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Under the Guidance of,  Prepared By, Mrs.K.Geetha . ,  Raviteja.A Assistant professor  Reg.No.10T21S0101 CMR COLLEGEOF PHARMACY KANDLAKOYA (V), HYDERABAD - 501401
CONTENTS ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
  Human DNA consists of about 3 billion bases, and more than 99 percent of those bases are the same in all people. The order, or sequence, of these bases determines the information available for building and maintaining an organism. DNA, or deoxyribonucleic acid, is the hereditary material,it is located in the cell nucleus (where it is called nuclear DNA), but a small amount of DNA can also be found in the mitochondria (where it is Called as mtDNA). INTRODUCTION
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STEPS IN DNA CLONING ,[object Object],[object Object],[object Object],[object Object],[object Object]
Cloning-: The production of identical copies of molecules, cells,  or replicating nucleic acid sequences from organisms from a single ancestor.  The DNA of interest can then be propagated in a foreign host cell (bacterial plasmid) This technology has been around since the 1970s, and it has become a common practice in molecular biology labs today.
[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Plasmids – vehicles for cloning Older cloning vector Newer cloning vector Tet r Amp r Ori pBR322 4361bp Ori pUC18 Amp r MCS LacZ
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Restriction enzymes nomenclature ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
DNA cloning requires restriction endonuclease and DNA ligase ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Plasmid vector + DNA fragment to be cloned Enzymatically insert DNA into plasmid vector Recombinant plasmid Mix E.coli cells with plasmids in presence of CaCl 2  Culture on nutrient agar plates containing ampicillin Cells that do not take up plasmids die on ampicillin plates Transformed E.coli cell survives Bacterial chromosome Independent plasmid replication Cell multiplication Amp R. Amp R. ORI
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A thermophilic (heat-loving) bacteria called  Thermus aquaticus  is the source of  Taq  DNA polymerase used in  PCR reactions. ,[object Object],[object Object]
A typical PCR protocol
Key features of DNA replication are used in DNA sequencing ,[object Object],[object Object],[object Object],[object Object]
Expression ,[object Object],[object Object],[object Object]
Expression ,[object Object],[object Object],[object Object],[object Object],[object Object]
Library Construction ,[object Object],[object Object],[object Object],[object Object],[object Object]
[object Object],[object Object],[object Object]
Genetically modified organisms   Introducing a foreign gene into an organism which can propagate creates a genetically modified organism. Transgenic sheep have been created. DNA fingerprinting   Hybridizing of genomic DNA with probes that recognize simple nucleotide repeats gives a pattern that is unique to an individual and can be used as a fingerprint. This has applications in forensic science, animal and plant breeding and evolutionary studies. Medical diagnosis   The sequence information derived from cloning medically important genes has allowed the design of many diagnostic test kits which can help predict and confirm a wide range of disorders. Gene therapy   Attempts to correct a genetic disorder by delivering a gene to patient are described as gene therapy.
Animals Can Be Cloned  by Several Methods ,[object Object],[object Object],[object Object],[object Object],[object Object]
Plants Can Be Cloned  from Single Cells ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Technology now exists to sequence everyone’s DNA Took just 4 months, $1.5 million to obtain the entire DNA sequence of James Watson.
Understanding the arrangement of genes may help understand disease
Genome resources for many organisms are available
Why is DNA Cloning Important? ,[object Object],[object Object]
Because of recent technological advancements cloning in the animals and humans has been an issue and is strictly banned, Scientists have made some major achievements with cloning, including the asexual reproduction of sheep and cows. There is a lot of ethical debate over whether or not cloning should be used. However, cloning, or asexual propagation has been common practice in the horticultural world for hundreds of years . Thus Cloning manipulate a novel gene in many different ways with the goal of uncovering its unique role in the cell .
 
 

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Cloning dna f inal

  • 1.  
  • 2. Under the Guidance of, Prepared By, Mrs.K.Geetha . , Raviteja.A Assistant professor Reg.No.10T21S0101 CMR COLLEGEOF PHARMACY KANDLAKOYA (V), HYDERABAD - 501401
  • 3.
  • 4. Human DNA consists of about 3 billion bases, and more than 99 percent of those bases are the same in all people. The order, or sequence, of these bases determines the information available for building and maintaining an organism. DNA, or deoxyribonucleic acid, is the hereditary material,it is located in the cell nucleus (where it is called nuclear DNA), but a small amount of DNA can also be found in the mitochondria (where it is Called as mtDNA). INTRODUCTION
  • 5.
  • 6.
  • 7. Cloning-: The production of identical copies of molecules, cells, or replicating nucleic acid sequences from organisms from a single ancestor. The DNA of interest can then be propagated in a foreign host cell (bacterial plasmid) This technology has been around since the 1970s, and it has become a common practice in molecular biology labs today.
  • 8.
  • 9.
  • 10.
  • 11.
  • 12. Plasmid vector + DNA fragment to be cloned Enzymatically insert DNA into plasmid vector Recombinant plasmid Mix E.coli cells with plasmids in presence of CaCl 2 Culture on nutrient agar plates containing ampicillin Cells that do not take up plasmids die on ampicillin plates Transformed E.coli cell survives Bacterial chromosome Independent plasmid replication Cell multiplication Amp R. Amp R. ORI
  • 13.
  • 14.
  • 15. A typical PCR protocol
  • 16.
  • 17.
  • 18.
  • 19.
  • 20.
  • 21. Genetically modified organisms Introducing a foreign gene into an organism which can propagate creates a genetically modified organism. Transgenic sheep have been created. DNA fingerprinting Hybridizing of genomic DNA with probes that recognize simple nucleotide repeats gives a pattern that is unique to an individual and can be used as a fingerprint. This has applications in forensic science, animal and plant breeding and evolutionary studies. Medical diagnosis The sequence information derived from cloning medically important genes has allowed the design of many diagnostic test kits which can help predict and confirm a wide range of disorders. Gene therapy Attempts to correct a genetic disorder by delivering a gene to patient are described as gene therapy.
  • 22.
  • 23.
  • 24. Technology now exists to sequence everyone’s DNA Took just 4 months, $1.5 million to obtain the entire DNA sequence of James Watson.
  • 25. Understanding the arrangement of genes may help understand disease
  • 26. Genome resources for many organisms are available
  • 27.
  • 28. Because of recent technological advancements cloning in the animals and humans has been an issue and is strictly banned, Scientists have made some major achievements with cloning, including the asexual reproduction of sheep and cows. There is a lot of ethical debate over whether or not cloning should be used. However, cloning, or asexual propagation has been common practice in the horticultural world for hundreds of years . Thus Cloning manipulate a novel gene in many different ways with the goal of uncovering its unique role in the cell .
  • 29.  
  • 30.