol after incubated for 4–5 min at RT. The disc was allowed to completely air dry for 1 h at RT. 3. RAPD PCR amplification with different enhancement agents Three commercially available primers RPI-08, RPI-09 and RPI10 (Accession Number AM 773773, AM 773315 and AM 750045, respectively) from Bangalore Genei, Bangalore and three randomly selected samples (BTA-4, GTA-5 and JJTA-2) were used in this study. The sample was selected based on quantity of DNA and ratio of A260/A280 (Table 3). DNA amplification was performed in Eppendorf gradient automatic thermal cycler using RAPD markers. Genomic DNA (25 ng) was amplified via the PCR reaction using 50 ll reaction volumes under the following conditions, 25 ll of 2· red dye PCR master mix (10 mM Tris– HCl, 50 mM KCl, 1.5 mM MgCl2, 25 units/ml Taq DNA polymerase, 0.2 mM dNTPs, 5% glycerol, 0.08% NP-40 and 0.05% Tween-20), 2 ll primer, 1 ll template DNA (25 ng/ll), 22 ll sterile DNase RNase Free water and different PCR enhancement agents namely BSA (400 ng/ll), DMSO (5% and 10%), formam-
ide (2.5%) and glycerol (5%, 10%, 15% and 20%) was added accordingly. Condition of thermal cycler was set at initial denaturation of the DNA at 94 C for 5 min, 08 cycles of 45 s at 94 C, 1 min at 55 C and 1.5 min at 72 C; 30 cycles of 45 s at 94 C, 1 min at 58 C and 1 min at 72 C and final extension at 72 C at 10 min and a hold temperature at 4 C. Control reactions without template DNA (negative control), without the enzyme (positive control) and without enhancement agents were also run in the experiments. All the experiments were repeated thrice to ensure reproducibility. Amplified DNA fragments with low range DNA ruler were separated by electrophoresis at 6–7 V/cm in TAE buffer for 3–4 h on 1.2% Agarose gels stained with Ethidium Bromide and photographed by Gel Documentation System (Alpha Innotech). Raw gel images were recorded and analyzed through Alphaview software. 4. Result 4.1. Isolation of DNA DNA was not recovered from young leaves of T. arjuna using both the modified CTAB based methods, some sticky yellow-
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Table 3 Result of RAPD PCR Amplification of T arjuna DNA, which was applied on FTA card before and after various modifications. SL
DNA isolation method
PCR performance
1. 2. 3. 4. 5. 6.
Leaves are pressed on FTA card Leaves are homogenize with PBS Leaves are homogenize with water Directly apply recovered DNA from described method of Haque et al. Haque et al. [11] on FTA Directly apply recovered DNA from the method of Vijay et al. Vijay et al. [50] on FTA Directly apply recovered DNA from modified Vijay et al. Vijay et al. [50] method on FTA
No amplification No amplification No amplification Amplified Amplified Amplified
ish brown compounds also precipitated during isolation and it is not appropriate for any downstream applications. When DNA was isolated using the HEPES based method, we have observed the presence of protein during spectrophotometric analysis. 4.2. Quantitative and qualitative analysis of DNA Quantity of recovered DNA by HEPES based method was relatively good (467.9 ± 82.87 ng/ll) after dissolving in 50 ll DNase RNase free water, with an enormous range of recovery maximum 997.15 ng/ll from GTA-1 encoded sample followed by GTA-2 (900.55 ng/ll) and minimum quantity of DNA (20.49 ng/ll) from JJTA-3. During spectrophotometric analysis of isolated DNA, low ratio of A260/A280 was observed (1.27 ± 0.090), which indicates the presence of higher amount of protein in all the samples (Table 2). 4.3. RAPD amplification of FTA Evaluating the results of PCR, obtained on the DNA isolated from various methods revealed that, the methods on which the leaves were pressed on FTA card and the leaves which were homogenized with PBS and water respectively were did not amplify i.e. no amplification was seen. The method in which the recovered DNA from modified CTAB based methods was directly applied to FTA card was amplified respectively. Along with all these, a new method in which the DNA precipitated after first phenol:chlorophorm:isoamyl alcohol precipitation of the modified CTAB based method was dissolved in DNase RNase free water and applied on FTA Card was amplified well (Table 3). 