The Basic Technique of Fingerprinting - Case Study Example

In this new Era, botanicals accurate recognition and uncovering of adulterants are the greatest challenges faced by the natural product companies. DNA fingerprinting refers to the technique used to reveal the specific DNA profile for a particular organism. This profile is as unique and as diverse from the other plant, as a separate fingerprint. It can differentiate plants from different species, families, and genera’s and from variety of cultivations. The DNA fingerprinting technique is basically employed to determine the genetic identity of the organisms. It is based on polymorphism reaction taking place on the bases of the genome at molecular level and a number of techniques are based on Polymerase Chain Reaction ( PCR) used for the amplification of DNA. The basic technique of fingerprinting was developed by geneticist Alec J. Jeffery of Great Britain, who was associated with University of Leicester. DNA profiling is also used in plants for identifying different characters, identification of gene diversity and for the management of biodiversity. Widely used techniques are Restrictive fragment length polymorphism, randomly amplified polymorphism DNA, Inter sample sequence repeats, Simple sequence repeats. However, a number of traditional techniques are being used for the identification of DNA fingerprinting but new innovations are being made in the field to make the process easier and precise. These traditional methods of DNA testing involved the comparison of random pieces of DNA but afterwards more reproducible and accurate analysis were conducted based on the comparison of gene sequencing in a particular DNA strand. Living cells, from flora and animals to human beings, are composed of basic building blocks namely DNA or deoxy-ribonucleic acid. It guides the production of enzymes or compounds that are required for growth or development of various traits of living being. It is a hereditary material which is passed from one generation to another for the production of specific types of characteristics as guided by the genetic material. Nearly every cell in an organism’s body is composed of same type of DNA. DNA is found the form of double helical strands of nucleotide. This DNA molecule is composed of three parts De-oxyribose Sugar, a phosphate group and the bases.(Khan, 2012) The genetic information is stored in DNA in the codes of these four chemical bases namely Adenine, Thiamine, Guanine and cytosine. The order of these bases determines the encoded genetic information. And all of these parts combine to form a single nucleotide structure. The sugar from one nucleotide links with the base of other nucleotide forming the structure of double helix. Meanwhile the bases links up with their complementary base present at the other side of the DNA strand that is Adenine bind with Thiamine and Cytosine binds with Guanine. The basic method of DNA fingerprinting is the extraction of DNA from the source and then qualitative and quantitative analysis of the fragment followed by further two steps. 1) PCR based: RAPD, ISSR, and SSR 2) Non-PCR based: RFLP First step is to extract the DNA from the plant tissue, mainly leaves are preferred. DNA is extracted from the source, very carefully while removing the cell wall and nuclear membrane and it is separated from the other components such as proteins, lipids and RNA. About 1 gm of leaf is taken in a mortar and piston and triturated to make a paste of powdered leaf with liquid nitrogen. This paste is mixed with extraction buffer containing, Ethyline Di- amine tetra- Acetate.(EDTA) which is used for the chellation of Mg ions present in the nucleases, CTAB is present which reacts with DNA and precipitates it and lyses the cell wall, NaCl can accelerate the breaking of insoluble complexes in the nucleic acid so it can be concentrated by ethanol precipitation and TrisHCL maintains the PH of the solution so that the chemicals which are released from the cell do not degrade instantly. Afterwards chloroform is added to remove cell free proteins and phenolic compounds which are extracted. The mixture is sent for centrifugation process. In this technique the mixture is rotated in fixed number of cycles per minute leaving behind the mixture with distinct layers of separated constituents. The supernatant liquid achieved after centrifugation is treated with isopropanol to precipitate (ppt) DNA. RNAse is an enzyme added afterwards to remove RNA from the sample. This sample needs to be stored at 4° C. DNA’s quantification and quality assessment is done afterwards using Ultraviolet-visible spectrophotometery, which is a