Genomics & Biotechnology, AP Biology 2014 VIEW IN SLIDE SHOW MODE

1. Genomics & Biotechnology, AP Biology 2014VIEW IN SLIDE SHOW MODE What is Genomics? SEQUENCING OF GENOMES, FOLLOWED BY APPLICATION OF THE DATA EITHER TO: FURTHER RESEARCH OF THE GENOME & ITS EXPRESSION Or TO ACHIEVE HUMAN GOALS

2. HOW TO USE THIS LESSON Work through the slides, exiting to run animations at the pasted websites or to review any diagrams and explanations. Each slide ends with several questions that you need to answer before moving on to the next slide or activity. Your knowledge of these answers will be assessed in a formative assessment.

3. Learning goals • Students will understand polymerase chain reaction (PCR) method & will be able to explain how PCR has accelerated biological discovery. • Students will understand methods for nucleic acid electrophoresis, blotting, and hybridization. • Students will understand methods for sequencing DNA. • Students will understand use of plasmid & phage vectors to clone genes, and they will be able to explain how restriction mapping & PCR analysis can be used to verify correct construction. • Students will understand methods for constructing & screening cDNA microarrays & will be able to explain the importance of microarrays for understanding gene expression and for individualizing medical diagnoses.

4. Learning Goal 1Students will understand the PCR method & will be able to explain how PCR has accelerated biological discovery. Watch these animations, then answer the questions http://www.hhmi.org/biointeractive/polymerase-chain-reaction-pcr http://www.hhmi.org/biointeractive/polymerase-chain-reaction http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter14/animation_quiz_6.html • What is the purpose of PCR? • What enzyme is used? • What is a primer? What is one reason for a primer? • What is the purpose for starting each cycle at 95˚C? For lowering to 77 ˚C? To 55 ˚C? • How many copies of a single DNA molecule can be made in 3 hours (after 30 cycles of PCR)? Skim this reference http://www.sciencemag.org/site/products/pcr.xhtml f. Describe 3 ways PCR has advanced discovery in biology.

5. Learning goal 1 (PCR) continued Run this animation, then answer questions below http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter14/animation_quiz_6.html g. What uses for PCR are described? • What is special about Taq polymerase (isolated from thermophilic bacteria from geysers) versus DNA polymerase from other cells? • Identify a 2nd reason a primer is essential to allow PCR. (think back to mechanisms of DNA replication & synthesis in the 5’ to 3’ direction) 5’ 3’-OH PO4—5’ C

6. By choosing PCR primers complementing the cloning site of a gene into a vector, PCR shows can be used to verify correct construction of a recombinant plasmid. The method also facilitates plasmid construction. www.addgene.org/plasmid_protocols/PCR_cloning/If restriction sites are added to the ends of the primers, PCR can be used to generate restriction PCR fragments that complement ends of a gene segment carrying a disease causing mutation generate products whose size show whether or not a person carries a disease causing allele (if the typical and atypical alleles have different sizes) fragments used to clone a gene into a vector cut to produce the same restriction enzyme “sticky ends”

7. If one or PCR primer is made to complement a disease-associated alleles sequence (the mutated section) but not the wildtype (typical) allele, then PCR fragments will have different lengths for the typical allele and disease allele, allowing determination whether a person is homozygoticdd or heterozygotic(Dd a carrier) for the disease causing allele. DD Dddd RR

8. The most important feature of PCR is that it very rapidly and cheaply copies DNA, allowing one or two copies to be amplified to billions within a few hours. PCR can be used for automated DNA sequencing. PCR can be used to replace RFLP analysis (in less time and with less complex & less expensive analysis) PCR can be used to add needed restriction fragments to genes so that you can use any vector’s multicloning site. PCR allows detection of disease causing alleles.

9. Kerry Mullis received the Nobel prize for his invention of PCR technique.

10. LG 2 Students will understand methods for nucleic acid electrophoresis, blotting, and hybridization. By comparing the relative migration of a nucleic acid fragment of unknown size & that of a fragment of known size, one can determine the size of the unknown fragment.