4.4. RAPD amplification using different PCR enhancer To optimize the PCR conditions, BSA (400 ng/ll), formamide (2.5%), DMSO (5% and 10%) and glycerol (5%, 10%, 15%, and 20%) was added to the PCR mixture. When amplification was carried out by Polymerases Chain Reaction using RPI primer-8, it was observed that BSA gives the best result. There was less noise in between the bands as compared to control. Five percent and 10% DMSO were satisfactory, while 2.5% formamide showed no amplification on sample GTA-05. In addition of glycerol to the PCR mixture, 5% glycerol gave good result while 10% was best. Fifteen percent and 20% glycerol was also good and satisfactory respectively, whereas in the control, sample GTA-05 was not amplified. When PCR amplification was carried out by using RPI primer-9, it was observed that BSA gives the best result. In the control, sample GTA-05
did not show amplification while 5% and 10% DMSO were less satisfactory. Sample GTA-05 and JJTA-02 did not show amplification with 2.5% formamide. While BSA gave the high intensity and a crisp pattern of bands. In addition of glycerol to the PCR mixture, sample GTA-05 did not show amplification with 5% glycerol while 10% glycerol gave the best result with this primer. 15% glycerol was good and sample GTA-05 and JJTA-02 did not show amplification with 20% glycerol. In the control, intensity of bands generated by sample GTA-05 was weak as compared to others. When PCR amplification was carried out by using RPI primer-10, it was observed that BSA gives the best result. In the 2.5% formamide, sample BTA-04 and JJTA-02 were weakly amplified while sample BT-Ar-02 did not show amplification. Sample GTA-05 was weakly amplified in control conditions, while 5% and 10% DMSO were satisfactory. In addition 10% glycerol was found to be good while sample GTA-05 was weak in 5% glycerol. 15% and 20% glycerol were found to be satisfactory while amplification in control were good (Figs. 1–6). 5. Discussion In this study, different isolation methods for recovering DNA from young leaves of T. arjuna were applied. Some sticky yellowish brown compounds also precipitated during isolating DNA from both modified CTAB based methods. Negative PCR results were observed from FTA plant saver cards when the sample was applied by pressing the leaves or apply after homogenized with PBS and DNase, RNase free water. Due to presence of secondary metabolites like ellagic acid, gallic acid, arjunic acid, arjungenin and their glucosides, arjunetin and arjunglucoside-I [2,29] T. arjuna is an important medicinal in Indian medicine system. We have also faced the same difficulty as described earlier [5] during the time of isolation of genomic DNA from T. arjuna after using a number of published protocols like to modify CTAB based methods and several FTA Plant saver card methods, along with various modifications. In CTAB methods the polyphenols are also isolated along with DNA from T. arjuna and converted to several products reacting with proteins [21], and the polysaccharides form complexes with nucleic acids forming highly viscous material inhibiting the DNA modification like restriction enzymes, DNA polymerase, ligase, etc. [45]. In FTA Plant Saver Card Method, polyphenols and polysaccharides did not wash out completely with the FTA purification reagent and inhibit DNA amplification during PCR. For the isolation of DNA from species like T. arjuna and T. tomentosa, several published protocols like CTAB based [6], high salt and PVP [34], high salt and sarcosyl, the combination of CTAB and SDS was tried earlier [5], glucose was also
Optimization of DNA isolation process and enhancement of RAPD PCR for low quality genomic DNA 1
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Figure 1 Comparative analysis of amplified fragments generated by RPI-8 primer using organic chemicals; Lane 1–3 (sample BTA-04, GTA-05 and JJTA-02, respectively) is the control, Lane 4–6 is 5%DMSO, Lane 7–9 is 10% DMSO, M is Low range molecular marker, Lane 10–12 is 2.5% Formamide and Lane 13–15 is BSA.