method to determine the purity and quality of the sample using absorbance of the discharged light from a source which subjects rays of specific wavelength. The normal value for the absorbance check is 260 – 280 wavelengths (λ) for DNA. If the absorbance value is 1.8, it shows highest purity, if more than 1.8 than RNA contaminant is also present and less than 1.8 shows protein presence. To make the bands of DNA visible a method named AGE is conducted. Clear single bands are required for the perfect results otherwise smear like image is seen. For PCR based methods DNA samples are diluted for PCR requirements. In this PCR based technique, the DNA extract is blended into a mixture of PCR buffer, primer, water, Taq polymerase enzyme. The mixture is than loaded into a PCR apparatus which is pre programmed to rotate in a fixed number of cycles under specified temperature, which is determined according to the technique. After these rotations the sample is subjected to electrophoresis, either AGE (Agarose gel electrophoresis) or PAGE depending upon the type of technique. Finally the staining is done to visualize the banding patterns. After DNA is isolated enough copies are replicated. Amplification of DNA is done by Polymerase Chain Reaction which involves the use of thermal cycling for the purpose. This method is an invitro method which is used to duplicates a DNA section into millions of copies from small amounts of DNA templates. It involves processes as DNA denaturing, Annealing and primer extension. This process involves the separation of double strands of DNA into a single strand by the application of temperature from 94 to 96 C for 1 to several minutes applied on the targeted DNA strand but higher temperatures may also be applicable. Then temperature is lowered to 50-65 C which allows the left and right primers to pair up with their complementary bases. These primers are designed to match the DNA region to be amplified. The duration for temperature application is from one to several minutes. Then temperature is again raised to 72 C so that Taq polymerase is allowed to bind with each primer site and then it synthesizes or extends the DNA strand.(CSHL, 2011) The number of cycles used in the PCR should be optimized; too many numbers of cycles can increase the amount and complexity of the non- specific product. Taq Polymerase is a DNA polymerase enzyme which is obtained from a bacterium Thermus Aquaticus and it is widely used in Polymerase chain reaction to amplify short segments of DNA. The use of thermostable Taq polymerase eliminates the need of any additional enzyme in the PCR during thermo cycling process thus, made PCR applicable for wide range of molecular biology applications. There are various techniques used for the DNA fingerprinting. Depending upon the requirements, cost and skills different techniques are employed RFLP is one of the major techniques to detect the variation in the DNA sequencing. The principle behind the technology is the comparisons of the band profiles generated after restriction enzyme digestion in DNA molecules. Diverse mutations affect the DNA, producing different fragments of variable lengths. DNA is first extracted and then digested with a chosen restricted enzyme which will cut the DNA in predictable way and giving reproducible sets of DNA fragments of different lengths called variable numbers of tandem repeats (VNTR). These VNTR’S are different in different plant species according to numbers and location of restriction enzyme recognition sites. The Southern Blot experiment is done in this technique. The restriction fragments are separated by electrophoresis mainly Agarose gel electrophoresis (IPGRI,2003) to detect the polymorphism. Hybridization and visualization by autoradiography or chemiluminesence, is done afterwards. The method is not usually preferred due to high cost, labor and professional’s extensive skill requirements. Randomly amplified DNA polymorphic markers is a PCR based genetic assay and it is most commonly used for primary assay. It differentiates between DNA sequencing of the two plant species and helps the search of random sequences required for amplification. DNA is cut and amplified using single primers at annealing temperatures that is 50 to 65 C. RAPD markers are decamer (10 nucleotide length) DNA fragments. RAPD does not require the knowledge of target DNA sequence and it, the identical primer will or will not amplify the segment of the DNA, depending on the sequence which is complementary to the position of the primer. If any mutations occur in the target DNA than no PCR product is produced resulting in different pattern of amplified DNA