11. Gel electrophoresis allows you to find the size of a piece of DNA by comparing it to the sizes of known pieces of DNA (called DNA size standards). DNA --negative charge --runs from negative pole to positive pose of gel. Longer pieces travel slower than shorter pieces. http://www.dnalc.org/resources/animations/gelelectrophoresis.html

12. LG3: Students will understand methods for sequencing DNA. Run a separate PCR reaction for each nucleotide. Include a small fraction of Taq polymerase enzyme blocking nucleotide analogue. All nucleotides are labeled for detection. At each analogue binding, the polymerase stops. By random chance it stops at each possible nucleotide many times. Read from shortest to longest. Sequence of replicated strand CACTCAGTGATG Sequence original strand GTGAGTCACTAC

13. The Ultimate test for whether you’ve correctly cloned a gene sequencinghttp://www.dnalc.org/resources/animations/sangerseq.html http://www.hhmi.org/biointeractive/dna/DNAi_human_genome_seq.htmlhttp://www.hhmi.org/biointeractive/dna/DNAi_sanger_sequencing.htmlhttp://www.hhmi.org/biointeractive/dna/DNAi_shotgun_seq.htmlThe human genome project used “real time sequencing” via PCR. 4 tubes—Taq polymerase, >99% normal nucleotide (ATP, e.g.), <1% altered nucleotide that acts as a reversible inhibitor of Taq. All nucleotides are labeled with a different fluorescent dye. One tube A-purple, another tube T-yellow, another tube C-pink, another tube G—green. DNA runs into a electrophoresis tube & as it migrates past a light sensor, the identify of the next nucleotide in the sequence is recorded.

14. Making & probing a Southern blot p395

15. Labeled Probe—single stranded piece of DNA OR RNA that is labeled (e.g., radioactive or colored)—here shown in yellow—will only complement the gene of interest. http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter14/animation_quiz_5.html southern blotting explained http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter14/animation_quiz_4.html probes explained

16. DNA fingerprinting (e.g., in a paternity case) is an application of blotting • http://highered.mcgraw-hill.com/sites/9834092339/student_view0/chapter17/dna_fingerprinting.html Genetic testing by restriction fragment polymorphisms is another application of blotting http://highered.mcgraw-hill.com/sites/9834092339/student_view0/chapter17/restriction_fragment_length_polymorphisms.html

17. Blots & labeled probes can be used to identify bacterial colonies transformed with a particular recombinant gene.

18. Learning Goal 4: Students will understand use of vectors & bacteria to clone genes. Level 2.0 2.0-1 Define or identify examples or descriptions. • Colony • Multicloning site • Plasmid • Recombinant DNA • Reporter gene • Resistance gene • Restriction enzyme • Selection • Sticky ends • Transformation 2.0-2 Label diagrams that describe gene cloning using bacterial plasmids. Level 3.0 3.0-1 Explain molecular events during construction, transformation, selection, & amplification of recombinant bacterial plasmid vectors. 3.0-2 Explain how restriction maps and PCR analysis can be used to verify correct construction of a recombinant plasmid vector. Level 4.0 Explain how PCR has simplified the process of plasmid cloning.

19. Tools of the trade: What is a vector?A vector is a piece of DNA that can shuttle DNA from one organism to another. Plasmid—small circular DNA element found in the cytoplasm of bacteria, some protists, and sometimes plants. In bacteria, these replicate when the host chromosome replicates. Ti plasmid—plasmid normally found in a bacterium causing plant tumors (galls). Ti plasmids allow high efficiency transformation of plants. Plants can be propogated asexually (by rooting cuttings), facilitating gene transfer in plant populations* do study guide #33

20. What is “cloning a gene”? Why do scientists clone genes? Cloning a gene is the process of placing a gene sequence in a piece of DNA or RNA called a vector, then transforming the vector into host cells: bacteria, viruses, or yeasts. Rapid reproduction of either of these hosts result in rapid copying (amplifying) the vector with its cloned gene with every round of host cell division.* http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter14/animation_quiz_1.html

