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The use of living things, biological systems and processes for the benefit of humans.

The use of living things, biological systems and processes for the benefit of humans. 1. Manipulating DNA. EL: To learn how to cut and paste DNA. Tools for manipulating DNA. 1. Cutting DNA. Restriction enzymes (molecular scissors) are found in prokaryotic organisms. Specificity.

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The use of living things, biological systems and processes for the benefit of humans.

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  1. The use of living things, biological systems and processes for the benefit of humans.

  2. 1. Manipulating DNA EL: To learn how to cut and paste DNA

  3. Tools for manipulating DNA

  4. 1. Cutting DNA • Restriction enzymes (molecular scissors) are found in prokaryotic organisms

  5. Specificity Restriction enzymes are specific: • The DNA and the enzyme need to be mixed together and incubated at a temperature that will result in maximum activity of the enzyme. • Each restriction enzyme will only cut the DNA at a specific sequence of A, G, T and Cs. We call this place a recognition site.

  6. Different restriction enzymes recognise specific recognition sites.

  7. Cutting Specificity • When DNA is cut with a restriction enzyme the resulting fragments are left with either a short overhang of single stranded DNA called a sticky end or no overhanging DNA which is called a blunt end (snake demo) EcoRI – leaves sticky ends GAATTC CTTAAG HpaI – leaves blunt ends GTT AAC GTTAAC CAA TTG CAATTG - site where enzymes cuts through the sugar phosphate backbone of the DNA strand.

  8. What’s in a name! • Restriction enzymes are named after the organism from which they were isolated. • E.g. Escherichia coli EcoRI • The Roman number indicates the order of discovery • If another letter is placed in front of the Roman number it signifies a particular strain of the bacterium. R = resistance

  9. Fragments are sorted by Gel Electrophoresis • This technique is used to separate out fragments, obtained by a restriction digest, of DNA according to their size (length in base pairs). • DNA fragments are separated into bands containing fragments of the same length by electrical separation in a gel matrix. • DNA molecules migrate to the positive electrode, when an electric field is applied to the gel matrix, as they are negatively charged. • This technique is used to isolate DNA fragments containing genes which are subsequently used to make recombinant DNA.

  10. Fragments are sorted by Gel Electrophoresis

  11. 2. Pasting • When two samples of DNA are combined using DNA ligases. • Any 2 DNA strands can be joined that have complementary exposed nucleotides (i.e. cut with same restriction enzyme).

  12. Activities • Watch DNAi animations • Complete activity 9.1 “Gel electrophoresis analysis” & 9.2 “Detecting traits in families….”

  13. Reflection Summarise in your own words – restriction enzymes, cutting, pasting and gel electrophoresis • What learning was new today? • What learning was revision or built on what I already know? • What did I find most challenging and what strategies will I put in place to help me? • What percentage of the class did I spend on task and how can I improve this if needed

  14. 2. Manipulating DNA EL: To learn how to copy and transfer DNA.

  15. 3. Copying (Amplification) of DNA using the Polymerase Chain Reaction “I was working for Cetus, making oligonucleotides (primers). They were heady times. Biotechnology was in flower and one spring night while the California buckeyes were also in flower I came across the polymerase chain reaction. It was the first day of the rest of my life”. Kary Mullis 1972 • The $300 million dollar man.

  16. Why PCR? • To amplify a small amount of DNA into an analysable quantity • E.g. crime scene, fossils etc

  17. PCR Tools • Taq DNA Polymerase – is an enzyme that works well at 72°C.

  18. PCR Tools Primers: • Synthetic short segments of DNA up to 25 nucleotides long. • Probe for a specific sequence or gene along a strand of DNA. • Hybridise with a sequence of bases on the template DNA through complementary base pairing. • Indicate to Taq DNA polymerase where to start building the complementary strand by extending the primer.

  19. Find the starting point for copying STR regions Select your primer Start region Sequence to be copied by extending the primer.

  20. Thermocycling machine Step 1. Denaturation • At this temperature the hydrogen bonds are broken resulting in two single strands of DNA. Step 1: Denaturing the DNA – 2 minutes T A C C G T A A A T G C C A T T Step 1: 92°C

  21. Step 2. Attachment of Primers T A C C G T A A A T G T A A A T G C C A T T Step 2: 55°C • Step 2: Primer annealing– 2 minutes • The temperature is lowered to allow the primers to bind (anneal) to their complementary bases on each of the single strands of DNA.

