Professor
Cell & Molecular Biology and Genetics
Sarojini Naidu Government Girls Post Graduate Autonomous College Bhopal - 462016
Cell Phone - 94244 17792 Email -
[email protected]
Professor H.K. Garg 17 Oct 2013
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Recombinant DNA is the formation of a novel DNA sequence by the combination of two DNA fragments
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These are taken from two different organisms
Professor H.K. Garg 17 Oct 2013
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The method by which DNA of the donor organism (target DNA) is cut into fragments with the help of Restriction Enzymes and insert one of these fragments into the DNA of the host.
Professor H.K. Garg 17 Oct 2013
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Hamilton Smiths (1970) at Johns Hopkins Medical School isolated the first RE that cut DNA at a very specific nucleotide sequence.
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Stanley Cohen & Herbert Boyer (1972) created Recombinant DNA.
Professor H.K. Garg 17 Oct 2013
Target DNA (Deoxyribonucleic acid) §
DNA is the hereditary material passed on from generation to generation.
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DNA is a long doublestranded chain of nucleotides
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DNA is made up of four nucleotides: A, C, G & T.
Professor H.K. Garg 17 Oct 2013
Target DNA (Deoxyribonucleic acid) §
A always pairs with T
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C always pairs with G
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Two complementary DNA strands will separate when heated, and will spontaneously pair together again when cooled.
Professor H.K. Garg 17 Oct 2013
Coning Vector •
Vector is a small piece of DNA into which a foreign DNA fragment can be inserted LacZ MCS
Ampr Ori
pBR322
pUC18
4361bp Ampr Tetr
Ori
Professor H.K. Garg 17 Oct 2013
Plasmid as a Coning Vector •
Plasmids are naturally occurring extrachromosomal DNA molecules.
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Plasmids are circular, double-stranded DNA.
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Plasmids can be cleaved by restriction enzymes, leaving sticky or blunt ends.
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Artificial plasmids can be constructed by linking new DNA fragments to the sticky ends of plasmid.
Professor H.K. Garg 17 Oct 2013
Restriction Enzyme •
Restriction Endonuclease is an Enzyme that cuts DNA (both single as well as double stranded) at specific nucleotide sequences known as restriction sites
Professor H.K. Garg 17 Oct 2013
Recognition site Cuts usually occurs at a palindromic sequence SmaI: produces blunt ends 5´ CCCGGG 3´ 3´ GGGCCC 5´ EcoRI: produces sticky ends 5´ GAATTC 3´ 3´ CTTAAG 5´
DON'T NOD DOGMA: I AM GOD NEVER ODD OR EVEN TOO BAD – I HID A BOOT RATS LIVE ON NO EVIL STAR NO TRACE; NOT ONE CARTON WAS IT ELIOT'S TOILET I SAW MURDER FOR A JAR OF RED RUM SOME MEN INTERPRET NINE MEMOS CAMPUS MOTTO: BOTTOMS UP, MAC GO DELIVER A DARE, VILE DOG MADAM, IN EDEN I'M ADAM OOZY RAT IN A SANITARY ZOO AH, SATAN SEES NATASHA LISA BONET ATE NO BASIL DO GEESE SEE GOD GOD SAW I WAS DOG DENNIS SINNED
Professor H.K. Garg 17 Oct 2013
DNA Ligase Enzyme •
Catalyze joining or recombine the two fragments of DNA - a process known as ligation.
Professor H.K. Garg 17 Oct 2013
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Both target DNA and the Vector are cut with a RE
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The Restriction Enzyme leaves sticky ends
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The sticky ends bind both the DNA together
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Ligase seals the DNA
Re-joined by Ligase Enzyme
Professor H.K. Garg 17 Oct 2013
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DNA is taken up by the Host Cells
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As the Host Cells grow, DNA also grow simultaneously.
Professor H.K. Garg 17 Oct 2013
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Three types of libraries can be developed :
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DNA Or Genomic Libraries
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cDNA Libraries from mRNA
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Expression Libraries to express foreign proteins
Professor H.K. Garg 17 Oct 2013
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A Genomic Library contains all the Chromosomal DNA of a cell
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DNA is purified and fragmented into pieces carrying thousands of bases.
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The size of fragments depends on the capacity of Vector to accommodate and propagate the DNA.
Professor H.K. Garg 17 Oct 2013
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A cDNA Library contains all the sequences of mRNA found in a cell
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The single stranded mRNA molecule is converted into double stranded DNA through the action of reverse transcriptase.
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They lack transcriptional control and intronic sequences found in genomic clones.
Professor H.K. Garg 17 Oct 2013
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Again a cDNA Library in a special form of vector that permits transcription of the incorporated cDNA.
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Eukaryotic proteins are formed in bacterial host cells that are normally not present in prokaryotes.
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Proteins can be made and purified more easily in bacterial cells.
Professor H.K. Garg 17 Oct 2013
Laboratory Applications •
Structure & Function of a gene in isolation and in varied juxtapositions.
Commercial & Therapeutic Applications •
Therapeutic Cloning
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Reproductive Cloning
Professor H.K. Garg 17 Oct 2013
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Therapeutic Cloning is performed to harvest embryonic stem cells. These cells are found inside the developing embryo and they can be used to develop tissues & organs.
Professor H.K. Garg 17 Oct 2013
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A cell is removed from the patient’s body. Its nucleus is taken out and inserted into an enucleated egg cell.
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Cell division is triggered either chemically or through electric shock. The resulting embryonic stem cells are then removed and used to treat the patient.
Professor H.K. Garg 17 Oct 2013
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Therapeutic Cloning can help in tissue or organ transplantation.
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The Embryonic Stem Cells can generate a genetically 100% compatible tissue or organ.
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The donation of tissues and organs not required in such cases.
Professor H.K. Garg 17 Oct 2013
Professor H.K. Garg 17 Oct 2013
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Reproductive Cloning refers to create an exactly identical or vegetative copy of an organism.
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It is performed using a technique known as SCNT - Somatic Cell Nuclear Transfer
Professor H.K. Garg 17 Oct 2013
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The ovum of the animal to be cloned is enucleated.
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Then a cell is taken from the same organism and its nucleus is removed.
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This nucleus is then transferred into the enucleated egg cell.
Sir Ian Wilmut who cloned a sheep - DOLLY 1996-2003
Professor H.K. Garg 17 Oct 2013
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Even after revelation of entire human genome, our understanding about human genome is quite trivial
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Only 1.5% of the entire genome contains genes. The rest, overlooked so far as ‘junk DNA’, may have also a bearing on the expression of genes.
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There is no such process like meiosis (crossing-over) which occurs during normal sexcycle.
“Genetic cloning is a powerful tool and the maturity, with which we tackle it, would decide the fate of human race.”
Thank You for the active listening
Professor H.K. Garg 17 Oct 2013
