RAPD Analysis

RAPD analysis of different Rice variety

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Steps containing:

1. Collection of Rice Varity
2. Rice grow in Plastic pot
3. Green leaves taken from the new rice plant
4. DNA isolation
5. DNA confirmation by Agarose gel electrophoresis
6. DNA quantification by UV Spectrophotometer
7. PCR of DNA
8. Finally PCR products run on agarose gel electrophoresis
9. Dendrogram among varity.
10. Collection of Rice Varity: Rice varity were collected from different parts of the country. Eleven rice vartity were collected
11. Rice was grown in Plastic pot: Collected rice verities then grow in plastic pot until they produced green leaves.
12. Green leaves taken from that rice plant.

Pic: RAPD Analysis.Photo Credit: freepik.

Table of Content

• DNA isolation:
• DNA confirmation by Agarose gel electrophoresis:
• DNA quantification by UV Spectrophotometer:
• PCR of DNA :
• PCR products run on agarose gel electrophoresis:
• Dendrogram among varity:

DNA isolation:

Juvenile and actively growing fresh leaf tissues were collected for the isolation of genomic DNA. Total genomic DNA was isolated from rice leaves following Phenol: Chloroform: Isoamyl alcohol purification and ethanol precipitation method (Rahman at el., 2007).

Materials:

Reagents:

Extraction buffer: Extraction buffer is made of combination of Sodium chloride, EDTA, Tris and sodium dodecyl sulfate.

Procedure:

1. At first, healthy portions of the youngest leaves were cut apart with sterile scissors and forceps
2. Leaves were then washed very well with sterile distilled water and ethanol and dried on fresh tissue paper to remove spore of microorganisms and any other DNA contaminants
3. Approximately 0.2-0.3 inch of leaf tissues were cut into small pieces (as small as possible to facilitate grinding) and taken into a 1.5 ml eppendorf tube
4. 400 µl extraction buffers were added
5. Leaves were grinned by homogenizer
6. Then 400 µl extraction buffers was added
7. Vortexed for 20 second
8. Incubated for 5 minutes at 65˚C
9. Again vortexed for 20 second
10. Incubated for 10 minutes at 65˚C
11. Centrifuged for 10 minutes at 14,000 rpm
12. 600 µl supernatant was transferred in a new tube
13. 600 µl P: C: I (Phenol: Chloroform: Isoamylalcohol = 25:24:1) added into it
14. Again centrifuged for 10 minutes at 14000 rpm
15. Again the upper aqueous layer (about 400 µl) was transferred into a new tube
16. 500 µl P: C: I ( Phenol: Chloroform: Isoamylalcohol = 25:24:1) was added into it
17. Centrifuged for 10 minutes at 14,000 rpm
18. 400 µl upper aqueous layer was transferred into a new tube
19. 40 µl sodium acetate was (1/10th of extract volume) added into it and mixed it gently
20. 1000 µl of absolute alcohol (2.5 times of the total extract volume) was added to it and mixed gently
21. Centrifuged for 25 minutes at 14000 rpm
22. The pellet was observed and the liquid portion of the tube was removed carefully
23. 1000 µl of 70% ethanol was poured into the tube
24. The aqueous portion was removed carefully and the pellet was air dried
25. After removing the liquid completely, the pellets were then air dried and dissolved in 50 µl of TE buffer
26. Finally, the DNA samples were stored at –20°C

Precaution:

All glassware, centrifuge tubes, glass pipettes, micropipette tips, distilled water and buffer solutions were properly autoclaved to keep away from DNase contamination. Forceps, scissors and tissue homogenizer sticks etc. were sterilized with absolute ethanol.

DNA confirmation by Agarose gel electrophoresis:

Though extracted genomic DNA was treated with RNAase, it often contains some amount of RNA and pigments, which might cause over estimation of DNA concentration during spectrophotometer reading. Therefore, the DNA samples were evaluated both qualitatively and quantitatively using agarose gel electrophoresis and spectrophotometer respectively.

DNA quantification by UV Spectrophotometer:

Use of spectrophotometer:

For more conformation, DNA was quantified through spectrophotometer. After washing the cuvette carefully with de-ionized water, 2 ml (2000 ml) de-ionized water was taken in 'blank' cuvette and the optical density was adjusted to zero.

