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Unit 4: DNA and the Molecules of Genetics

Unit 4: DNA and the Molecules of Genetics. A) Composition of DNA. 4 different nucleotides: Composed of a phosphate group, a deoxyribose sugar and a nitrogen base. Phosphate group Deoxyribose sugar Nitrogen base A=Adenosine G=Guanine T=Thymine C=Cytosine. Deoxyribonucleic Acid. Purine.

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Unit 4: DNA and the Molecules of Genetics

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  1. Unit 4: DNA and the Molecules of Genetics

  2. A) Composition of DNA • 4 different nucleotides: Composed of a phosphate group, a deoxyribose sugar and a nitrogen base Phosphate group Deoxyribose sugar Nitrogen base A=Adenosine G=Guanine T=Thymine C=Cytosine • Deoxyribonucleic Acid Purine Pyrimidine

  3. A) Composition of DNA • Chaff’s Rules: • #T + #C = #A = #G • A bonds to T and G bonds to C • Franklin and Wilkins used x-ray diffraction which suggested along and skinny structure that had 2 parallel components, was helical • Watson and Crick – put all information together in 1953 to get final structure of DNA

  4. B) Structure of DNA • Nitrogen base pair structures Purine 2 rings Pyrimidine 1 rings

  5. B) Structure of DNA • Important features of DNA structure • Right handed double helix • Helice are antiparallel • Helice are held together with hydrogen bonds between nitrogen bases

  6. B) Structure of DNA 5’ end • What does 5’ and 3’ mean? • 3’ and 5’ refer to the carbon that is exposed on the sugar • DNA is extended on the 3’ carbon 5’ 4’ 1’ 3’ 2’ Phosphodiester bond 5’ 4’ 1’ 3’ 3’ end 2’

  7. C) RNA • Ribose sugar rather than deoxyribose • Single stranded sequence of nucleotides • Several types: • mRNA – Messenger RNA • tRNA – Transfer RNA • rRNA – Ribosomal RNA • (we will go into these more later) • Nucleotides A, U, G, C • U (Uracil) is used in place of T • RNA contains codons – sets of 3 nucleotides that represent (translate into) amino acids (amino acids make up proteins

  8. D) Central Dogma of Biology Nucleus Cytoplasm Replication Transcription Translation DNA RNA Protein

  9. E) Replication • DNA undergoes semi-conservative replication • The 2 original strands are split and 2 new strands are made • DNA unwinds and nucleotides are being incorporated into new DNA

  10. Nucleotides added to 3’ end of new strand

  11. Nucleotides added to 3’ end of new strand

  12. E) DNA Replication • The small short fragments are called Okazaki fragments – joined together by DNA ligase • DNA Helicase: unwinds DNA helix • DNA Polymerase: adds nucleotides to the 3’ end of the DNA template – also proofreads and corrects error

  13. E) DNA Replication • Practice Replication of DNA: • Old strand: ATG CTA GT • New strand: TAC GAT CA • Try this on you own: • Old strand: TGA CTG ACT AG • New strand: ACT GAC TGA TC

  14. F) Transcription • Synthesis of mRNA • RNA polymerase binds to and separates DNA strands at certain locations (indicated by promoters) • RNA polymerase uses on strand of DNA as a template to make the complimentary strand of mRNA • After the mRNA is created, it goes through processing in the nucleus • Non-coding sections of mRNA (introns) • After splicing mRNA, it travels to the cytoplasm where it interacts with a ribosome to make proteins

  15. G) Sources of DNA • Prokaryotic DNA • A bacteria typically contains only one long, circular DNA molecule • Usually super coiled, folded like a twisted rubber band • Very little spacer (unused) DNA • Extra smaller rings of DNA floating around in cytoplasm – called plasmid • R plasmids contain antibiotic resistance genes • Plasmids can be exchanged between bacterial cells • Regulation: RNA polymerase binds at the promoter regions (gene is on), if something is bound to promoter region the gene is “turned off” because polymerase can not bind and begin transcription.

  16. G) Sources of DNA • Eukaryotic DNA • Packaged in to chromosomes • Generally several chromosomes per cell • Each chromosome is a single, linear, very long molecules of DNA coiled around protein (histones) • Total amount of DNA does not relate to an organism’s complexity • Much of eukaryotic DNA is non-coding, meaning it does not translate to a protein

  17. G) Sources of DNA • Eukaryotic DNA • Regulation: • Does not contain operators like bacterial DNA, • Does not contain promoter region • Enzyme transcribes entire gene until it reaches a terminator region • Genes are usually “on” and expressed at a low level • Expression is increased or decreased with enhancers and silencers – called transcription factors • In DNA is coiled around histones then polymerase can not access DNA to transcribe – essentially “turned off”

  18. G) Sources of DNA • Viral DNA • No cell structure, not living • Consist of protein coat and DNA or RNA • Viral DNA or RNA is relatively short • Once inserted into viable cell, genes turned on

  19. Lab 4b - Background • Why is DNA so important to science & to life in general? • How is the structure of DNA related to its function? • What is the “Central Dogma” concerning DNA? • How has the DNA being used in this lab been isolated?

  20. Today: 12/04/2013 • Your background must be completed today • You should be able to finish the DNA spooling and storage section today, assuming you correctly created your solutions yesterday. • Keep everything cold! • You need to have your trash from earlier experiments cleaned up today, or I’ll dock 10 points from this current lab.

