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DNA

DNA. How is the expression of genes controlled in prokaryotes? What are some ways the expression of genes are controlled in eukaryotes? What are histones?. DNA Technology. Meet Dolly. Biotechnology. The manipulation of organisms or use of living things as technology

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DNA

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  1. DNA • How is the expression of genes controlled in prokaryotes? • What are some ways the expression of genes are controlled in eukaryotes? • What are histones?

  2. DNA Technology Meet Dolly

  3. Biotechnology • The manipulation of organisms or use of living things as technology • i.e. genetic engineering, manipulating genes for practical purposes

  4. Studying One Gene • If we want to study a particular gene in depth, it is cumbersome to use the entire DNA molecule • Much easier if we can make multiple copies of that one gene to focus on

  5. Gene Cloning • We will first look at the overview of cloning a particular gene, then go into it in detail • The goal is to create multiple copies of a single segment of DNA

  6. Step 1A • Isolate a plasmid from a bacterial cell

  7. Step 1B • Isolate the DNA we wish to clone

  8. Step 2 • Insert gene into plasmid Recombinant DNA

  9. Step 3 • Reinsert Plasmid into Bacteria Recombinant Bacterium

  10. Step 4 • Plasmids replicate independently, reproducing the gene of interest

  11. Step 5 / Step 6 • Identify the bacterial plasmids that did in fact clone the gene • Use the gene! • Can use copies of the gene itself • Can use the protein products of the gene

  12. Why Is This Useful? • We can insert genes into other organisms • i.e. in agriculture we can introduce pest-resistance to crops • Alter bacteria to accomplish a task • Create proteins for medicines and other uses • Create Human Growth Hormone to treat short kids

  13. Restriction Enzymes • Cut DNA at specific places (recognize target sequences) • Used to combat foreign DNA in nature • Create restriction fragments • Creates the same fragments every time

  14. Sticky Ends • Doesn't cut at the same spot on both strands • Leaves single stranded edges called sticky ends • These two ends can be resealed by DNA ligase • Or new DNA can be inserted between

  15. Recombinant DNA using Restriction Enzymes

  16. DNA • What is a restriction enzyme? • What are sticky ends? • What is a plasmid? • What are some of the uses of genetic engineering?

  17. A More Detailed Look at Cloning • We use a plasmid containing 2 useful genes • 1 – ampicillin resistance • 2 – lacz gene • Called a cloning Vector • Easy to insert in bacteria

  18. Restriction Enzyme Targets lacz Gene • A restriction enzyme recognizes and cuts a segment of the lacz gene • Also cuts DNA containing gene of interest into small fragments

  19. Mix Plasmids with Our DNA • Sticky ends of plasmid can base pair with sticky ends of DNA • Also end up with plasmid-plasmid combos and DNA-DNA combos etc. • Seal Plasmid and DNA using DNA ligase

  20. Some Plasmids take in the DNA, some Don't • DNA is inserted in the middle of the lacz gene if DNA is taken by plasmid • Amp gene is intact either way

  21. Introduction of Plasmids to Bacterial Cells • Recall transformation, bacteria will take up plasmids • The bacteria do not have the lacz gene • Some bacteria take in plasmids with our DNA • Some take in unaffected plasmids

  22. Plate the Bacteria • We place the bacteria on a plate containing ampicillin and X-gal • Only bacteria containing the plasmid can grow (the ampicillin resistance allows their survival) Bacterial Colonies

  23. What Is X-Gal? • X-gal reacts with galactosidase to create a blue product • The product of the lacz gene breaks down galactosidase • If the lacz gene is intact – no blue • If there is foreign DNA then lacz gene is interrupted and bacteria are blue White Blue

  24. Checking our Agar Plate • Blue colonies have taken in foreign DNA in their plasmids • White colonies have the plasmid – but no foreign DNA is in the plasmid and the lacz gene is intact

  25. Isolate Our Gene of Interest • The foreign DNA may not have been the gene we care about! • We must use nucleic acid probe (a short segment of complementary DNA) to find the gene of interest • Attach fluorescent protein to probe

  26. Making the Bacteria Express the Gene • We can express the gene in the bacteria, but sometimes we need to insert a promoter as well • Called an expression vector • The promoter tells the prokaryotic RNA polymerase to transcribe the gene

