Cloning and Sequencing Explorer Series

2. Cloning and Sequencing Explorer Series

3. Instructors 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 Essy Levy, M.Sc. essy_levy@bio-rad.com Leigh Brown, M.A. leigh_brown@bio-rad.com

4. Partnering with Bio-Rad Bellarmine University Louisville, Kentucky Prof. Dave Robinson Dr. Joann Lau US Department of Energy Joint Genome Institute Walnut Creek, Ca Geospiza Inc. Seattle, Wa Sandra Porter

5. Why TeachCloning 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. Appropriate courses • Molecular Biology • Recombinant DNA Techniques • Biotechnology • Molecular Evolution • Bioinformatics • Advanced Cell Biology • Advanced Genetics • Advanced Plant Biology • Independent Research

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

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

10. Genomic DNA Extraction DNA Precipitation DNA Quantitation GAPDH PCR Nested PCR Degenerate primers Exonuclease Gel Electrophoresis DNA Gel Interpretation Band Identification Standard Curve Use 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

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

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

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

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

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. 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 • Use of degenerate primers in the initial PCR reaction may also result in some non-specific amplifications

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

19. Initial PCR Nested PCR 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

20. PCR results1% agarose gel loaded with 20 µl initial PCR samples and 5 µl nested PCR samples. 1 2 3 4 5 6 7 8 9 Arabidopsis Green bean Lamb’s ear pGAP MW I N I N I N I N 2000 bp- 1500 bp- 1000 bp- 500 bp-

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

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

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

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

26. 1 2 3 4 5 6 7 8 9 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 pJet vector GAPDH inserts Digested and undigested DNA were electrophoresed on a 1% TAE agarose gel

27. Setting up Sequencing Reactions • Add sequencing primers to DNA (10 minutes) • Load 96-well plate • Send sealed plate off to JGI for sequencing • JGI will post sequence on the web

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

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. Sequencing • 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

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

32. iFinch Landing Page

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

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

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

36. FinchTV Sequence Chromatograms

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

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

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

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. Student Authors Great for a resume!

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

44. Watch the Webinar Playback Planning Guide available for download

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