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14. Molecular Genetic Methodologies II.pdf

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McGill University
Biology (Sci)
BIOL 200
Richard Roy

Naveen Sooknanan McGill Fall 2011 Molecular Genetic Methodologies II: E. coli and bacteriophage lambda were, and still are, key elements in molecular biology. Replicational, transcriptional and translational mechanisms are nearly identical to higher eukaryotic organisms, although the molecules used in these mechanisms are different Purified components such as proteins from these sources have helped a great deal in biological procedures Paul Berg was able to take exogenous fragments of a desired DNA source and place them into heavily engineered DNA fragments called plasmids o This ushered in a new era of recombinant DNA technology and the birth of biotechnology o Many drugs such as insulin and many other hormones are available thanks to recombinant DNA technology used to treat diabetes, dwarfism, haemophilia, etc. Cloning, biologically speaking is the ability to reproduce identical organisms from one source. It was in the late 60s and early 70s that the first successful cloning experiment was carried out on an animal As a frog embryo developed into blastomeres which have not yet been determined, cloning was carried out by splitting each of these blastomeres which then each developed into one frog Cloning can also be used for different purposes; taking the nucleus of a differentiated call and placing it into an innucleated site will cause nuclear reprogramming during which the nucleus becomes reprogrammed to the needs of the site. Dolly, the cloned sheep, was created from the nucleus of a mammary gland o Unfortunately, she passed away early due to reason scientists are still trying to fully understand It is also possible to clone DNA fragments of a genome using recombinant DNA technology. This can be useful for identifying mutant phenotypes. The Antennapedia gene from fruit flies can be mutated such that legs grow where its antenna should be For biotechnology, milligram amounts of DNA are needed (rather than the micrograms needed previously) which means the desired DNA strand needs to be replicated and mass produced o The DNA fragment it placed into a plasmid and is allowed to replicate in order to quickly increase the purified amount of DNA available Restriction enzymes, such as EcoR1, are used to cut DNA at specific palindromic sites known as restriction sites These produce either blunt or tapered cut which produce sticky ends These overhangs can then bind to a restriction site of the same enzyme on the polylinker region of the plasmid o The polylinker is a only region of the plasmid where these restriction sites occur only once per enzyme 1Naveen Sooknanan McGill Fall 2011 Plasmids conventionally have some sort of antibiotic resistance, in this case, then amp gene which codes for ampicillin resistance Once useless bits of a DNA fragment are removed and the useful part is inserted into a plasmid by using the same restriction endonuclease for both the plasmid and the DNA fragment, the new specialized plasmid is called a vector In order to insert the desired DNA fragment in a specific direction, 2 restriction enzymes must be used to make 2 different sticky ends Using the 2 same endonucleases on a plasmid will allow insertion of the fragment in a directional manner By taking a desired DNA sequence and fragmenting it, it is possible to insert each of these fragments into a plasmid creating a DNA library of vectors These vectors can then be propagated when needed A genomic library contains DNA is chromosomal form, containing exons, introns, repeated sequences and other noncoding regions o This way, it is possible to organise and entire genome in a library A cDNA library uses DNA which has be reverse transcribed from an mRNA molecule o This allows us to look at either the mRNAs present in a given cell or the proteins coded by
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