The Identification of Bambusa Sp

The identification of Bamboo using various PCR and Sequencing Techniques Abstract Often the wrong(p) bamboo species is sold to unsuspecting customers at shops. This offer have a disastrous effect on their garden. Three separate and unknown Bamboo leaf samples were taken and were ask to be distinguished familialally from one another. Using ITS-PCR desoxyribonucleic acid amplification techniques, the ITS locality DNA was amplified and used in PCR-RFLP and RAPD PCR in order to determine the genetic identity of each sample. Sequencing was performed, and results allowed us to distinguish between samples (to a certain extent. ) IntroductionBamboos be a group of woody perennial green installs (Wikipedia et al. 2006) that are found in many parts of the world. There are 91 genera and about 1,000 species of bamboo (Wikipedia et al. 2006). They are found in diverse climates, from cold mountains to hot equatorial regions. Bamboo is a passing desirable plant grown for many reasons in plan tations and gardens around the world. Many reasons it is grown are that it is a beautiful ornamental plant with unique properties. Bamboo is also an extremely strong plant that is light it is used in many building applications for floorboards, and is also often used in article of furniture making.There are a number of taller growing species that are effective at blocking out the eyes of pepping toms and nosy neighbors. There are both main forms of bamboo, each form describing the way in which the bamboo itself spreads. These are known as clumping (monopodial) and reckoningning (sympodial) forms. (Wikipedia et al. 2006) Clumping bamboo species tend to spread underground slowly. Running bamboo species are highly variable in their tendency to spread this is related to both the species and the soil and climate conditions. Some can send out runners several metres a year, season others can stay in the same general area for long periods.If neglected, they can be invasive over time and c an cause problems by moving into adjacent areas. The reputation of bamboo as being highly invasive is often exaggerated, and situations where it has taken over large areas is often the result of years of untended or neglected plantings. Many invasive bamboo species are often sold, unsuspectingly to people, who plant them without realizing this. The result of this is the complete takeover of ones garden. Some species of bamboo can grow at a tremendous rate, some at over 36inches (90cm) a day, providing it is provided with ideal conditions (OneEarth, 2006).Plant Biosecurity breaches often occur when bamboo plants are imported with incorrect or glowering labeling, often in an attempt to bring illegal ornamental species in to the country for indoor use. This black market operation is a serious menace to native species of plants, and, if a threatening sympodial bamboo species is imported and planted in place of a monopodial (which is preferred, as they do not spread), serious damage to native forests and grasslands can occur (NGIA, 2006). Some of the techniques that can be used to identify to a species level are PCR-ITS, RAPD, and PCR-RFLP.These will be used to identify our unknown samples of bamboo. ram To identify, to a species level, using nucleotide analysis and sequencing techniques, three unknown samples of bamboo. Materials For DNA extraction 3 Unknown Bamboo Samples (Leaves) Mortar and Pestle Liquid Nitrogen Quiagen Dneasy DNA origin Kit Centrifuge tubes Pipettes and Tips Ice and Esky Quantification of DNA Well Combs (10uL) rise up UV Transilluminator Agarose Tris Borate EDTA Ethidium Bromide Loading Dye Centrifuge Tubes Gel Tank (To run agarose gel electrophoresis) Pipettes and TipsFor ITS based PCR 5uL of extracted DNA 5x Reaction buffer MilliQ (Ultra Pure Water) DNTPs (dATP, dGTP, dCTP, dTTP) PCR Machine MgCl2 Centrifuge Tubes Pipettes and Tips For RAPD-PCR ITS-PCR DNA product 5x Reaction buffer MilliQ (Ultra Pure Water) MgCl2 Primers OPM-01 and OPM- 17 Wells Well Combs (10uL) UV Transilluminator Agarose Tris Borate EDTA Ethidium Bromide Loading Dye Centrifuge Tubes Gel Tank (To run agarose gel electrophoresis) Pipettes and Tips For ITS-RFLP ITS-PCR DNA product Enzymes Hha1 and Rsa1 buffer Red (Rsa1) Buffer C (Hha1) MilliQ (Ultra Pure Water)Wells Well Combs (10uL) UV Transilluminator Agarose Tris Borate EDTA Ethidium Bromide Loading Dye Centrifuge Tubes Gel Tank (To run agarose gel electrophoresis) Pipettes and Tips Methods DNA Extraction and Purification Quiagen Dneasy Kit ITS-RFLP ITS Region is a particular sequence of DNA which is present in all organisms. It is a region, in between each greenness sequence, contains DNA that is highly conserved and unique amongst a particular species, and is thus not used to translate into proteins. Enzymes are used to restrict or fare the DNA at certain points.The location of the cuts depends on nucleotide sequence that the enzyme recognizes. The number of nucleotides in sequence determi nes size of the restricted piece of DNA in base pairs (BP). ITS-PCR This is done to amplify the ITS region DNA which is highly conserved and unique to each individual species Primers ITS 1 and ITS 4 are used because the ITS region (18s, 5. 8s and 28s regions) are common in all organisms. The region in between the 18s and 28s is the region that is highly conserved and unique to any given species. Added to Master coalesce (containing buffer solution) PCRd ITS Region DNA is amplified out RAPDRAPD Primers OPM-01 and OPM-17 are added to the ITS-PCR DNA product and where are given a genetic fingerprint of the DNA. HOW, WHEN, WHAT, WHERE, WHO? What was done? comfortable detail for repetition by others Results (facts only) (2) HOW, WHEN, WHAT, WHERE? What was found? Presentation of results as simply and clearly as possible Figures to present data and concepts clearly and concisely (a moving-picture show is worth 1000 words) Types of figures photographs, drawings, tables, graphs Numerical data as tables or graphs (graphs preferred) Text to point out trends (not repeat information in figures) Discussion (3) WHY, WHAT, WHO?What does it mean? reading of results relative to the hypothesis or aim Comparison with work of others References (6) WHO? List of all references cited in text http//www. bonsai-bci. com/species/bamboo. html Sabrina Caine Last limited accessed 01/06/06 http//en. wikipedia. org/wiki/Bamboo wikipedia last modified 27/05/06 accessed 01/06/06 http//www. 1earth. com. au/collect/wicker_furniture. html last modified 27/05/06 accessed 01/06/06 1Earth Antiques and Appraisals http//www. ngia. co. nz/news/507bamboo. php Nursing and Garden Industry Association (NGIA) Wellington, New Zealand Accessed 01/06/06 Updated