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Figure 2 Comparative analysis of amplified fragments generated by RPI-9 primer using organic chemicals; Lane 1–3 (sample BTA-04, GTA-05 and JJTA-02, respectively) is the control, Lane 4–6 is 5% DMSO, Lane 7–9 is 10% DMSO, M is Low range molecular marker, Lane 10–12 is 2.5% Formamide and Lane 13–15 is BSA.
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Figure 3 Comparative analysis of amplified fragments generated by RPI-10 primer using organic chemicals; Lane 1–3 (sample BTA-04, GTA-05 and JJTA-02, respectively) is 2.5% Formamide, Lane 4–6 is BSA, M is Low range molecular marker, Lane 7–9 is control, Lane 10–12 is 5% DMSO, Lane 13–15 is 10% DMSO.
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Figure 4 Comparative analysis of amplified fragments generated by RPI-8 primer using glycerol; Lane 1–3 (sample BTA-04, GTA-05 and JJTA-02 respectively) is 5% glycerol, Lane 4–6 is 10% glycerol, Lane 7–9 is 15% glycerol, Lane 10–12 is 20% glycerol, M is the Low range molecular marker and Lane 13–15 is control.
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Figure 5 Comparative analysis of amplified fragments generated by RPI-9 primer using glycerol; Lane 1–3 (sample BTA-04, GTA-05 and JJTA-02, respectively) is 5% glycerol, Lane 4–6 is 10% glycerol, Lane 7–9 is 15% glycerol, Lane 10–12 is 20% glycerol, M is the Low range molecular marker and Lane 13–15 is control.
Figure 6 Comparative analysis of amplified fragments generated by RPI-10 primer using glycerol; Lane 1–3 (sample BTA-04, GTA-05 and JJTA-02, respectively) is 5% glycerol, Lane 4–6 is 10% glycerol, Lane 7–9 is 15% glycerol, Lane 10–12 is 20% glycerol, M is the Low range molecular marker and Lane 13–15 is control.
used as a reducing agent in standard CTAB protocol [30], and protocol for other species of Terminalia [51] sticky, viscous and colored DNA were isolated by above-mentioned methods which inhibiting the activity of DNA modifying enzymes. Followed with this we acquired the published method [5], which isolated adequate quantity of genomic DNA from young leaves of T. arjuna. This also revealed us with low ratio of A260/A280, which indicates the presence of higher amount of protein during spectrophotometric analysis. In previously reported protocols for isolation of DNA from T. arjuna, commercial DNA isolation kit was used [41]. Young leaves of T. arjuna were homogenized with PBS and Water and applied to the FTA card along with direct application by pressing, all secondary metabolites like ellagic acid, gallic acid, arjunic acid, arjungenin and their glucosides, arjunetin and arjunglucoside I [29], cell particles and protein gets accumulated on the FTA card which does not wash off with FTA purification reagent and creates problems during DNA amplification. FTA card is the method which conserves DNA for a long time at room temperature with certain limitations of the application. CTAB based DNA isolation process has been successfully applied to a wide range of plant species containing secondary metabolites. In Commiphora spp., polyphenolic and polysaccharides residuals were also precipitated during isolation of genomic DNA from young leaves by described the conventional procedure and highly viscous sticky like non-amplifiable DNA was obtained, a simplified protocol for isolation of high quality genomic DNA from Commiphora spp., was described [11]. On the other hand this method is not appropriate for isolation of genomic DNA from T. arjuna, because of residual polyphenolic and polysaccharides makes the procedure
problematic and renders the DNA unsuitable for digestion by restriction enzyme and amplification by Taq DNA polymerase [34,24]. The isolated DNA from modified CTAB methods was directly applied to the FTA Plant saver card. The FTA purification reagent purified the DNA well which gave better results on amplification. DNA amplified via CTAB based methods could not amplify because of residual polyphenol and polysaccharide hindrances. When these DNA was applied on FTA card, the remaining residues got washed off by FTA purification reagent and DNA got trapped well on the FTA card matrix resulting DNA amplified appropriately. Modified CTAB methods, we picked up the DNA after first precipitation and directly applied to the FTA card followed by washing with FTA purification reagent and