12th Class Biology Molecular basis of inheritance

  • question_answer 1) What is DNA finger printing? Mention is applications. 

    Answer:

    DNA FINGER PRINTING Definition. Technique of using DNA fragments, resulting from restriction endonuclease enzyme cleavage to identify particular individuals, is called DNA finger printing. Introduction. DNA finger printing technique was developed by Alee Jffery (1985, 86) at Leicester University, United Kingdom. Inheritance of DNA is very stable. Every person has specific pattern of DNA sequence which shows a combination of DNA sequence of both mother and father. Study of DNA finger prints helps in establishment of identity of a person, identification of criminals in case of a murder or rape, and paternity test in case of disputed parentage. This technique helps in identification even when the stains on victims clothes etc. are even several years old. In India, first test of DNA finger printing was done in June, 1989 for setting a disputed parentage in Madras. Laboratory for DNA finger printing is situated in Hyderabad at the Centre for Cell and Molecular Biology (CCMB). Paternity dispute cases are much more common in India and most of them are referred to CCMB for DNA evidence. Southern Blotting technique of DNA finger printing: VNTR (Variable number of tandem repeats) technique involved Southern blot hybridization using radiolabelled VNTR as a probe. The steps were: (a) For DNA finger printing, DNA is isolated from anybody cell or even from blood stains, semen stains or hair roots. (b) DNA sample is first digested with a restriction endonuclease enzyme and the digested sample is then gel electrophoresed. (c) DNA bonds in the gel are denatured into single strands by treating them with alkali solution. (d) Gel is laid on the top of a buffer saturated filter paper, placed on a solid support (e.g. glass plate), with its two edges immersed in the buffer. (e) A sheet of nitrocellulose membrane is placed on top of the gel and a stack of paper towels is placed on the top of this membrane (acts as an absorbent tissue). (f) A weight of about 0.5 kg is placed on the top of pack of paper towels. Leave this arrangement for a few hours or overnight. The buffer solution is drawn up by the filter paper wick, and passes through the gel to the nitrocellulose membrane and finally to the paper towels. While passing through the gel, the buffer carries with it single stranded DNA molecules, which bind to the nitrocellulose membrane, while the buffer passes through it to the paper towels.   Soluthern blotting technique of DNA finger printing.   (g) Next day, paper towels are removed and discarded. (h) Nitrocellulose membrane with single stranded DNA bands is incubated at 80°C for the 2-3 hours to fix the DNA bands permanently on the membrane. (i) This membrane now has a replica of DNA bands from agarose gel and can be used for hybridization with specific labelled DNA or RNA probes. (j) The membrane may then be washed to remove any unwound DNA and X-ray film is exposed to the hybridized membrane to get autoradiograph. Significance: DNA finger printing shows polymorphism of DNA which is used for identification of a personous with much more certainty that has hitherto been possible through techniques of blood groups since the number of blood groups available becomes a limitation. The technique has shown the possibility of two persons having same pattern of DNA finger prints is very remote (exception monozygotic i.e., identical twin.) ?             The number of repeats m VNTR shows high degree of polymorphism. ?             The size of VNTR differs from 0.1 to 20 kb. In autoradiogram after hybridization with VNTR probe several bands of varying sizes appear. ?             Alec Jeffreys used satellite DNA as probe that indicated high degree of polymorphism and was called as VNTR (variable number of tandem repeats). ?             VNTR belongs to a class of satellite DNA called as mini-satellite. ?             PCR (polymerase chain reaction increases the sensitivity of technique). ?             If an inheritable mutation 4s observed in a population of high frequency, it is called as DNA polymorphism.    

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