DNA Cloning and Manipulation notes

DNA Cloning and Manipulation notes -...

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DNA Cloning and Manipulation 1. Define a cloning vector, insert DNA, recombinant DNA molecule, and clone a. Cloning vector i. A small molecule of DNA capable of self-replication ii. A vector is a delivery agent b. Insert DNA  i. DNA ligase links the cloning vector and DNA to be cloned c. Recombinant DNA molecule i. Composite DNA molecules comprising of covalently linked segments from  two or more sources of DNA d. Clone i. Identical DNA molecule 2. Describe restriction endonucleases and explain the characteristics of recognition sites,  types of ends generated, what is meant by compatible ends, and how these enzymes  are used to produce recombinant DNA a. Restriction endonucleases i. Purpose 1. Restricts foreign DNA  ii. Three types 1. Types I. II. And III a. Types I and III i. Type I  1. Cleaves at random sites that can be 1,000  base pairs from the recognition sequence
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ii. Type III 1. Cleave DNA about 25 bp away from  recognition site iii. Both require ATP to move along the DNA b. Type II restriction enzymes i. Purpose in cells 1. Protect bacteria from DNA ii. Recognition Sites 1. Usually 4-6 bp long 2. Palindromic (fg. 8-18) 3. High in G and T concentration iii. Cut sites 1. Cleaves within palindrome (of recognition site) 2. Types of cuts a. Make staggered cuts on the two strands, leaving two to  four nucleotides of one strand unpaired i. These unpaired strands are called “sticky ends” b. No staggered cuts 1. Leaves blunt ends iv. Joining of cut sites 1. DNA ligase is used to join similarly digested cloning vectors – that  is, a vector digested by the SAME restriction endonuclease 2. To join blunt ends
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a. Generate sticky ends from blunt ends by inserting synthetic 
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This note was uploaded on 12/08/2009 for the course BMB 462 taught by Professor Staff during the Spring '08 term at Michigan State University.

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DNA Cloning and Manipulation notes -...

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