Indian Journal of Experimental Biology Vol. 47, November 2009, pp. 921-924
Protocol for improved extraction and PCR amplification of genomic DNA from liverwort, Plagiochasma appendiculatum Arvind Soni & Anil Kumar* Plant Genomic Lab, Molecular Biology and Genetic Engineering Division, National Botanical Research Institute, Luckonw 266 001, India Received 9 February 2009; revised 22 June 2009
A simplest method was followed for isolation of high quality genomic DNA form thallus of Plagiochasma appendiculatum that contained large quantities of polyphenols, terpenoids, tannins, contamination of high amount of RNA and polysaccharides. The method involved a modification of CTAB procedure using PVP (1%) and LiCl (4M) solution to remove polyphenols and RNA and some other binding proteins. The present protocol was found suitable for restriction enzyme digestion and random amplified polymorphic DNA (RAPD) analysis. Keywords:
CTAB, Genomic appendiculatum
DNA,
PCR,
Plagiochasma
With the advent of fast, reliable techniques of DNA studies by PCR (Polymerase chain reaction) and DNA sequencing, plant systematics have began to use DNA sequences data to study relationship between various plant species, thereby complementing the enormous amount of morphological data currently available on taxa ranging from vascular plants to bryophytes and algae. Liverworts are the thalloid green plants attached to the substratum of moist, shady and humid places with the help of simple, delicate hair-like projection called rhizoids which are the direct sources of contamination by means of microscopic organism like algae, fungi etc. During extraction process, DNA of these microscopic organisms interferes or degrades DNA when stored for long time which makes it contaminated and undesirable for amplification. Extraction of DNA from bryophytes is very difficult due to the presence of polyphenols, terpenoids1 and some other binding substances2. _______________ *
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These compounds make the DNA unmanageable in pipetting and inhibit the activity of Taq polymerase during PCR amplification3. We have tried many reported protocols in bryophytes (liverworts) and other higher plants described earlier4-18. But we could not amplify DNA of P. appendiculatum. Therefore to overcome these limitations, certain modifications are required for obtaining the purified genomic DNA. The present study deals with the modified form of StenØien (1999)1 protocol in bryophytes. Specifically, polyvinyl pyrrolidone (PVP) is used in the extraction buffer to remove polyphenols19 and lithium chloride for selective precipitation of RNA and other binding proteins. RNase treatment is also required for the removal of remaining small fragments of RNA, which are not visible on gel electrophoresis and inhibit the amplification cycle20. The present study was attempted to isolate purified genomic DNA from P. appendiculatum for restriction and amplification. The thallus of Plagiochasma appendiculatum was rinsed in ddH2O to remove the microscopic organism like algae, fungi and foreign plant tissue, checked by seeing under the microscope and stored at -80°C for further use. Chemicals used⎯ Extraction buffer containing 3%, CTAB, 50 mM, Tris-HCl (pH 8.0); 1.4 M, NaCl; 10 mM, EDTA (pH 8.0); 1%, β-mercaptoethanol (v/v); 1%, PVP (w/v); chloroform-isoamylalcohol 24:1 (v/v); isopropanol was used. LiCl (4M; Sigma), ribonuclease A (10 mg/ml; Bangalore Genie), TE buffer- 10 mM Tris-HCl (pH 8.0), EDTA (1 mM), deoxyribonucleotides (dNTPs; Bangalore Genie), Taq polymerase and its 10X buffer (Bangalore Genie), random primer (10 mM; Operon), TBE buffer (0.5X) were used. DNA extraction⎯ Prepared the extraction buffer in 30 ml polypropylene centrifuge tubes by adding 12 ml 2X CTAB buffer, 1% β-mercaptoethanol and 100 mg PVP mix and kept on ice. Thallus (1 g) of P. appendiculatum stored at -80°C and ground finely by using motor and pestle in the presence of liquid nitrogen. Transfer frozen ground thallus to above extraction buffer in tubes and kept into water bath at 68°C for incubation for overnight. The tubes were
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removed from water bath and allowed to cool at room temperature. Chloroform (8 ml) was added in the tubes, mixed by inversion to form an emulsion and centrifuged at 4000g for 10 min at 24°C. Transfer aqueous supernatant to autoclaved corex tubes using pipette. Isopropanol (0.7 vol) was added to precipitate DNA and observed threads at this step. Stored the tubes at -20°C for 5 h. After that, the tubes were centrifuged at 5000g for 15 min at 4°C and discarded the supernatant carefully without disturbing the pellet stuck at the bottom of tubes. Wash the pellet with ethanol (70 %), centrifuged again at 5000g for 5 min at 4°C and discarded the ethanol. The pellet was dried at 37°C in incubator for 15 min and then dissolved the pellet in dH2O (700 μl). LiCl (1/3 vol; 4 M) was added for RNA precipitation and some DNA binding proteins that inhibit the PCR reaction. These tubes were stored at -20°C for 1h, centrifuged the tubes at 5000g for 10 min and separated out the supernatant in new Ependroff tubes. To these tubes 2 μl of RNase A (10mg/ml) was mixed and incubated in water bath at 37°C for 1 h. Then equal amount of saturated phenol was added and again centrifuged at 5500g for 10 min. The supernatant was taken into new Ependroff tubes, added equal volume of phenol-chloroform and spun at 5500g for 10 min. The supernatant was taken into new Ependroff tubes, added equal volume of chloroform and spun again at 5500g for 10 min. The supernatant was separated and added 0.7 volumes of cold isopropanol and store at -20°C for 5 h. Centrifuged the tubes at 6000g for 15 min at 4°C, discarded the supernatant carefully and collected DNA pellet stuck at the bottom of tubes. Wash the pellet by 70 % ethanol, centrifuged again at 7000g at 4°C, discarded the ethanol and kept the tubes in incubator at 37°C for 15 min to dry the pellet. Dissolve the pellet in TE buffer (100 μl) and store DNA at -20°C until used. RAPD-PCR procedure⎯ The random primer kits of OPA, OPB and OPD (OPERON Technologies) were used for RAPD analysis. PCR was carried out in 20 μl volumes using DNA (20ng), dNTPs (2 mM of each of four nucleotides: Fermentas), 10X Taq buffer, 5 pmol primer, and 1 unit Taq DNA polymerase (Bangalore Genie). PCR conditions were initiated at 92°C followed by 44 cycles of denaturation at 92°C for 1 min, annealing at 42°C for 1 min and extension at 72°C for 1:30 min, followed by final extension of 5 min.
