Biology 212 General Genetics Spring 2007Lecture 14 Recombinant DNA IReading: Chap. 6 pp. 224-233Lecture Outline:1. Restriction enzymes2. Gel electrophoresis and Southern blot3. Polymerase chain reaction (PCR)4. DideoxysequencingLecture:- Recombinant DNA encompasses a number of different methods for altering and detecting DNA. A recombinant DNA molecule consists of a foreign DNA inserted in a vector.- Many of these methods depend on use of E. coli enzymes such as DNA polymerase I as specific tools.1. Restriction enzymes- discovered in bacteria, blue-green algae- site-specific, most recognize 4-6 bp sequence- are endonucleases--cut within a DNA molecule-----G GATC C------------------------C CTAG G-------------------Recognized by restriction enzyme BamHICleavage produces 4 bp overhangUses of restriction enzymesFragment DNA of an organism into smaller piecesSeparate by electrophoresis Insert into vector to create a recombinant DNAto study particular region2. Gel electrophoresis and Southern blotAgarose gel electrophoresis- Separate molecules in electric field- Smaller molecules travel faster- Larger molecules travel more slowly- DNA is negatively charged and is attracted toward the positive poleConstruct a physical map of DNA (restriction map) by:1- digesting DNA with restriction enzymes- separating DNAs by gel electrophoresis- determining sizes of fragments by comparison to known fragments- physical mapping data mostly additiveExample:Construct restriction map of linear DNA: EcoRI BamHI 4 kb 6 kb 2 kbSouthern blot: Transfer of DNA fragments from a gel to a membrane for hybridization with a probe. Specific DNA fragments can be identified in a mixture.Fig. 6.27Hybridization:- Based on base pairing of complementary DNA or RNA sequences- Prepare hybridization "probe", a DNA or RNA sequence to gene of interest, usually tagged with a radioactive nucleotide- Incubate with Southern blot, wash off excess- Hybridizing fragments detected by autoradiography3. Polymerase chain reaction (PCR)2- method for reproducing DNA- means to obtain large quantities of a specific DNA sequence- requireso small amount of starting DNA o forward and reverse primerso deoxynucleotide triphosphateso thermostable DNA polymeraseo appropriate buffer conditions- involves multiple cycles (chain reaction)Process of PCRa. Denaturation: heat at 94˚Cb. annealing 45-65˚Cc. extension 72˚CSpecial applications of PCR:Forensics: reproduce DNA from as little as a single cellDNA diagnostics: very little starting sample needed to perform a DNA test to diagnose a genetic disease or identify carriers4. Dideoxy DNA sequencingmethod for determining order of bases on DNAuses modified nucleotides as chain terminators in a DNA synthesis reactionFig. 6.29 compare structure of deoxy and dideoxynucleotideRequirements of reaction:- Primer- Template- Mixture of deoxynucleotides- Dideoxynucleotide- Synthesize chains of various lengths - Terminate at dideoxynucleotide (for example, ddA)- Carry out similar reactions for all four nucleotides- Separate products by gel electrophoresis- Smallest products travel farthest--sequence closest to primer is at bottom of gelAdvances in DNA sequencing technology- Automated process- Fluorescent tags to distinguish 4 different bases- Scan gel during electrophoresis- Produce colored trace- Sequence data can be analyzed and assembled using high speed