segments on gel. Amplified segments are separated by gel electrophoresis and polymorphism is detected by the presence or absence of bands of particular sizes. The polymorphism is primarily due to variation in the primer sites of annealing. It is an inexpensive method than the RFLP but the drawback is it lacks specificity due to low annealing temperatures and easier reaction conditions. SSR or microserellites are a class of repetitive DNA elements which show high degree of polymorphism. They are arranged in tandem arrays consisting of 5-50 copies and are abundant in plants. They are isolated using hybridized probes following their sequence. Amplified product is separated by gel electrophoresis and then visualized by silver staining autoradiography or via automation after fluorescently labeling the primer. Advantage of SSR is that the amount of DNA required is less than RFLP but separate SSR primers are needed for each species. The technique is more robust and efficient than RAPD. It is variation of SSR that requires simple sequence repeat primers to amplify regions between their target sequences. It is based on SSR for the amplification of inter-SSR DNA sequence. Using the technique unlimited number of primers can be achieved of di, tri, tetra and penta-nucleotide variations. The method provides highly reproducible results with higher levels of polymorphism. Amplified Fragment Length Polymorphism (AFLP) is variation of RFLP but is PCR based method. Sequences are selectively amplified using primers. This is more useful method than RFLP and RAPD. It is reproducible and cost effective. Recognition of 19 different diseases of soya bean is done by the use of fingerprinting technique. Various fingerprinting methods are being done on soya beans to determine the length polymorphism of SSR in the specie. Chromosomal maps of the genome are being produced using DNA markers to identify and label genes controlling the economically favorable traits of the plant. These also help in isolation of these genes and their transference to the cultivars by the application of biotechnological tools and produce resistance to diseases and production of quality traits. (Soybean Genomics,2011) Specific information about the genesis of plants, in terms of family, genus and specie can be determined by DNA fingerprinting which is helpful in identification of the plants and also provides a new perspective in terms of growth and development and disease origin of plants. As there is a growing trend in physicians to use botanicals as complementary and alternative medicine proper testing is required according to FDA guidelines to ensure identity, purity, potency and strength. This use opens a wide range of application of DNA fingerprinting in botanicals. A large number of lethal side effects are also associated with the consumption of botanicals which can be determined by using the technique and afterwards such products can be avoided. Thus it is becoming a powerful tool for the authentication and quality control of herbs. By genetically modifying the DNA structures scientist are producing plants which are resistant to certain diseases or environmental factors. Genetical engineering allows the plants to survive in less than favorable environments and make it possible for them to adapt to the existing environment. Moreover, information about the molecules involved in plant health helps the scientist to identify and engineer the genes that regulate such healthy processes within plants and help the plant regenerate faster. BASIC OUTLINE: INTRODUCTION Introduction of Plant fingerprints New and Traditional Techniques Introduction Of Deoxy Ribonucleic Acid METHOD: DNA Extraction Quantification and Quality Assessment PCR (1) DNA denaturing (2) Annealing (3) Primer Extension ENZYME TECHNIQUES 1) Restriction Fragment Length Polymorphisms (Rflps) 2) Randomly Amplified Polymorphic DNA (Rapds) 3) Simple Sequence Repeats: 4) Inter Simple Sequence Repeats 5) Amplified Fragment Length Polymorphism (AFLP): EXAMPLE CONCLUSION Significance of Plant Fingerprinting REFRENCES: Cold spring harbor labortarory CSHL, (2011), DNA learning Centre, http://www.dnalc.org/resources/animations/pcr.html IPGRI and Cornell University, DNA-based technologies Restriction fragment length polymorphisms, Available at:http://www2.bioversityinternational.org/Publications/Molecular_Markers_Volume_1_en/molmarkers/PDF/RFLPs.pdf Top of FormKhan, F. A. (2012). Biotechnology fundamentals. Boca Raton, FL: CRC Press. Virginia Tech,(2011),Soybean Genomics: Crop genetics at Virginia Tech < Available at> http://www.cropgenetics.cses.vt.edu/soybeangenomics/index.html Bottom of Form Read More