Amplified product were separated in agaorse gel (1.4 %) stained with ethidiun bromide and visualized under ultraviolet (UV) light. The image of gel was taken by multiimager TM 3400 (Alpha Innotech Co.) The quantity of purified genomic DNA of P. appendiculatum was detected by agaorse gel electrophoresis (to determine the intactness of DNA) and also DNA digested with the restriction enzymes. The digestion was performed by using approximately 1 μg of DNA and 1 unit of restriction enzyme and incubated in 37°C for 4 h. It was completely digested, as observed by characteristic “smearing”, absence of the high molecular weight genomic DNA (30-50 μg/g) and RAPD reaction only at 20 ng of DNA. Usually the amount of template DNA for amplification of genomic DNA ranges from 10-50ng. Higher amounts of template DNA usually increase the yield of non-specific PCR products, but present modified protocol was required only 20 ng to obtained reproducible and comparable amplified bands. (Fig. 1) Mostly 100mM of dNTPs were used in reported protocols that give inaccuracy, non-reproducible and incomparable bands, but modified protocol required only 20mM of dNTPs to obtained reproducible bands for the RAPD analysis and saves the loss of dNTPs that may be used in further reactions. The purity of DNA is also influenced by the presence of secondary metabolites such as
Fig. 1⎯ Amplified DNA band using RAPD-PCR procedure [Lane M- EcoRI and HindIII double digested λ-DNA Marker; Lane 1-crude DNA; Lane 2- Purified DNA; Lane 3 & 4-Genomic DNA digested with EcoRI and HindIII; and Lane 5-7-amplified DNA with random primer OPA-12, OPB-12 and OPD-5, respectively].
NOTES
polysaccharides, polyphenols, and tannins21 which inhibits the enzyme activity during PCR. Bryophytes contain high amount of polyphenols, which inhibit activity in PCR. Cell disruption by grinding in liquid nitrogen and incubate in extraction media with detergent results in lysis of the cell containing the polyphenols. Even in the presence of PVP (a solid polymer that has high molecular weight and water soluble), polyphenols adhere to DNA and forming coloured matrix around them. PVP forms a complex with polyphenols through hydrogen bonding and allowing them to separate from DNA and reduces the level of polyphenols from product19. So that, PVP was used for the removal of secondary metabolites from DNA and increased the quality and purity in the present protocol. Contamination with RNA occurs with isolation of genomic DNA because abundant amount of cellular RNA precipitate concurrently with DNA. Several reports have indicted that the presence of RNA can repress PCR amplification and lead to nonreproducible and unreliable DNA amplification patterns in RAPD analysis2, 22-24. So, in the present study precaution was taken to remove RNA from genomic DNA and added 4M of lithium chloride. Larger RNA molecules (rRNA and mRNA) selectively precipitated and separated out from DNA. After RNA precipitation by LiCl, RNase (10 mg/ml) was used to remove small fragments of ribonucleosides which are not usually detectable on gel electrophoresis and they contribute to the absorbance of 260 nm, yielding misleading high flurometeric reading or serves as primer in the thermal cycle reaction25. The random amplified polymorphic DNA analysis described by Williams et al26 is a method for creating genomic fingerprinting of plants species. During PCR annealing temperature is one of the important factors to give the non-specific PCR products. In the present study, the assay was done at the temperature ranging from 36°- 42°C and obtained reproducible and reliable bands at 42°C in case of P. appendiculatum for RAPD analysis to attain comparable results (Fig. 2). The protocol described for the isolation of purified DNA from P. appendiculatum is reliable and stable. DNA, thus obtained, can be amplified by PCR requiring only 20 ng of DNA. So, the present protocol is effective for DNA extraction and RAPD analysis in P. appendiculatum.
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Fig. 2⎯ Representing the optimization of annealing temperature. [EcoRI and HindIII double digested λ-DNA Marker (lane M). Stepwise optimization of different annealing temperature ranging from 36°C (lane 1); 38°C (lane 2); 42°C (lane 3); and 44°C (lane 4)].
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