Genetic Engineering Methods: DNA Techniques and Enzymes - PDF

Genetic engineering (3) DNA purification Column chromatography DNA purification with silica Plasmid extraction Size difference Conformational difference DNA manipulation (Enzyme) Cloning Enzymes Ligases join nucleic acid molecules together. Polymerases make copies of molecules...

Genetic engineering (3) DNA purification Column chromatography DNA purification with silica Plasmid extraction Size difference Conformational difference DNA manipulation (Enzyme) Cloning Enzymes Ligases join nucleic acid molecules together. Polymerases make copies of molecules. Pol I, Taq, RT Nucleases are enzymes that cut, shorten, or degrade nucleic acid molecules. Modifying enzymes  DNA ligase is a enzyme that can link together DNA strands that have double-strand breaks (a break in both complementary strands of DNA). – Naturally DNA ligase has applications in both DNA replication and DNA repair. – Needs ATP DNA ligase has extensive use in molecular biology laboratories for genetic recombination experiments ATP Endonuclease Restriction and Modification Enzymes Restriction enzymes protect cell from invasion from foreign DNA – Destroy foreign DNA – Must protect their own DNA from inadvertent destruction Restriction enzymes: recognize specific DNA sequences and cut DNA at those sites – Widespread among prokaryotes – Rare in eukaryotes – Protect prokaryotes from hostile foreign DNA (e.g., viral genomes) – Essential for in vitro DNA manipulation Restriction and Modification Enzymes Three classes of restriction enzymes – Type II cleave DNA within their recognition sequence and are most useful for specific DNA manipulation (Figure 11.1a) Restriction enzymes recognize inverted repeat sequences (palindromes) – Typically 4–8 base pairs long; EcoRI recognizes a 6-base-pair sequence Sticky ends or blunt ends Restriction and Modification Enzymes Modification enzymes: protect cell’s DNA for restriction enzymes – Chemically modify nucleotides in restriction recognition sequence – Modification generally consists of methylation of DNA Restriction enzymes & DNA methylation Restriction enzymes Table 9.1 Selected Restriction Enzymes Used in rDNA Technology  Electrophoresis Equipment Power supply Gel tank Cover Electrical leads  Casting tray Gel combs Figure 11.2a © 2012 Pearson Education, Inc. Agarose Electrophoresis Loading Electrical current carries negatively- charged DNA through gel towards Buffer positive (red) Dyes electrode Agarose gel Power Supply  Terminal deoxynucleotidyl transferase Agarose Electrophoresis Running Agarose gel sieves DNA fragments according to size – Small fragments move farther Gel running than large fragments Power Supply UncutBlue CutBlue UncutWhite CutWhite /Hind III / BstE II Artificial Tailing Students assignment Modification enzyme Alkaline phosphatase Polynucleotide kinase Terminal deoxynucleotidyl transferase DEPHOSPORYLATED VECTOR Figure 11.5 Foreign DNA Cut with restriction enzyme Add vector cut Sticky with same ends restriction enzyme Vector Add DNA ligase to form recombinant molecules Cloned DNA Introduction of recombinant vector into a host © 2012 Pearson Education, Inc.  Insert the naked DNA into a host cell 1.Transformation * treatment make cells competent to accept foreign DNA (CaCl2 make pores in cell membrane) 2. Electroporation *use electrical current to form microscopic pores in the membranes of cell 3. Protoplast fusion – yeast, plants and algal cells 4. Microinjection 5. Gene gun Figure 9.5b TRANSFORMATION OF LIGATION PRODUCTS The process of transferring exogenous DNA into cells is call “transformation” There are basically two general methods for transforming bacteria. The first is a chemical method utilizing CaCl2 and heat shock to promote DNA entry into cells. A second method is called electroporation based on a short pulse of electric charge to facilitate DNA uptake. Transformation CHEMICAL TRANSFORMATION WITH CALCIUM CHLORIDE TRANSFORMATION BY ELECTROPORATION  Some possible products of the transformation reaction: Bacterial cell Genomic DNA Plasmid w/ insert Plasmid w/o insert No plasmid Ampicillin Ampicillin resistant No ampicillin resistant resistance Functional LacZ Nonfunctional No LacZ gene Blue colony on LacZ LB+X-gal+amp. No growth on White colony on Ampicillin LB+X-gal+amp. Recovery period Allow the cell to regain strength and start to multiply Necessary to allow expression of the antibiotic resistance gene on the plasmid Add 250 µl sterile Luria Broth to both tubes using a sterile pipette Move tubes to water bath at 37oC for 5 min.