NIS - BIOLOGY

Lecture 57 – Lecture 58DNA Technology

Ozgur Unal

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Genetic Engineering

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What comes to your mind when you hear the term “Genetic Engineering”?

Genetic Engineering

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Genetic engineering is the technology that involves manipulating the DNA of one organism in order to insert exogenous DNA (DNA of another organism).Example: Researchers have inserted a gene for a bioluminescent protein called green fluorescent protein (GFP) into various organisms.GFP is naturally present in jellyfishes in the north Pacific OceanSome organisms have been genetically engineered to synthesize GFP.Figure 13.3 mosquito larvae

Genetic Engineering

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Selective breeding is used to produce plants and animals with desired traits.Similarly, genetic engineering can be used to increase or decrease the expression of specific genes in selected organisms.Genetic engineering has many applications human health, agriculture etc.But how are these engineering processes achieved?

DNA Tools

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An organism’s genome is the total DNA present in the nucleus of each cell.Human genome contains around 25,000 thousand genes.In order to study a specific gene, DNA tools can be used to manipulate DNA and to isolate genes from the rest of the genome.

DNA Tools

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Restriction Enzymes:Some types of bacteria contain powerful defenses against viruses.These cells contain proteins called restriction enzymes that recognize and bind to specific DNA sequences and cleave the DNA within that sequence.A restriction enzyme, also called endonuclease, cuts the viral DNA into fragments after it enters the bacteria.There are many different types of restriction enzymes.

DNA Tools

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Restriction Enzymes: EcoRIOne restriction enzyme that is widely used by scientists is EcoRI.EcoRI specifically cuts DNA containing the sequence GAATTC.EcoRI cuts this sequence such that it produces complementary sticky ends.Not all restriction enzymes create sticky ends!http://highered.mcgraw-hill.com/olc/dl/120078/bio37.swf

DNA Tools

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Gel Electrophoresis:An electric current is used to separate the DNA fragments according to the size of the fragments in a process called gel electrophoresis.Check out Figure 13.5!!When an electric current is applied, the DNA fragments move toward the positive end of the gel.The smaller fragments move faster than the larger ones.Portions of the gel containing each band can be removed for further study.

Recombinant DNA Technology

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After DNA fragments have been separated by gel electrophoresis, fragments can be removed and combined with DNA fragments from another source recombinant DNALarge quantities of recombinant DNA are needed to study themIn order to transfer the recombinant DNA into a bacterial cell, scientists use a carrier (also called vector) Plasmid or VirusPlasmids are small, circular and double stranded DNA molecules that occure naturally in bacteria and yeast.

Recombinant DNA Technology

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If a plasmid and a DNA fragment obtained from another genome have been cleaved by the same restriction enzyme, the ends of each DNA fragment will be complementary and can be combined.An enzyme normally used by cells in DNA repair and replication, called DNA ligase, joins the two DNA fragments chemically. Check out Figure 13.6!!

Gene Cloning

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Gene cloning is used to make large quantities of recombinant plasmid DNA.Some bacterial cells take up the recombinant plasmid DNA into them through a process called transformation.Bacteria can be transformed using electric pulsation or heat.Large quantities of identical bacteria, each containing the inserted DNA molecules, can be produced through a process called cloning.Check out Figure 13.7!!

DNA Sequencing

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The sequence of the DNA nucleotides of most organisms is unknown.Knowing the sequence is a valuable is information to have.Check out Figure 13.8 for the steps in DNA sequencing technique!!

Polymerase Chain Reaction

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Once the sequence of a DNA fragment is known, a technique called the polymerase chain reaction, PCR, can be used to make millions of copies of a specific region of a DNA fragment.PCR is extremely sensitive and can detect a single DNA molecule in a sample.Follow the steps of PCR from the book and check out Figure 13.9!!http://www.dnalc.org/resources/animations/pcr.htmlhttp://www.maxanim.com/genetics/PCR/PCR.htmCheck out Table 13.1 for the differences in the DNA manipulation techniques we learned so far.