This document provides an overview of DNA cloning including: 1. The basic steps in DNA cloning including isolation of vector and gene source DNA, insertion into the vector, and introduction into cells. 2. Uses of polymerase chain reaction and restriction enzymes in cloning. 3. Applications of cloning such as recombinant protein production, genetically modified organisms, DNA fingerprinting, and gene therapy.

2. Under the Guidance of, Prepared By, Mrs.K.Geetha . , Raviteja.A Assistant professor Reg.No.10T21S0101 CMR COLLEGEOF PHARMACY KANDLAKOYA (V), HYDERABAD - 501401

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

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.

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

15. A typical PCR protocol

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.

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

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 .