• Similarly, the 'test' cuvette was filled with 2 ml of de-ionized water and 2 ml of each DNA sample was added, and uniformly mixed with the help of micropipette.
• The absorbance of each sample was measured at 260 nm.
• The cuvette was again rinsed with de-ionized water and wiped with tissue paper and absorbance readings for the other samples were taken in the same way and recorded according to the following formula:
• DNA Conc. (ng/ μl) = Absorbance ×( Volume of distilled water (μl)/ Amount of DNA sample (μl) )× C.F (0.05) × 1000
• Where, C.F means conversion factor.

PCR of DNA :

2µl genomic DNA was added with 8µl PCR cocktail and finally total volume was 10 µl. During the experiment, PCR buffer, dNTPs, and primer solutions were thawed from frozen stocks, mixed by overtaxing and placed on ice. DNA Samples were also thawed out and mixed gently. The primers were pipetted first into PCR tubes compatible with the thermocycler used (0.2 ml). For each DNA sample being tested, a pre mix was then prepared including, in the following order: buffer, dNTPs, DNA template and sterile distilled water. Taq DNA polymerase enzyme was then added to the pre mix. The pre mix was then mixed well and aliquoted into the tubes containing primers. The tubes were then sealed and placed in a thermo cycler and the cycling was started immediately.

Precautions:

• Care was taken during pipetting all the chemicals of the reaction mix.
• Care was taken during use of the enzyme Taq DNA polymerase.
• Taq DNA polymerase, primer, reaction mix were always kept on ice.

PCR products run on agarose gel electrophoresis:

The amplified products were separated electrophoretically on 1.4% agarose gel. The gel was prepared using 1.4g agarose powder and 100 ml 1X TBE buffer. Agarose gel electrophoresis was conducted in 1X TBE buffer at 120 V for 1.5 hr. Two DNA ladder were used . The sizes of the ladders were 100bp and 1kb respectively. After electrophoresis, the gel was taken out carefully from the gel chamber and transferred in a prepared ethidium bromide solution for staining. Staining was done for 20 minutes.

Dendrogram among varity:

A dendrogram is a type of tree diagram showing hierarchical clustering — relationships between similar sets of data. They are frequently used in biology to show clustering between genes or samples, but they can represent any type of grouped data.

References:

Areas, R.G.B.M., Paiva, D.M., Souza, S.R., and Fernandes, M.S. (2006). Genetic similarities of rice landraces according to morphology, RAPD markers and grain protein content. Bragantia, 65: 19-28.

Arif, M., Kousar, S., Bajwa, M.A., Arif, A., and Zafar, Y. (2005). Genetic diversity among rice genotypes of Pakistan through random amplified polymorphic DNA (RAPD) analysis. Pakistan Journal of Botany, 37: 585-592.

Barooah, D., and Sarma, R.N. (2004). Genetic diversity analysis of traditional Sali rice (Oryza sativa L.) germplasm of Assam through RAPD markers. Indian Journal of Genetics and Plant Breeding, 64: 5-8.

Baruah, A. and Sarma, R.N. (2004). Genetic polymorphism of rice (Oryza sativa L.) from north-east India as revealed by RAPD assay. Res. Crops, 5: 220-226.

BBS. (2011). Agriculture Wing. Bangladesh Bureau of statistics, Ministry of planning, Government of the People ‘Republic of Bangladesh, Dhaka. 37P.

BBS. (2005). Hand book of Agricultural Statistics Bangladesh Bureau of Statistics, p: 14. Ministry of Planning, Government People’s Republic., Bangladesh

Chakravarthi, B.K., and Naravaneni, R. (2006). SSR marker based DNA fingerprinting and diversity study in rice (Oryza sativa L). African Journal of Biotechnology, 5: 684-688.

Chapco, W., Ashton, N W., Martel, R K., Antonishishyn, N ., & ,Crosby , W L. (1992). A feasibility study of the use of random amplified polymorphic DNA in the populationgenetics and systemic of grasshoppers. Genome, 35: 569-574.

ChengJun, W., ZaiQuan, C., XingQi, H., ShouHua, Y., KaiMing, C., and ChongRong, S. (2004). Genetic diversity among and within populations of Oryza granulata from Yunnan of China revealed by RAPD and ISSR markers. China Plant Science, 167: 35- 42.