DNA Fingerprinting is a method which is based on the features (morphological) and this method is only restricted to human beings for giving the identity to a person at the molecular level. This method helps to find the genetic material identity of an individual.
It is based on polymorphism which held on the basis of the gene sequence. This technique was discovered by a famous geneticist Sir Alec J. Jeffrey’s of the University of Leicester in the UK (United Kingdom), while the study of the gene of myoglobin (a protein that store energy in muscle cells. Earlier this method is only announced for human genetic analysis but later on the method also introduce the study of plants (botany). DNA Fingerprinting is used in plants just only to protect the biodiversity, identification markers for traits, gene diversity, and variation identification and for other things also. The popular techniques used with plants are RFLP (restriction fragment length polymorphism).ISSR (inter-simple sequence repeat), SSR (simple sequence repeat), RAPD (random amplified polymorphic DNA) etc.
Method to do DNA Fingerprinting in plants:
Firstly we will take a sample of DNA from plant cells, extraction of quantification and quality is done. Further, there are two steps as follow:
- Polymerase chain reaction-based (RAPD, ISSR, and SSR)
- Non- polymerase chain reaction (RFLP)
Firstly we will learn about PCR based method this include: a diluted form of DNA is mixed with master mix comprising a buffer, primer, liquid(water), dNTPs and Taq- polymerase enzymes in the Eppendorf tube of polymerase chain reaction. Then the mixture is set in PCR. PCR is programmed in a number of cycles and temperature according to the technique. After the cycles, the sample is transferred to electrophoresis, it can be PAGE and AGE (depend on method). For revealing the pattern on the plate stain is done.
Isolation of DNA from plant tissue can be done in a number of ways. Basic steps of removal of the cell wall and nuclear membrane around the DNA and separating of cell components like debris, proteins, lipids or RNA from DNA without giving harm to the integrity of the cell. In the year 1980 scientists Murray and Thompson discovered the CTAB method. Isolation of DNA from plants tissue is mainly from leaves. A mass of leaf about 1 gm is taken use in the powder form and piston using liquid nitrogen. Quickly transfer the powder of leaf to centrifuge tube (because it can lead to phenolic oxidation) it contains the extraction which contains buffer CTAB, NaCl, EDTA, TrisHCL. CTAB method cut the cell wall at the same time it makes DNA complex and does precipitation of it. EDTA also included in buffer extraction to chelate magnesium ion, a cofactor for many nucleases. The insoluble complex is made by breaking NaCl and ethanol precipitation concentrates the nucleic acid. Polysaccharides which are present in cell extract are removed by NaCl. Stabilization of chemicals and maintaining of Ph is done by TrisHCL. After this, the mixture treated with chloroform for centrifugation. Isoamyl alcohol used to remove cell extract protein and other contents. For precipitation of DNA we use ice cold isopropanol, it further treated with ethanol (70%). To prevent RNA contamination we add RNase enzyme and then sample stored for further use.