TAILIEUCHUNG - Lecture Biology - Chapter 17: Biotechnology

In this chapter, you will learn to understand the importance of plasmids and viruses to genetic engineering. Know the natural function of restriction endonucleases and how a normal bacterial cell protects its DNA from their activity. Understand how “sticky ends” are formed and their importance to gene technology. Describe how a chimeric genome is constructed,. | Biotechnology Chapter 17 DNA Manipulation The molecular biology revolution started with the discovery of restriction endonucleases -Enzymes that cleave DNA at specific sites These enzymes are significant in two ways 1. Allow a form of physical mapping that was previously impossible 2. Allow the creation of recombinant DNA molecules (from two different sources) DNA Manipulation Restriction enzymes recognize DNA sequences termed restriction sites There are two types of restriction enzymes: -Type I = Cut near the restriction site -Rarely used in DNA manipulation -Type II = Cut at the restriction site -The sites are palindromes -Both strands have same sequence when read 5’ to 3’ DNA Manipulation Type II enzymes produce staggered cuts that generate “sticky ends” -Overhanging complementary ends Therefore, fragments cut by the same enzyme can be paired DNA ligase can join the two fragments forming a stable DNA molecule C C G G T A T A A T A T C C G G T A T A A T A T C G A A T T C G A A T T C G T T A A C G T T A A C G A A T T DNA ligase joins the strands. DNA duplex G A T T C G A A T T Sticky ends Restriction sites EcoRI Recombinant DNA molecule Restriction endonuclease cleaves the DNA EcoRI EcoRI EcoRI DNA from another source cut with the same restriction endonuclease is added. Restriction endonuclease cleaves the DNA Sticky ends Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. C A Gel Electrophoresis A technique used to separate DNA fragments by size The gel (agarose or polyacrylamide) is subjected to an electrical field The DNA, which is negatively-charged, migrates towards the positive pole -The larger the DNA fragment, the slower it will move through the gel matrix DNA is visualized using fluorescent dyes Restriction endonuclease 1 cut site Restriction endonuclease 2 cut site Reaction 1 Reaction 2 Reaction 3 Restriction endonuclease 3 Short segment Long segment Medium segment Medium segment Mixture of DNA

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