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Download Chapter 12: Biotechnology 1. Recombinant DNA What is
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Chapter 12: Biotechnology 1. Recombinant DNA 2. Recombinant DNA Applications 1. Recombinant DNA What is “Recombinant DNA”? The joining of DNA from different sources. This can happen in nature (in vivo)… • the transfer of DNA involving bacteria or viruses …or in the laboratory (in vitro) • the cutting & splicing of DNA fragments by molecular biologists The term “recombinant DNA” generally refers to laboratory kind… 1 Recombinant DNA in Nature Bacteria can acquire DNA from an outside source by a process known as transformation • chemical treatment of bacterial cells followed by a brief heat treatment can induce the cells to internalize & retain the DNA Plasmids Bacteria are transformed more commonly by small, circular DNA fragments called plasmids • taken up from outside or transferred from one bacterium to another Restriction enzyme recognition sequence 1 Recombinant DNA in the Laboratory GAATTC CTTAAG DNA Restriction enzyme cuts the DNA into fragments G CTTAA 2 The cutting and splicing of DNA in vitro involves the use of restriction enzymes (RE’s): A AT TC G Sticky end Addition of a DNA fragment from another source A AT TC 3 G Two (or more) fragments stick together by base-pairing 4 G A AT T C C T TA A G G A AT T C C T TA A G DNA ligase Pastes the strand 5 Recombinant DNA molecule G CTTA e.g. A EcoR I cuts at: ..GAATTC.. ..CTTAAG.. Hind III cuts at: ..AAGCTT.. ..TTCGAA.. There are many different RE’s, each cutting a different sequence 2 “Cloning” a Gene Human cell E.coli 1 Isolate DNA from two sources 2 Cut both DNAs with the same restriction enzyme DNA Plasmid Gene V DNA “sequence of interest” is inserted into a plasmid & “carried” inside bacteria Sticky ends 3 Mix the DNAs; they join by base-pairing 1) cut plasmid and DNA to be cloned with same RE 2) ligate fragments together using DNA ligase enzyme 4 Add DNA ligase to bond the DNA covalently Recombinant DNA plasmid Gene V 5 Put plasmid into bacterium by transformation Recombinant bacterium 3) transform bacteria, select for “clones” with plasmid **In this form the “DNA of interest” can be easily multiplied and purified** 6 Clone the bacterium Bacterial clone carrying many copies of the human gene PCR Amplification The creation of recombinant DNA molecules in the laboratory requires large amounts of the DNA fragments to be combined: • the PCR (Polymerase Chain Reaction) technique is routinely used to generate huge numbers of identical DNA fragments • involves in vitro DNA replication to amplify desired DNA sequences exponentially 2. Recombinant DNA Applications 3 Useful Applications Involving Recombinant DNA Technology DNA sequencing, fingerprinting • involves the separation of DNA fragments by size using gel electrophoresis Commercial Production • using recombinant bacteria, yeast to make “lots” of a protein (e.g., insulin) Gene therapy • replacing defective genes (still experimental) Transgenic organisms (GMO’s) • putting “desirable” genes into animals, plants Gel Electrophoresis – Mixture of DNA molecules of different sizes – Longer molecules Power source Gel + Shorter molecules + Completed gel DNA fragments of different lengths generated by RE’s or other methods are separated by size by “running a gel” • used in DNA sequencing, DNA fingerprinting and numerous other techniques Transgenic Organisms Potato plant containing pest resistance gene Salmon containing extra growth hormone gene **Transgenic organisms contain a foreign gene** 4 Key Terms for Chapter 12 • recombinant DNA • transformation • plasmid • restriction enzymes • PCR • gel electrophoresis • transgenic Relevant Review Questions: 2-4, 6 5