  27. cDNA • Introns are a pain for prokaryotes • Sometimes it's necessary to make DNA without the introns first • Use reverse transcriptase to make cDNA from mRNA

  28. cDNA library contains only the segments that code for a gene In fact only codes for genes transcribed – useful for studying genes expressed in brain cells for example Genomic Library vs. cDNA library • Collection of all of the segments of the DNA that is separated by restriction fragments • The library will have multiple copies of each gene • Some genes are split between two segments

  29. Polymerase Chain Reaction (PCR) • Allows us to quickly make many copies of a segment of DNA • Very specific, due to use of specific primers that recognize each gene • Need only small amount of DNA to replicate

  30. PCR • Heat the DNA to separate the strands • Cool strands and allow DNA primers to bind to DNA • DNA polymerase synthesizes new strand • Repeat

  31. Why is PCR So Amazing? • From a small amount of DNA we can make millions of copies • Important in solving crimes with DNA, determining paternity etc. • Useful for a lot of other biotech processes

  32. Gel Electrophoresis • Separates DNA, Proteins etc. based on charge and size • For DNA, all molecules have the same charges, so separates DNA by length of strand

  33. Restriction Fragment Analysis • Cut pieces of DNA with restriction enzymes • The same DNA with the same enzymes will produce the same fragments every time • Show up as bands on gel electrophoresis

  34. Southern Blotting • The full genome has too many genes to use simple gel electrophoresis (get too many bands) • But we can use Southern Blotting to identify only the genes we care about

  35. Southern Blotting • We add radioactively labelled DNA to our gel electrophoresis • We can figure out A) if the DNA segment we are interested in is present and • B) What size fragment the segment is located on • C) How many times the gene is present

  36. Restriction Length Polymorphisms (RFLPs) • Recall that human's have DNA that is 99.9% similar • So how can we compare DNA? • By identifying locations in the genome where people often differ • If two people differ in a nucleotide that is part of a restriction site, then only one of the people will have their DNA cut by that restriction enzyme

  37. RFLPs

  38. Finding Genes in Genomes • In situ hybridization • Use a radioactive probe that can base pair with the gene • i.e. we can see if a gene from a mouse is present in humans

  39. The Human Genome Project • Working version of genome worked out in 2000 • “Final Draft” in 2003 • Not a single individual – there are many places where nucleotides differ • Available on the Internet

  40. Genetic Linkage Mapping • As discussed earlier, we can figure out the order of genes by the frequency of recombination • Genes that are further apart are more likely to be separated during crossing over

  41. Getting the Whole Genome • Cut the genome into tons of little pieces • These pieces are identifiable restriction fragments • Then order fragments by how they overlap • Must first clone DNA so we have copies

  42. Chromosome Walking Each segment overlaps, so we can use the end of one segment to probe for the next segment

  43. DNA Sequencing (The Basics) • We take a strand of DNA and make copies of it • The DNA is added to a solution containing everything necessary for DNA replication • Primer, DNA Polymerase, A, T, G and C nucleotides • One more ingredient in each batch

  44. Dideoxy Nucleotides! • Special nucleotides that are missing another OH group • ddA nucleotides are added to the DNA • If a dd nucleotide is added DNA replication ends • No phosphodiester bond can be made Each dd nucleotide is labelled with a fluorescent color

  45. Synthesize New DNA • The DNA is replicated, BUT replication ends as soon as a dd nucleotide is added • We end up with a bunch of different length strands, each labelled by the dd nucleotide on the end

  46. DNA Segments Are Separated By Size • DNA is run through a machine – smaller segments get through faster • A computer reads the color at the end • Tells us the order of the nucleotides

  47. Much Faster than the Sanger Method • This revolutionized the Human Genome Project • Sanger method – have 4 batches, introduce 1 dd nucleotide to each batch • Use gel electrophoresis 4 separate times to determine the length of the strands ending in A, C, G and T

  48. We Can Use cDNA to Identify Which Genes Are Expressed • Separate genes (by gene cloning and hybridization) • Make cDNA and label it • Mix cDNA and each gene to see if they match • Can tell which genes are in that cDNA

  49. Microassay

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