Chapter 16 - Chapter 16: Recombinant DNA and Biotechnology...

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Chapter 16: Recombinant DNA and Biotechnology I. DNA in vitro a. “Cloning” i. Restriction digestion – restriction enzymes 1. Cut the target gene out in test tube 2. Now several different pieces ii. Gel electrophoresis 1. Separate pieces of DNA fragments iii. Ligation 1. Reconnect the pieces of DNA II. Restriction Digestion a. Restriction Enzymes (endonucleases) i. Cut any double stranded DNA into smaller pieces 1. Break phosphodiester bond 2. Break sugar phosphate backbone ii. Look for certain palindrome sequence 1. Recognition sequence (restriction site) 2. 4-7 bp 3. smaller sequence = more oftenly cut iii. Naturally occurring defense system 1. Cut up foreign DNA (viruses) iv. Bacteria protect their own DNA by methylation 1. Recognize methylation so enzymes don’t eat their own DNA v. Example: EcoRI, named after bacteria that naturally produce them (E.coli) 1. E = genus 2. co = species 3. R = strain 4. I = order of identification vi. Cut sticky ends or blunt ends 1. Sticky ends leave single strand part at end a. Jagged, like staircase 2. Sticky ends can rejoin if ends are complementary 3. Blunt ends can rejoin any other end vii.Each restriction enzyme cuts in same way regardless of whether or not its different DNA III. Gel Electrophoresis a. Separate fragments by size b. Electric field to porous gel c. Negatively charged DNA moves towards positive end of gel (cathode) d. Smaller fragments move farther than larger ones e. DNA fingerprint i. Everyone has random fragment lengths between restriction sites so each person’s fragment length will be different
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ii. This happens because these genes are polymorphic (multiple alleles) IV.Ligation a. Still nick in DNA because no phosphodiester bonds between fragments that come together b. Come together, H bonds formed
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This note was uploaded on 01/27/2011 for the course BIOSC 0160 taught by Professor Bledsoe during the Spring '08 term at Pittsburgh.

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Chapter 16 - Chapter 16: Recombinant DNA and Biotechnology...

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