finally DNA amplified well. As PCR Enhancers, different organic compounds are added to PCR Mixture, which makes DNA available for different enzymatic reactions from low quality DNA. A variety of chemical additives like DMSO, bovine serum albumin (BSA), glycerol, spermidine, formamide, polyethylene glycol 6000 (PEG 6000), gelatin, tween-20, nonidet P40 (NP40) and Triton-X100 [22,40,1,49,52] known to enhance amplification were utilized to optimize amplification. Pretreatment of the DNA with restriction enzymes or BSA and several other proteins also enhanced the PCR amplification. Treatment with these proteins may remove PCR inhibitors in the DNA samples extracted from the paraffin blocks. Epstein–Barr virus DNA in paraffin-embedded gastric carcinoma tissue was detected effectively by PCR, after pretreatment of the extracted DNA with a restriction enzyme, BamHI or EcoRI [42]. The effects of BSA were observed on extracts of bacterial fractions from chicken, cow, hog, horse, and sheep manure,
Optimization of DNA isolation process and enhancement of RAPD PCR for low quality genomic DNA as well as extracts of filter retentates collected from the River, Ocean, Bay, Marsh, Freshwater Creek, and Canal, which all inhibited PCR, due to interference from unknown inhibitors. BSA may be protecting the Taq DNA polymerase [8,17]. This may also inhibit endogenous protease activity [35]. The wellknown function of Phenols are binding of proteins by forming hydrogen bonds with peptide bond oxygen’s, and the BSA has been widely used for isolation of organelles and enzymes from plants [21]. BSA acts as stabilizing negotiator for proteins in PCR mixture, which may enhance amplification by stabilizing the DNA polymerase [14]. Equivalent performances were observed when concentration-dependent combination of the known PCR additives betaine, DMSO, and DTT was carried out resulting in a cost-effective PCR enhancer solution compared with commercial enhancers [36]. Five percent DMSO and 500 lg/ml of gelatin additives under different conditions in a matrix were shown the best result, against no band was observed at 10% DMSO which interpreted the negative effect of DMSO at high concentration [28]. DMSO reduces the secondary structure of DNA, enable strand separation, which can affect Taq polymerase activity. Formamide may enhance Taq activity and inhibit DNase activity. BSA may bind color compounds (melanin, bile salts, humic substances) and anions, which inhibit Taq polymerases and act as substrate for proteases respectively. Glycerol enhanced association between enzyme and template [9,17,40]. Organic solvents affect the thermal stability of the primers and activity of DNA polymerase [33], thereby increasing the specificity of amplification [49]. Organic solvents have the capability to destabilize DNA in solution due to effect of thermal stability [20,7]. Glycerol is used in the storage buffer of thermo stable enzymes because it is used by organisms and cellular systems to maintain biological activity under extreme conditions. The addition of betaine with glycerol to PCR reaction enhance the specificity of the primer [9] and reduce the formation of secondary structures caused by GC-rich regions [13]. Glycerol can also lower the strand separation temperature of DNA, thus facilitating amplification [27]. The restriction digestion-based assay to assess an increase in mutant PCR products in a simple, efficient and high-throughput fashion was performed. The presence of 2% DMSO has a significant adverse effect on PCR fidelity that falls below the threshold for detection [12].
Acknowledgment Financial support from the Government of Madhya Pradesh, India is highly appreciated. The authors wish to thank Prof. P.K. Verma, Director General, MPCST and Dr. R.K. Singh, Resource Scientist & Group Head, Advance Research Instrumentation Facility, MPCST for their kind consideration and encouragement to complete this work. The authors also wish to thank Mr. Amit Sen for his help during this work. References [1] H. Ahokas, M.J. Erkkila, PCR Methods Appl. 3 (1993) 65–68. [2] M. Bajpai, A. Pandey, S.K. Tewari, D. Prakash, Int. J. Food Sci. Nutr. 56 (2005) 287–291. [3] K. Bone, Clinical Applications of Ayurvedic and Chinese Herbs, Phytotherapy Press, Warwick, Queensland, Australia, 1996, pp. 131–133.
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