  22. Step 3: Extension T A C C G T A A A T G G C Taq Taq A A T C T T Step 3: 72°C G G T A A A T G C C A T T • Step 3: DNA synthesis – 1 minute • Taq DNA polymerase extends the DNA strand from the primers using the base pairing rule.

  23. And you can repeat the three step cycle over and over!

  24. PCR song • http://www.youtube.com/watch?v=dD3faDLEvmY&feature=related

  25. Fluorescent jellyfish 4. Transferring Jellyfish Plasmid • Because DNA is the same in all organisms, we should be able to take a piece of DNA from one organism and put it into another organism. • You can change the way an organism looks or behaves! • This process of taking DNA from one organism and putting into another is called transformation. Fluorescent Jellyfish and plasmid DNA is cut with the same restriction enzyme.

  26. Vectors • Gene inserted into a vector that will carry the gene into the desired organism. • Common vectors are: • Viral vectors (eg. Adenovirus and retorovirus) – must have disease symptom genes removed first! • Liposome vectors – small circular molecules surrounded by phospholipid bilayer • Plasmid vectors – small circular piece of bacterial DNA. Plasmids are used as vectors in bacterial transformations.

  27. Plasmids are not naturally attracted to bacteria! Bacterium

  28. Transformation of Bacteria with a Recombinant DNA Plasmid Making the bacteria more ‘attractive’ to plasmids Plasmids are now attracted to the bacteria Bacterium CaCl2solution

  29. The Transformation • Now give the bacteria some food and the right temperature to reproduce. • Any bacteria with the plasmid inside will start making the jelly protein, that results in fluorescence. HEAT SHOCK

  30. Activity • In pairs, complete Activity 12.2 “finding a gene” • Quick check qu 1-3 (pg 426), 4-6 (pg 431) • Ch 12 review qu 3, 4, 5, 6, 7, 8, 12

  31. Reflection Summarise in your own words – copying (include PCR) and transferring • What learning was new today? • What learning was revision or built on what I already know? • What did I find most challenging and what strategies will I put in place to help me? • What percentage of the class did I spend on task and how can I improve this if needed

  32. 3. Applications of DNA manipulation EL: To explore the uses of DNA manipulation.

  33. Gene Sequencing Gene sequencing is identifying the nucleotide order in asegment of RNA or DNA. A G G A C T C A T G G A G A A G A A C T TT . . . Our genome has been sequenced. We have 3,100,000,000 base pairs, what a big book!

  34. Gene Cloning • Making identical copies of sequences of DNA that code for proteins using plasmids • extract plasmid from bacteria • Cut plasmid DNA and DNA of the gene to be inserted with same restriction enzyme • Paste 2 pieces of DNA using DNA ligase to create a recombinant plasmid. • Add recombinant plasmid to bacterial culture, where some are taken up and replicate (called transformation) • Isolate and analyse bacteria containing recombinant plasmids. PRACTICAL APPLICATION: Production of human growth hormone

  35. DNA Profiling • Compares base sequence of 2 or more individuals • Short tandem repeats (STRs) and variable nucleotide tandem repeats (VNTRs): non-coding sections of DNA repeated many times between genes • E.g. GAGAGAGAGAGAGA • There are more than 10,000 STR loci in one set of human chromosomes!

  36. DNA Profiling • The repeat is present in all members of the population, but the number of repeats varies among individuals and is inherited. • DNA profiling allows us to view these patterns in our DNA. • Uses PCR and gel electrophoresis – smaller fragments will migrate further on the gel.

  37. DNA Profiling

  38. DNA Profiling • Loci of STR regions found to vary from person to person with a high frequency • 13 are used in America, but only 9 are used in Australia – why?

  39. Activities • Genetic Engineering: A model (Biol: The Common Threads, pg 175) • DNA fingerprinting (Biol: The Common Threads, pg 179) • Quick check qu 7-10 (pg 433), 11-14 (pg 437), 15-17 (pg 443), 18-21 (pg 448), 22-24 (pg 455), 25&26 (pg 457) • Ch 12 ch review qu 9, 10, 11 (pg 461)

  40. Reflection Summarise in your own words gene sequeencing, cloning and DNA profiling • What learning was new today? • What learning was revision or built on what I already know? • What did I find most challenging and what strategies will I put in place to help me? • What percentage of the class did I spend on task and how can I improve this if needed

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