1 / 32

Molecular Biology Lab Announcements - Week Overview

This week's lab involves a transformation lab overview and homework problem set 4 due in lab. Exam 2 preparation, group presentations, and review sessions are scheduled. Details on the Summer Scholar Program and a review of the last lecture on DNA and RNA are provided.

charla
Download Presentation

Molecular Biology Lab Announcements - Week Overview

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Announcements Pick up lab overview for transformation lab this week. Homework-problem set 4- due in lab this week. 3. Look over Ch. 11, problems 4, 5, and 8 for exam 2. 4. Group B presentations are coming up 10/29, 30; start thinking of topics and deciding on sources. Group A did very well - pressure is on! 5. Review session in class Wednesday. Bring your questions! 6. Exam 2 next week: 10/17, 18, and 20. 15 multiple choice and 7 written (exam 1 had 18 multiple choice and 9 written). Exam is at CLAS testing center, available 3 days this time. Hours are 9-9 Thursdays, 9-5 Fridays, and 3-7 Sundays. Bring a pencil, bluebook, calculator. 7. Summer scholar program - research opportunity in summer, $2400 stipend. Need to find a faculty member to sponsor you. Often need to volunteer in the lab spring semester. Application includes a formal written proposal; deadline mid-Feb.; need 56 credits completed by start of summer and be returning next fall to CMU.

  2. Review of Last Lecture Evidence that DNA is genetic material Structure of DNA/RNA: 5 different bases, 2 different sugars, phosphates History: the race to determine the structure of DNA was VERY competitive; 2 key pieces of data = Chargaff’s base compostion analysis and X-ray diffraction studies

  3. Outline of Lecture 20 I. Structure of DNA II. Analytical analysis of nucleic acids III. Replication of DNA How is DNA organized? 1 single chain, 2 chains, 3 chains? How does the structure allow for replication, expression, storage and mutation?

  4. I. The DNA Double Helix DNA structure • Double helical • major, minor grooves • right-handed • bases are 3.4 Å apart (10 Å = 1 nm) • 10 bases/turn • Complementary Base Pairing • through H bonds: A=T, GC • Antiparallel Strands • 5’ to 3’ • 3’ to 5’ Discussion of original paper in class Friday

  5. Right- and Left-handed DNA

  6. Base-Pairing in DNA A=T GC

  7. Structure of RNA Sugar: ribose, not 2-deoxyribose Bases: uracil, not thymine Organization: single-stranded, not double-stranded How is genetic information in DNA expressed? First step is transcribing RNA from DNA - single-stranded RNA is generated using DNA as a template

  8. Reading DNA Strands Single strand of DNA: 5’-AGCATTCG-3’ 3’-TCGTAAGC-5’ Complementary strand of above, usually written 5’ to 3’: 5’-CGAATGCT-3’ Double-stranded fragment is written: 5’-AGCATTCG-3’ 3’-TCGTAAGC-5’

  9. Learning Check The sequence of the dwarf gene in garden peas is as follows: 5’ - A G C T A C G T -3’ 3’ - T C G A T G C A -5’ Write the RNA sequence transcribed from the top strand of DNA, 5’- 3’.

  10. II. Analytical analyses of nucleic acids Denaturation/Renaturation Determining the Tm allows for an estimate of the base composition of a DNA sample 1 2 Which DNA has higher GC content and why?

  11. Nucleic Acid Hybridization Transcription of 1 strand of DNA 3 G G T T G G G C C A A C C C A C G C T T G C G A 2 1 U U U G C G C T T T G C G C A AA C G C G 3 Add RNA to denatured DNA; allow to hybridize Heat - denature A C G C T T G C G A G G T T G G G G G T T G G G C C A A C C C A C G C T 2 1 C C A A C C C T G C G A U U U G C G C Hybrid T T T G C G C A A A C G C G 3 A AA C G C G

  12. Nucleic Acid Gel Electrophoresis

  13. What makes nucleic acids acidic? Base Pairing Rules

  14. Points to know about DNA structure • Note how many hydrogen bonds are in the base pairing: • If 2, then the pair is AT • If 3, then the pair is GC • Recall that A and G are purines with 2 rings, while T and C are pyrimidines with 1 ring; also T has a CH3 group on its ring.

  15. III. DNA Replication How is genetic information replicated accurately at each cell division? Could each strand of the DNA double helix act as a template for the complementary strand? At each cell division, 109 base pairs are replicated. If error rate is 10-6 , then 3000 errors/cell division - TOO many.

  16. DNA Replication is Semiconservative

  17. Other Theoretical Possibilities

  18. Separation of Nucleic Acids by CeCl Gradient Centrifugation

  19. Meselson-Stahl Experiment DNA Labeling with 15N Subsequent Generations Labeled with 14N Cesium Chloride Gradient Banding

  20. Expected Results From Conservative or Dispersive Reproduction If Conservative: Two bands, heavy and light, in 1st and 2nd generations If Dispersive, one smeary band in 1st and 2nd generations

  21. Expected Results if Semiconservative These results were obtained. A related experiment was performed in plants (Fig. 12.5)

  22. Bacterial DNA Replication begins at a Single Origin and Proceeds Bidirectionally Origin of Replication

  23. DNA Polymerase I can Synthesize DNA • Arthur Kornberg et al. (1957) discovered the enzyme in E. coli • Requires template DNA strand, primer, MgCl2, and 4 dNTPs • Monomers added 5’ to 3’

  24. 5’ to 3’ Addition of Monomers

  25. DNA polymerases I, II and III • pol I • most abundant (400/cell) • RNA primer removal • pol II • unknown abundance • DNA repair? • pol III • low abundance (15/cell) • DNA replication

  26. Problems of DNA Synthesis • Unwinding • Tension must be relieved • Priming • Antiparallel strands • RNA primer removal • Backbone joining • Proofreading

  27. Steps of DNA Synthesis • Denaturation and Unwinding • Priming and Initiation • Continuous and Discontinuous Synthesis • Including Proofreading and Error Correction • Removal of Primer • Ligation of nicks in backbone

  28. Steps of DNA Synthesis:Denaturation and Unwinding of DNA • DnaA, DnaB, DnaC proteins are helicases which bind origin and separate strands • Single-strand binding protein (SSBP) keeps strands apart • DNA gyrase, a type of DNA topoisomerase, cuts to relax supercoiling

  29. Initiation of Synthesis • RNA Primase makes RNA primer on DNA template • DNA Polymerase III extends primer with DNA • DNA Polymerase I removes RNA primer, replaces with DNA

  30. Directionality of DNA synthesis

  31. Proofreading occurs as polymerase moves along; if incorrect base pairing, base is removed and replaced.

  32. Continuous and Discontinuous Synthesis • Continuous • on Leading Strand. • Discontinuous • on Lagging Strand • creates Okazaki • fragments. • DNA ligase joins • nicks in backbone.

More Related