Lecture 57 2005 - Lecture 57: Recombinant DNA and cloning...

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1 Lecture 57: Recombinant DNA and cloning Today’s lecture: Text pp. 923-924, 969-979 Preprinted lecture notes 115-132 Download from http://www.mbi.ufl.edu/facilities/msg/bch4024-notes.html Office hours: Wednesday (4/27) 1-3 pm LG-126 Prep questions and all lecture notes (with additional notations) will be posted by Friday Enzymes for DNA analysis and molecular cloning ¾ Restriction endonucleases fragment the DNA into defined segments ¾ DNA Ligase joins DNA fragments ¾ DNA polymerase Synthesizes DNA based on a template Fills in gaps in DNA ¾ Reverse transcriptase Copies RNA into DNA Æ “cDNA” ¾ Ribonucleases (endo and exo) ÆÅ ¾ RNA polymerase ¾ Terminal transferase For radiolabeling ¾ Polynucleotide kinase For radiolabeling ¾ Alkaline phosphatase To remove phosphate; 3’ or 5’ 5’ 3’
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2 Restriction endonucleases ¾ Cleave DNA in a sequence-specific manner ¾ Evolved as a defense mechanism against infection by foreign DNA ¾ Different restriction enzymes in different organisms ¾ Protect host DNA by modification-methylation ¾ Nomenclature: EcoRI ¾ Many restriction enzymes recognize “palindromic” sites EcoRI cuts: 5’—GAATTC—3’ 3’—CTTAAG—5’ To yield: 5’—G-OH and 5’—ATTC—3’ 3’—CTTAA—5’ HO-G—5’ ¾ EcoRI will not cut at this site if the Adenine is methylated, as a means of protecting its own DNA from being digested. 5’—GAA m TTC—3’ will not be cut by EcoRI 3’—CTTA m AG—5’ Genus Species Strain Enzyme number Digestion of DNA ¾ Digestion of a particular DNA gives specific, “invariant” fragments of characteristic size ¾ Example: 3.2 Kbp DNA 5’--------ATGAATTCCA--------GCCGAATTCTTT---------3’ 3’--------TACTTAAGGT--------GCCGTTAAGAAA--------5’ EcoRI Restriction fragments of 2.0, 0.7, 0.5 Kbp CTAGAATTCTA GATCTTAAGAT EcoRI Restriction fragments of 1.9, 2.2 Kbp (Linear!) 700 bp 2000 bp 500 bp T C G A 4.1 Kbp Circular DNA 1 6 0 b p 1900 bp 600 bp
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3 Size-separation of DNA molecules and fragments by Electrophoresis Rate of DNA MIGRATION is inversely proportional to length ¾ Size of DNA ¾ Concentration of Agarose ¾ Conformation of DNA ¾ Applied voltage (+) (-) Circular + Linear Linear λ fragment with insert Linear 3.2 Kbp λ fragment λ HinD III Digest 0.5 2.3 4.4 23 C L
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This note was uploaded on 06/07/2011 for the course BCH 4024 taught by Professor Allison during the Fall '08 term at University of Florida.

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Lecture 57 2005 - Lecture 57: Recombinant DNA and cloning...

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