21. Tools of the trade: Bacterial plasmid vectors Bacterial plasmids commonly carry antibiotic resistance genes that allow only cells that contain the plasmid to survive selection. Many different genetically modified plasmids are available for purchase. These can be purchased to code particular antibiotic resistance (for selection)and to allow cloning of genes with different terminal restriction sequences at the multi-cloning site. Weakness: Plasmids an’t carry whole genes with regulatory regions & introns (genes too large); bacterial promoters don’t bind normal eukaryotic transcription factors, so expression may not be regulated normally if used in gene therapy. bacterial hosts don’t carry out alternative splicing.* The multicloning site is located inside a Reporter Gene. http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter14/animation_quiz_2.html

22. Before you can clone a gene, you must remove it from the chromosome and place it into a vector that can transfer it into a host cell. Restriction enzymes allow you to cut sequences out of DNA or to prepare vectors for insertion of new DNA.* Alternatively, PCR primers can be used to copy a gene and add the correct restriction sites to its ends to allow it to be cloned into a vector.

23. Tools of the trade: Restriction enzymesRestriction enzymes cut DNA at specific recognition sequences and generate “sticky” (complementary single stranded ends) fragments. These are needed to insert genes into vectors. PCR primers can be used to create restriction sites to facilitate adding a gene to any needed vector. http://highered.mcgraw-hill.com/sites/9834092339/student_view0/chapter17/restriction_endonucleases.html

24. Note restriction enzymes recognize and cut sequences having the same sequence in the 5’3’ direction on both complementary strands. • The ends are sticky because they complement the ends of any other piece of DNA cut with the same restriction enzyme. • The complementary base pairs align and H bond. • Some restriction enzymes will not cut DNA that has been modified by methylation (so heterochromatin is hard to cut).

25. Parts of a plasmid • Antibiotic resistance gene (codes an enzyme that destroys an antibiotic normally lethal to bacteria)—allows only cells transformed with the plasmid with this resistance gene to survive in the presence of that antibiotic. • Reporter gene (allows cells having plasmids with inserted genes to be distinguished from cells whose plasmids are not carrying a cloned gene) e.g., beta galactosidaseLacZ—keep the host that are not blue in presence of Xgal substrate • Multi-cloning site inside the reporter gene—restriction enzyme cutting sequences allow the gene of interest to be inserted—if a gene is inserted, the reporter gene is inactivated.*

26. Once cut out of the original chromosome, the gene must be prepared for transfer into the new host cell. • It must be ligated into a piece of DNA, a vector, that can transfer it into the new host. • If the vector and gene are cut with the same restriction enzyme, the enzyme ligase will join them together.*

27. The recombinant plasmids are transformed into competent bacterial hosts, then as the bacterial hosts replicate, they copy (amplify) the plasmid and the cloned (copied) gene. Not all of the transformed cells will contain a plasmid with an insert (some have no plasmid, some have a plasmid without an insert), so negative selection (with a drug to which only plasmid bearing cells are resistant) and positive selection (only cells without reporter gene activity) are collected. These occur as colonies on a petri culture plate.

28. How do you transform bacteria? Make bacteria competent for plasmid uptake. That is, grow them to log phase—that is, to the point at which the population is growing exponentially and no limiting factors like competition for food or space are present. Then, Use CaCl2 to burst small holes in the bacterial cell walls and add plasmids. Heat over optimal temperatures for 90 seconds to activate heat shock proteins (SOS) proteins to repair the holes.

29. What is a colony? Clones of the same single bacterium that survived selection in the antibiotic. Pick the transformed, insert carrying colonies, then grow large numbers of these. Test them for accuracy of insertion (correct direction) and accuracy of sequence (correct size and nucleotide sequence)

30. Phages may also be used as vectors to transfer DNA into bacteria. • Phages are viruses specific to bacteria. • These are usually used in constructing DNA libraries (covered later in the chapter).

