Lecture 5: Genetics and Synthetic Biology - DNA Replication and Essay Prompt - Prof. Arthu, Study notes of Biology

Download Lecture 5: Genetics and Synthetic Biology - DNA Replication and Essay Prompt - Prof. Arthu and more Study notes Biology in PDF only on Docsity! Lecture 5: Introduction to Genetics, DNA Replication A few comments on synthetic biology: (essay due on 10/13!) The Institute of Biological Engineering Announces the 2009-2010 Bioethics Essay Contest for undergraduate and graduate students Submit a 1000 to 1200 word, original essay on the following bioethics topic by October 15, 2009, to be part of this year’s contest. TOPIC: “In the context of synthetic biology, how should the public’s concerns regarding safety and ethics be heard and integrated?” Five semifinalists will be selected and invited by January 7, 2010, to read their winning essays at the 2010 Annual Meeting of the Institute of Biological Engineering (IBE) to be held March 4-6, 2010, in Cambridge, MA. The final ranking of the essay will be determined after the reading of the essays at the Annual Meeting. The top three winners will receive cash awards of $150 for first place, $100 for second place, and $75 for third place. The first place essay will be reviewed for publication in the Journal of Biological Engineering, the official journal of IBE. Entries should be submitted in Word or PDF format to the following email address: essay@ibe.org Undergraduate and graduate students may enter the contest. By submitting, you are agreeing to attend the 2010 IBE Annual Meeting and provide your own travel expenses if you are selected as a semifinalist. IBE will provide complimentary Annual Meeting registration for all five semifinalists. All essays must include title, author’s full name, program of study, degree you are seeking, name of the institution, daytime telephone number, email address, and mailing address. Entries will be submitted to a plagiarism-checking service. IBE reserves the right to disqualify any entry judged as non-original or any entry over 1200 words. All winning essays will become the property of IBE. IBE is not responsible for any possible difficulties in submission. Only winners will be notified of results. Register for the 2010 IBE annual meeting at: www.ibe.org Registration for the meeting will begin in October. The Institute of Biological Engineering www.ibe.org •  What is your definition of synthetic biology? •  In general, synthetic biologsts engineer artificial, generally microbial life •  Drug design/delivery •  New or modified fuel sources Use of a Punnett Square to predict outcomes •  Next experiment: crossing F2 plants with each other, to distinguish between F2 •  Green F2 plants crossed with other green F2 plants yielded only green peas •  Yellow F2 plants crossed with other yellow F2 plants: 1/3 only yellow, 2/3 had a mixture of yellow and green in a 3:1 ratio (just like F2 results) •  Overall, from the F1 x F1 cross, ¼ were pure green, ¼ were pure yellow, and ½ were impure yellow •  A Punnett square lists all possible combinations and allows a simple representation of these findings •  Y/Y: homozygous dominant; Y/y: heterozygous; y/y: homozygous recessive •  What ratios would you expect if you cross a homozygous recessive plant (a/a) with a heterozygous plant? •  Punnett squares are suitable for predicting results from single gene crosses •  Things get more complicated in predicting potential outcomes for multiple genes •  Now assume that there are two genes being investigated: pea color (Y: YELLOW, y: green) and seed shape (R: ROUND, r: wrinkled) •  First cross: P x P: Y/Yr/r x y/yR/R •  Results: F1 were all YELLOW and ROUND •  This is intuitive, but a Punnett square helps: P Yr yR YyRr 16/16 ROUND YELLOW Outcomes for multiple genes •  Second cross: F1 x F1: Y/yR/r x Y/yR/r •  Results: F2 were 9/16 ROUND YELLOW, 3/16 ROUND green, 3/16 wrinkled YELLOW, and 1/16 wrinkled green •  Again, a Punnett square helps to understand how we got these numbers, but is complicated (we won’t go through the whole square) F1 YR Yr yR yr YR YYRR YYrR yYRR yYrR Yr YYRr YYrr yYRr yYrr yR YyRR YyrR yyRR yyrR yr YyRr Yyrr yyRr yyrr 9/16 ROUND YELLOW 3/16 ROUND green 3/16 wrinkled YELLOW 1/16 wrinkled green Outcomes for multiple genes, cntd. Probability applied to genetics •  We can apply the same principles to genetics… •  Suppose that there are two animals of genotypes •  A/a, b/b, C/c, D/d, E/e •  a/a, B/b, c/c. D/d. E/E •  What is P(A/a, b/b, C/c. d/d. E/E)? A/a: ½; b/b: ½; C/c: ½; d/d: ¼; E/E: ½; total = ½ x ½ x ½ x ¼ x ½ = 1/64 •  What is P(a/a, b/b, c/c. d/d. e/e)? 0 •  When might you use such analysis in a biology lab? Replication allows the duplication of DNA •  When and why would you need to duplicate DNA? •  Single-strand binding proteins stabilize DNA unwound by helicases •  Leading strand is synthesized continuously by DNA polymerase •  Lagging strand is synthesized discontinuously •  Primase synthesizes a short RNA primer, which is extended by DNA polymerase to form Okazaki fragments •  RNA is replaced by appropriate DNA •  DNA ligase joins fragments together to complete strand •  Can you think of problems that unwinding DNA might cause in the helical DNA? DNA polymerase DNA polymerase ligase RNA primer primase helicase SS binding proteins topoisomerase Okazaki fragment Proofreading mechanisms provide faithful replication •  Can you estimate the replication error rate for mammalian DNA? 1 in ~106-107 •  Several proofreading mechanisms exist in the 5’-3’ direction •  Base pair addition is based on binding kinetics [R] + [L] [RL] (k+: association rate constant; k-: dissociation rate constant) d[RL]/dt = k+[R][L] – k-[RL] •  Higher association rate if base pairs are complementary •  After nucleotide binding, but before covalent addition to the chain: •  Polymerase undergoes a conformational change. •  Incorrectly bound bases have a higher rate of dissociation. •  After covalent binding: •  A 3’-OH site on the strand is required for extension •  Mismatched base makes this site inaccessible •  Exonuclease (separate catalytic site in polymerase) clips off mismatched bases until proper 3’-OH site is exposed k+ k- Double-checking the PCR result: gel electrophoresis •  Polymerase-based proofreading occurs, but there may still be errors •  Gel electrophoresis allows you to test for a DNA product •  Basic principle: load DNA and a dye that binds to DNA at the top of an agarose gel •  Also run a ladder with fragments of known DNA size (why?) •  Apply a current across the gel •  DNA migrates from negative to positive electrodes due to negative charge of sugar phosphate backbone http://users.ugent.be/~avierstr/principles/pcr.html Double-checking the PCR result: sequencing •  Gel electrophoresis tested whether you had a product or not •  Sequencing tests the accuracy of the product •  Similar to PCR, EXCEPT •  Only one primer •  Some dNTPs are replaced with fluorescent ddNTPs (3’-H instead of 3’-OH). What effect would this have on synthesis? http://users.ugent.be/~avierstr/principles/seq.html Double-checking the PCR result: interpreting sequencing data •  Where might you expect inaccuracy? What might you do to resolve this? http://users.ugent.be/~avierstr/principles/seq.html