  21. Today: • Using your cell phone/mobile device, log into: • m.socrative.com • Room number: 266770 • If you do not have a capable device, get an ipad from me.

  22. Today: 12/05/2013 • You should complete your lab & notebook write up by the end of class today. • You need to have your trash from earlier experiments cleaned up today, or I’ll dock 10 points from this current lab. • Conclusion Questions:

  23. Conclusion Questions: • What was the purpose of adding the 5 MNaCl solution to the salmon DNA? • What was the purpose of adding 95% ethyl alcohol to your DNA solution, but making sure not to mix the alcohol into the DNA solution? • Why is this experiment required for using DNA? Why couldn’t the prepared salmon testes solution simply have been used? Do NOT do the “Data Analysis & Conclusion” questions posed on page 67

  24. Today: 12/06/2013 • Page 68 – Complete Step one of the Pre-Lab • Page 83 – Complete the procedures for gel prep and storage

  25. Today: 12/09/2013 • Complete making your gels (page 82) • Read background on page 69 & be ready to explain the concepts to Mr. Young prior to testing • Using a well plate prepare: • Well #1 – water • Well #2 – water + ethidium bromide • Well #3 – salmon DNA • Well #4 salmon DNA + ethidium bromide * Each solution will be 10 microliters

  26. Today: 12/12/2013 • Complete Yeast DNA Extraction – Lab 4c, page 68 • Read over & plan out how to do the DNA Extraction from Bacteria – Lab 4h, pages 79-81 • Electrophoresis gels must be transferred out of the pour containers. • What needs to be on your samples: Name of material & concentration/%/etc., initials of the individual that made it & the date that it was made. • Clean up the lab materials. • If you are still interested in going on the AAI trip in the Spring, please put you name on the lab white board

  27. Yeast DNA Extraction – Important Notes 8. Place the used glass stirring rod in the EtOH container in the fume hood. 9. The protease is in a powder form in the top door shelf of the 4ºC fridge. Keep the protease container open as little as possible & return it to the fridge when you are not using it. 10. When you are using the microcentrifuge tubes, place them in used glass tubes so that you can use the controlled centrifuge. 13. Our centrifuge can only reach 6,500 rpm, not 14,000 rpm 20. We will not be using a hair drier. Instead, place your open microtube in the flowing fume hood.

  28. Final Lab: 12/16/2013 • Background: Summarize provided backgrounds on pages 69/70 (Lab 4d), and page 85 (Lab 4j). • Hypothesis: Answer the following: Which type of DNA that you isolated will have the most bands/base pairs? Why do you think this is? • Conclusion: Tape in your print out of your DNA gel • How is this technique useful in the real world? • What errors occurred and what caused these errors? • What do you like/think needs improvement in this class now that you have completed a full semester? • What would you like to do/see next semester?

  29. Today: 12/16/2013 • Check for the presence of DNA in your isolated samples: • Well #1 – 100 µl Water • Well #2 – 100 µl Ethidium Bromide (toxic) • Well #3 – 50 µl Water + 50 µl Ethidium Bromide • Well #4 – 50 µl Salmon DNA • Well #5 – 50 µl Salmon DNA + 50 µl Ethidium Bromide • Well #6 – 50 µl yeast DNA • Well #7 – 50 µl yeast DNA + 50 µl Ethidium Bromide • Well #8 – 50 µl bacterial DNA • Well #9 – 50 µl bacterial DNA + 50 µl Ethidium Bromide

  30. Today: 12/16/2013 • Prepare all of your samples to be put into the gels in 1.7 ml or 2.0 ml microfuge tubes, label them, and place them in the fridge for tomorrow • You need: • Salmon DNA • Yeast DNA • Bacterial DNA • Lambda DNA uncut • Lambda DNA ladder

  31. Today: 12/17/2013 • Lanes in your wells: • Sample Loading dye • Salmon DNA • Yeast DNA • Bacterial DNA • Lambda DNA 100 bp ladder • Lambda DNA uncut • Suspect DNA

  32. Today: 01/06/2014 • New Lab Rules: • Pick up materials only when you need them & know what you are supposed to be doing with them (how much you need, what container it is supposed to go in, etc.) • Return the materials to the table where you found them. • All student products (gels, solutions, plates, etc.) must be labeled with the following. If they are not, they will be throw away: • What is the product (Example: 50X TAE) • Who made this product (Example: MSY) • When was it made (1/6/2014)

  33. Today: 01/06/2014 • Prepare an agarose gel for each member of your group (page 83). • You can be preparing TAE buffer or agarose gel, there is no reason to back up around the analytical balances. • Expected Knowledge: Textbook pages 120-125

  34. DNA Identification Lab • Background: You background should cover the following: • What is a restriction enzyme? • What is/are its role(s) in the natural world? • Why are restriction enzymes used in labs? • In this lab we will be using EcoRI & HindIII. What recognized sequence & cuts do each make in DNA? Make sure to include 5’ and 3’ labeling. • What is agarose? • DNA has what charge & will move towards which end (+/-) during gel electrophoresis?’ • The gels we will be using are 0.8% agarose. Why is the concentration of agarose important? If the concentration was too high, what would the consequences be? • How is the DNA that travels the furthest in a lane different than the DNA that didn’t travel as far?

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