31. Review: How is a gene cloned into a plasmid vector then amplified in host bacterial cells?Run: http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter14/animation_quiz_1.htmlRun http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter14/animation_quiz_2.html

32. Now that you’ve obtained colonies that carry plasmids with inserted genes, how do you confirm that the gene is the correct gene, is oriented correctly, and is not damaged in sequence? Use Restriction fragment analysis OR PCR analysis to confirm size and orientation. Use either DNA sequencing or high stringency probe hybridization (only very accurate complementary base pairing probes adhere at high temperature and salt concentration)

33. Restriction fragment analysis allows determination of whether a host carries plasmids having the size and orientation and overall structure needed. Plasmids isolated from selected colonies are cut with restriction enzymes matching sites in either the plasmid OR the insert OR both. Because the sequence of both are known (restriction maps), it is possible to predict the sizes of DNA fragments expected. Gel electrophoresis is used to separate the fragments and determine their size. http://www.dnaftb.org/24/problem.html http://www.phschool.com/science/biology_place/biocoach/red/mapping.html

34. Often mutations in genes interupt restriction enzyme recognition sites, creating restriction fragment polymorphisms (RFLPs). By cutting DNA with restriction fragments and comparing size to known expected sizes, a person’s DNA can be tested to see whether he carries a disease causing allele. P 393. For example, this disease allele would produce smaller pieces of DNA than the typical allele.

35. how to use RFLP analysis to test for presence of sickle cell allele When a fragment of DNA carrying the hemoglobin gene is cut with the restriction enzyme DdeI, it produces 2 short pieces (175 bp and 201 bp and 1 large piece of DNA), but the sickled allele produces a larger 376 bp fragment because it is missing one DdeI site. Run Activity: Analyzing DNA Fragments Using Gel Electrophoresis RunInvestigation: How Can Gel Electrophoresis Be Used to Analyze DNA?

36. Analyze your ability to interpret restriction maps Go to Lab Bench at the Campbell’s website. Work part II of #6 molecular biology & answer the questions that go with it. You should write these into your study guide, rather than typing them into the online lab report.

37. Genomic Libraries—asingle tube of viral supernatant (like lambda λ phage DNA) or of bacteria, like E. coli, contains vectors whose inserts include every sequence in the genome, cut into fragments. Use it to obtain a gene, ready to use. 1 Isolate genomic DNA (total DNA) from an organism’s cells (for example, from crushed muscle) 2 Cut the DNA with a restriction enzyme, generating many fragments with the same sticky ends e.g., when a “6 cutter enzyme”--like EcoR1--whose restriction site contains 6 nucleotides is used, the human genome is cut into pieces 20K base pairs long. 3 Cut the vector (usually λ phage with the same R enzyme), then ligate in the entire collection of inserts. 4 infect bacterial hosts (transduction), select, and freeze the entire culture until time to screen it for a certain gene.

38. Genomics involves sequencing entire genomes, then applying the info—use genomic libraries to get the DNA fragments used in sequencing.

39. cDNA Libraries—asingle tube of viral supernatant (like lambda λ phage) or E. coli contains vectors whose inserts include DNA complementary to RNA being expressed in the cell at the time RNA was collected. Use it to compare gene expression in the same organism, but in different differentiated cell types, in the same cell type under different conditions, etc. • Obtain total RNA Or mRNA from cells • Add retroviral reverse transcriptase to reverse transcribe the RNA molecules into complementary DNA (cDNA)—no introns, shorter than genomic DNA inserts + whole transcript on a single fragment • Cut cDNA’s with same Restriction enzyme as the λDNA, then ligate, transduce into bacteria, keep the culture until ready to screen for particular genes.

40. DNA microarrays allow rapid screening for genes important in phenotype. http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter14/animation_quiz_3.htmlcDNA

41. RUN: http://www.hhmi.org/biointeractive/genomics/sm_microarrays.htmlWatch http://www.hhmi.org/biointeractive/genomics/gene_chips.html Describe 3 different ways DNA microarrays are useful in either basic scientific research or in personalized medicine.