BIOS E-1a Lecture 15 112811 annotated

BIOS E-1a Lecture 15 112811 annotated - Lecture 15 Genetic...

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Unformatted text preview: Lecture 15: Genetic Engineering Reading: Chapter 13 1 2 • Recombinant DNA technology – Use of laboratory techniques to isolate and manipulate fragments of DNA • Recombinant DNA contains DNA from 2 or more sources • Once inside a host cell, recombinant molecules are replicated to produce identical copies or clones 3 Gene cloning • Procedures that lead to the formation of many copies of a particular gene • W h y ? – Want copies of a gene for study or use – Obtain lots of gene product: mRNA or protein 4 Gene cloning 5 Step 1 in gene cloning • Vector DNA acts as a carrier for the DNA segment to be cloned • When a vector is introduced into a living cell, it can replicate making many copies • Common vectors are plasmid or viral • Also need the gene of interest from chromosomal DNA 6 Step 2 • Insert chromosomal DNA into vector • Cut DNA using restriction enzymes or restriction endonucleases – Made naturally by bacteria as protection against bacteriophages – Cuts at specific known restriction sites – Most restriction sites palindromic – May produce sticky ends – DNA ligase must be used to permanently link DNA • Result may be – Re-circularized vector with no gene of interest inserted – Recombinant vector with gene of interest inserted 7 Generating a recombinant vector 8 Step 3 – actual cloning • Goal for recombinant vector to be taken up by bacteria – Some will take up a single plasmid – Most cells fail to take up a plasmid • Vector carries a selectable marker – Presence of antibiotics selects for cells expressing amp R gene: contains plasmid – amp R gene codes for β-lactamase that degrades ampicillin, which normally kills bacteria • After treatment, only cells with the plasmid will grow on plates treated with ampicillin – To eliminate recircularized vectors from further examination, lacZ gene part of vector – Insertion of chromosomal DNA disrupts lacZ gene – lacZ codes for β-galactosidase which cleaves colorless X-Gal into a blue dye • Recircularized plasmids will form blue colonies • Recombinant vectors will form white colonies 9 10 Introducing a recombinant vector into a host cell 11 DNA library • Treatment of chromosomal DNA with restriction enzymes yields tens of thousands of different fragments • DNA library: collection of many recombinant vectors each with a fragment of chromosomal DNA • 2 types of common DNA libraries – Genomic: inserts derived from chromosomal DNA – cDNA: use reverse transcriptase to make DNA from mRNA of interest (complementary DNA) - lacks introns so simpler to use 12 DNA library Electrophoresis • Technique that is used to separate macromolecules, such as DNA and proteins, on a gel • Can be used to separate molecules based on their charge, size/length, and mass 13 1 2 3 Higher-mass molecules Lower-mass molecules Samples Gel – + – + Load samples of DNA fragments into wells at the top of the gel....
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BIOS E-1a Lecture 15 112811 annotated - Lecture 15 Genetic...

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