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GENETIC ENGINEERING and MOLECULAR CLONING

Working with DNA

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Prokaryotic vs Eukaryotic

Visualizing DNA
A karyotype is the number and appearance of chromosomes in the nucleus of a eukaryotic cell - It is also used to describe the complete set of chromosomes in a species, or an individual organism.

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Visualizing DNA

DNA Extraction

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DNA Extraction

Visualizing DNA

http://library.thinkquest.org/12022/ani-l6b.gif

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Visualizing DNA

Agarose Gel Electrophoresis

Genomic DNA MW MW bp

1000 800 500 200

Genomic DNA after restriction enzyme digestion

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Recombinant DNA Technology

Gene Cloning or Molecular Cloning or Genetic Engineering or Genetic Manipulation or rDNA Technology (Recombinant DNA Technology)

REVOLUTIONIZED BIOTECHNOLOGY

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So what REALLY is a GMO?

genetically modified organism (transgenic organism)

a GMO is an organism that contains a gene introduced intentionally by man (through genetic modification /engineering techniques)

Genome: similar to a pile of books

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Hybrid or crossing of genomes

Genetic Engineering

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Manipulating DNA: Ingredients

Restriction enzymes (a.k.a. restriction endonucleases) Cut DNA Ligase Joins DNA strands together Plasmid DNA Circular bacterial DNA

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Plasmid DNA
Bacterial DNA Plasmid DNA

plasmid DNA molecule that is separate from, and can replicate independently of, the bacterial chromosomal DNA DNA

1973
Stanley Cohen and Herbert Boyer created a transgenic bacterium containing the gene for frog ribosomal RNA using recombinant DNA technology

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Transforming the host cell

E.coli gene ligate Vector

transgene
gene Transform E.coli

rec vector

Transforming the host cell

GM E. coli

Recombinant vector/ plasmid DNA multiplies

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The Dawn of Biotechnology

1977 Genentech, Inc. reports the production of the first human protein manufactured in a bacteria: somatostatin, a human growth hormone-releasing inhibitory factor.

www.gene.com

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Manipulating DNA

The Dawn of Biotechnology

1975 1977 Frederick Sanger developed rapid DNA sequencing method (dye-terminator sequencing)

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Sequencing DNA: DNA extension

DNA polymerase

http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html

Copying of the DNA

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Sequencing DNA: Using dideoxynucleotides

DNA

Phosphodiester bond

Nitrogenous Bases

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Sequencing DNA: DNA on Gels

dGTP ddCTP dATP dTTP

G C GAA C G C T TAAC G CAG G T G T T G C GAT G T

DNA Polymerase

Sequencing DNA: Using fluorescence

dGTP ddNTP dATP dCTP

G C GAA C G C T TAAC G CAG G T G T T G C GAT G T

DNA Polymerase

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Sequencing DNA: Reading the sequence using a computer

http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html

The Dawn of Biotechnology

1978 Louise Brown, the first test tube baby was born in England

http://newsimg.bbc.co.uk/media/images/ 39315000/jpg/_39315173_203louisebirth 3.jpg

http://www.babble.com/CS/photos/babble/images/26 08/360x240.aspx

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The Dawn of Biotechnology

1981 1982 First transgenic mice and fruit flies 1983 First transgenic plants Kary Mullis invented the PCR technique

Polymerase Chain Reaction

Kary B. Mullis, working for Cetus Corporation, developed the Polymerase Chain Reaction in 1983. The technique allows for the rapid synthesis of DNA fragments. In about an hour, over 1 million copies of a DNA strand can be made For his development of PCR, he was co-awarded the Nobel Prize in chemistry in 1993. Mullis is currently doing HIV and AIDS research The technique has been invaluable to the development of biotechnology and genetic engineering. Its real world applications include gene mapping and forensic science.

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Kary Mullis: Surfer-Scientist

www.revolt.it

PCR

library.thinkquest.org

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PCR

library.thinkquest.org

PCR

library.thinkquest.org

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PCR

library.thinkquest.org

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The Dawn of Biotechnology

1990 Beginning of the Human Genome Project, the international effort to map all of the genes in the human body, was launched. Estimated cost: $13 billion.

Sequencing the Human Genome Using a Shotgun

http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html

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The Dawn of Biotechnology

1994 FDA approved the sale of the first genetically modified food, the FLAVR SAVR tomato (produced by Calgene) 1996 The cloned sheep Dolly was born at Scotlands Roslin Institute

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The Dawn of Biotechnology

2001 Working draft of the human genome sequence published by HGP consortium (Nature) and Celera Genomics (Science) 2002 Working draft of the rice genome sequence published (indica rice was completed by China) 2003 Human Genome Project completed

Major Applications of Genetic Engineering

Hon-Ming Lam, 2005

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Major Applications
Genetic Testing and Screening Cloning Gene Therapy Genetically Modified Crops

Five Major Applications

Genetic Testing and Screening Cloning Somatic Gene Therapy Germ-Line Therapy Genetically Modified Crops

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Genetic Testing and Screening

Screening for genetic defects e.g., sickle cell anemia, cystic fibrosis, Huntingtons disease Diagnosis and Prognosis Family planning Identification of individuals (parentage, forensics, wild animal poaching, etc.)

Amniocentesis or Chorionic Villus Sampling

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Five Major Applications

Genetic Testing and Screening Cloning Somatic Gene Therapy Germ-Line Therapy Genetically Modified Crops

Cloning
Cloning of genes: recombinant DNA Cloning in microorganisms: by the use of recombinant DNA for making medicinal products (e.g., insulin, etc.) Cloning of plants: vegetable propagation, tissue cultures Cloning of animals: Dolly, Polly Cloning of human?

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Cloned Plants

Cloned Sheep

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Some issues in human cloning

As an alternative to in vitro fertilization Bring back a dead loved one Reproduce oneself For experiments Act as an organ reserve

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Adenosine Deaminase Deficiency (SCID)

Cystic Fibrosis

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Under Virginia's 1924 Eugenic Sterilization Law (based on the case of Emma, Vivian, and Carrie): 7,000 people were surgically sterilized between 1924 and 1979 because they are feeble-minded, mentally defective epileptics

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Vitamin A Enriched Rice

Transgenic rice producing -carotene (precursor of vitamin A) Rice lacks vitamin A Rice is a major food for Asia and Africa 250,000 children go blind each year because of insufficient vitamin A

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Vitamin A Enriched Rice

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