Chapter 20: Biotechnology: DNA Technology & Genomics

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1 Biotechnology

2 Chapter 20: Biotechnology: DNA Technology & Genomics The BIG Questions How can we use our knowledge of DNA to: o Diagnose disease or defect? o Cure disease or defect? o Change/improve organisms? What are the techniques & applications of biotechnology? o Direct manipulation of genes for practical purposes. Biotechnology Genetic manipulation of organisms is not new. o Humans have been doing this for thousands of years. Plant & animal breeding Biotechnology Today Genetic Engineering o Manipulation of DNA o If you are going to engineer DNA & genes & organisms, then you need a set of tools to work with o This unit is a survey of those tools Bioengineering Tool Kit Basic Tools o restriction enzymes o ligase o plasmids / cloning o DNA libraries / probes Advanced Tools o PCR o DNA sequencing o gel electrophoresis o Southern blotting o microarrays Cut, Paste, Copy, Find Word processing metaphor o Cut restriction enzymes o Paste ligase o Copy plasmids bacteria transformation PCR o Find Southern blotting / probes 1

3 Cut DNA Restriction enzymes o Restriction endonucleases o Discovered in 1960s o Evolved in bacteria to cut up foreign DNA ( restriction ) Protection against viruses & other bacteria Bacteria protect their own DNA by methylation & by not using the base sequences recognized by the enzymes in their own DNA Restriction Enzymes Action of Enzyme o Cut DNA at specific sequences Restriction site o Symmetrical palindrome o Produces protruding ends Sticky ends Many different enzymes o Named after organism they are found in EcoRI, HindIII, BamHI, SmaI Biotech Use of Restriction Enzymes Paste DNA Sticky ends allow: o H bonds between complementary bases to anneal. Ligase o Enzyme seals strands Bonds sugar-phosphate bonds. Covalent bond of DNA backbone. 2

4 Copy DNA Plasmids o Small, self-replicating circular DNA molecules. Insert DNA sequence into plasmid. Vector = vehicle into organism. o Transformation Insert recombinant plasmid into bacteria. Bacteria make lots of copies of plasmid. o Grow recombinant bacteria on agar plate. Clone of cells = lots of bacteria. o Production of many copies of inserted gene. DNA RNA Protein Trait Recombinant Plasmid Antibiotic resistance genes as a selectable marker Restriction sites for splicing in gene of interest Selectable Marker Plasmid has both added gene & antibiotic resistance gene If bacteria don t pick up plasmid then die on antibiotic plates If bacteria pick up plasmid then survive on antibiotic plates Selecting for successful transformation o Ampicillin becomes a selecting agent. o Only bacteria with the plasmid will grow on amp plate. LacZ is a Screening System Make sure inserted plasmid is recombinant plasmid. o LacZ gene on plasmid produces digestive enzyme lactose (X-gal) blue blue colonies o Insert foreign DNA into LacZ gene breaks gene lactose (X-gal) blue white colonies o White bacterial colonies have recombinant plasmid. 3

5 Biotechnology: PART 2 What if you don t have your gene conveniently on a chunk of DNA ready to insert into a plasmid? You have to find your gene of interest out of the entire genome of the organism DNA Libraries Cut up all of nuclear DNA from many cells of an organism. o Use a Restriction enzyme Clone all fragments into many plasmids at same time. o Shotgun cloning. Create a stored collection of DNA fragments. o Petri dish has a collection of all DNA fragments from the organism. Find your gene in DNA library Locate Gene of Interest o To find your gene you need some of gene s sequence. If you know sequence of protein Can guess part of DNA sequence. back translate protein to DNA If you have sequence of similar gene from another organism Use part of this sequence. 4

6 DNA Hybridization o Find gene in bacterial colony using a probe Short, single stranded DNA molecule Complementary to part of gene of interest Tagged with radioactive P32 or fluorescence o Heat treat genomic DNA Unwinds (denatures) strands o DNA hybridization between probe & denatured DNA Problems A lot of junk! o Human genomic library has more junk than genes in it Introns, introns, introns! o If you want to clone a human gene into bacteria, you can t have INTONS! Solution Don t start with DNA Use mrna o Copy of the gene without the junk! But in the end, you need DNA to clone into plasmid How do you go from RNA DNA? o Reverse transcriptase! 5

7 cdna (copy DNA) Libraries Collection of only the coding sequences of expressed genes o extract mrna from cells o reverse transcriptase RNA DNA from retroviruses o Clone into plasmid Applications o Need edited DNA for expression in bacteria Human insulin. 6

8 Ch 20: Advanced Techniques: Electrophoresis & RFLPs Gel Electrophoresis Separation of DNA fragments by size o DNA is negatively charged Moves toward + charge in electrical field o Agarose gel Swimming through Jello Smaller fragments move faster Measuring Fragment Size Compare bands to a known standard. o Usually lambda phage virus cut with HindIII. Nice range of sizes with a distinct pattern. RFLP Restriction Fragment Length Polymorphism. o Differences in DNA between individuals. 1

9 Polymerase Chain Reaction (PCR) What if you have too little DNA to work with? o PCR is a method for making many copies of a specific segment of DNA o ~only need 1 cell of DNA to start It s copying DNA in a test tube! What do you need? o template strand o DNA polymerase enzyme o nucleotides o primer What do you need to do? o in tube: DNA, enzyme, primer, nucleotides o heat (90 C) DNA to separate strands (denature) o cool to hybridize (anneal) & build DNA (extension) The Polymerase Problem Heat DNA to denature it o 90 C destroys DNA polymerase o Have to add new enzyme every cycle Almost impractical! Need enzyme that can withstand 90 C o Taq polymerase o From hot springs bacteria Thermus aquaticus 2

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