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2. 2 Cloning and Sequencing Explorer Series

3. 3 Stan Hitomi Coordinator – Math & Science Principal – Alamo School San Ramon Valley Unified School District Danville, CA Kirk Brown Lead Instructor, Edward Teller Education Center Science Chair, Tracy High School and Delta College, Tracy, CA Bio-Rad Curriculum and Training Specialists: Sherri Andrews, Ph.D. sherri_andrews@bio-rad.com Leigh Brown, M.A. leigh_brown@bio-rad.com Instructors

4. 4 Partnering with Bio-Rad Geospiza Inc. Seattle, Wa Bellarmine University Louisville, Kentucky Prof. Dave Robinson Dr. Joann Lau

5. 5 Why Teach Cloning and Sequencing Series? Students guide the research process and make decisions about their next steps Encompasses a myriad of laboratory skills and techniques commonly used in research Students generate original data that may lead to publications in GenBank Students formulate scientific explanations using data, logic, and evidence Students understand research is a process rather than a single experiment giving students a real-life research experience with both its successes and challenges

6. 6 Appropriate courses Molecular Biology Recombinant DNA Techniques Biotechnology Molecular Evolution Bioinformatics Advanced Cell Biology Advanced Genetics Advanced Plant Biology Independent Research

7. 7 Laboratory Overview Microbial Culturing Antibiotic Selection Sterile Technique Genomic DNA Extraction DNA Precipitation DNA Quantitation GAPDH PCR Nested PCR Degenerate primers Exonuclease Gel Electrophoresis DNA Gel Interpretation Band Identification Standard Curve Use Cloning Direct PCR cloning Transformation Ligation PCR Purification Size Exclusion Chromatography PlasmidMiniprep Restriction Enzyme Digestion Gel Electrophoresis Sequencing Automated sequencing Bioinformatics Sequence Data Editing Contig Assembly Intron-Exon Prediction

8. 8 Student use the following techniques Micropipetting DNA extraction Gel electrophoresis & interpretation Polymerase chain reaction DNA purification Restriction enzyme digests Microbiological sterile technique Preparing competent bacteria DNA ligation Heat-shock transformation Plasmid DNA isolation Sequence analysis BLAST searching GenBank submission

9. 9 Output of the lab is a tangible product  Publication in Genbank database Students as Authors of Sequence Data

10. 10 DNA Preparation and PCR Amplification of GAPDH Students choose plant tissue Two rounds of PCR –Round 1: Use of degenerate primers –Round 2: Nested PCR Genomic DNA Extraction DNA Precipitation DNA Quantitation GAPDH PCR Nested PCR Degenerate primers Exonuclease Gel Electrophoresis DNA Gel Interpretation Band Identification Standard Curve Use

11. 11 Benefits of using plants Large number of species Lots of diversity Phylogenetic approaches Avoid ethical concerns associated with animals No pre-approval

12. 12 What is a Housekeeping Gene? Highly conserved genes that must be continually expressed in all tissues of organisms to maintain essential cellular functions. Examples: GAPDH Cytochrome C ATPase ß-actin

13. 13 Why use GAPDH? Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) Enzyme of glycolysis Structure and reaction mechanism well-studied Multitude of sequences Highly conserved

14. 14 Host Cell Endosymbiotic event Most algae gene duplication Land plants gene duplication Mesostigma (small group of green algae) GAPC GAPAGAPA/B GAPC/CP Gene Families

15. 15 DNA Extraction Use young, fresh plant-tissue DNA extraction at room temperature Time requirement ~30 minutes Does not require DNA quantification

16. 16 PCR Reactions Initial Nested Color-coded PCR primers (hallmark of Bio-Rad PCR kits) Two positive controls Arabidopsis pGAP (plasmid DNA) One negative control

17. 17 Use of degenerate primers in the initial PCR reaction may also result in some non- specific amplifications Initial PCR is done with degenerate primers Degenerate primers are a mix of primers with variable sequences designed to recognize the GAPDH genes of different plant species

18. 18 Nested PCR amplifies only regions within the GAPDH gene Nested PCR is more specific

19. 19 Using Nested PCR to increase your final PCR product There is more PCR product from the nested PCR reactions since there is more specific template DNA to start from Results: intense, bold band on agarose gel DNA template: Genomic DNA DNA template: Initial PCR products Initial PCRNested PCR

20. 20 PCR results 1% agarose gel loaded with 20 µl initial PCR samples and 5 µl nested PCR samples. ArabidopsisGreen beanLamb’s earpGAP MW ININININ 123456789 500 bp- 1000 bp- 1500 bp- 2000 bp-

21. 21 Choose best PCR products for ligation Transformation and selection Plasmid Miniprep preparation Microbial Culturing Antibiotic Selection Sterile Technique Cloning Direct PCR cloning Transformation Ligation PCR Purification Size Exclusion Chromatography PlasmidMiniprep Restriction Enzyme Digestion Gel Electrophoresis Ligation, Transformation and Plasmid Minipreps

22. 22 Ligation and Transformation Column purification (10 minutes) Blunt-end PCR product & ligate to pJet vector (30 minutes) Preparation of competent bacteria cells (30 minutes) Efficient heat-shock transformation (15 minutes)

23. 23 Picking colonies for plasmid minipreps Each colony is a clonal growth (clones) from one transformed bacteria

24. 24 Plasmid minipreps Isolate plasmid DNA (40 minutes) Restriction digest (1 hour) Electrophorese to confirm inserts (20 minutes)

25. 25 2.5 kb > 2.0 kb > 1.5 kb > 1.0 kb > 0.5 kb > Analysis of plasmid digests pJet vector GAPDH inserts Bgl II Digest

26. 26 Example of a miniprep digestion with Bgl II Lane 1: 500 bp molecular weight ruler Lanes 2, 4, 6, 8: minipreps digested with BglII Lanes 3, 5, 7, 9: undigested minipreps Different sizes of inserts suggests different GAPDH genes were cloned in this ligation Inserts can vary from 0.5–2.5 kb depending on plant species 123456789 Digested and undigested DNA were electrophoresed on a 1% TAE agarose gel pJet vector GAPDH inserts

27. 27 Setting up Sequencing Reactions Add sequencing primers to DNA (10 minutes) Load 96-well plate Send sealed plate off to sequencing facility for sequencing

28. 28 pJet cloning vector Setting up Sequencing Reactions Always need to sequence reverse, complementary strand GAPDH gene of interest

29. 29 Why use multiple sequencing primers? Typical sequencing reactions yield 500-600 bases of sequence. If the GAPDH insert is longer a single set of sequencing primers will not lead to the full sequence.

30. 30 Sanger method of sequencing 4 fluorescent dyes- 1 for each base DNA fragments separated by CE Fragments separated in sequential order iFinch screens out low quality sequence Sequencing

31. 31 Bioinformatics Two month subscription to genetic analysis software from Geospiza Data is stored on iFinch server Data can be accessed 24/7

32. 32 iFinch Landing Page

33. 33 What does iFinch do? Upload and store DNA sequence data Examine the quality of the sequences Screens for GAPDH & vector sequences

34. 34 # sequences per folder (4) Folder for each student group iFinch Data Folders

35. 35 http://www.geospiza.com/finchtv.html FinchTV Free application for viewing and editing chromatograms

36. 36 FinchTV Sequence Chromatograms

37. 37 Data Analysis Contiguous sequence Need to examine all 4 sequences (2 forward, 2 reverse) Determine overlap and align sequences Run the CAP3 program to assemble the sequence fragments to a full-length “contig” or contiguous sequence

38. 38 Students compare 3 sequences What is the accurate sequence? Usually requires going back to chromatograms Assembling the full-length contig Contiguous sequence

39. 39 Example of an alignment Compare between groups before publishing data

40. 40 Searches a DNA/protein database for published sequences that are similar to your sequence Basic Local Alignment Search Tool, or BLAST BLAST Searches

41. 41 Additional Analysis Assemble full-length contig Perform BLAST searches Identify and remove intronic regions Six-frame translation Align multiple contigs from the same species Preparing sequence for publication

42. 42 Student Authors Great for a resume!

43. 43 Try iFinch http://www.geospiza.com/ifinchBioRad.html http://classroom1.bio-rad.ifinch.com/Finch/ Username: BR_guest Password: guest Tutorial movies available

44. 44 Watch the Webinar Playback Planning Guide available for download

45. 45 Webinars Enzyme Kinetics — A Biofuels Case Study Real-Time PCR — What You Need To Know and Why You Should Teach It! Proteins — Where DNA Takes on Form and Function From plants to sequence: a six week college biology lab course From singleplex to multiplex: making the most out of your realtime experiments explorer.bio-rad.com  Support  Webinars