1 Cloning and Sequencing Explorer Series 2 Instructors

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Cloning and Sequencing Explorer Series 2

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 Leigh Brown, M. A. leigh_brown@bio-rad. com 3

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

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 Gen. Bank • 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 5

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

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

Student use the following techniques 8 • • • • 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 Gen. Bank submission

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

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

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

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 12

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

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

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

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

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 17 • Use of degenerate primers in the initial PCR reaction may also result in some nonspecific amplifications

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

Using Nested PCR to increase your final PCR product Initial PCR DNA template: Genomic DNA 19 Nested PCR DNA template: Initial PCR products • 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

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

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

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

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

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

Analysis of plasmid digests Bgl II Digest GAPDH inserts 2. 5 kb > 2. 0 kb > 1. 5 kb > 1. 0 kb > 0. 5 kb > p. Jet vector 25

Example of a miniprep digestion with Bgl II 1 2 3 4 5 6 7 8 9 p. Jet vector GAPDH inserts Digested and undigested DNA were electrophoresed on a 1% TAE agarose gel Lane 1: 500 bp molecular weight ruler Lanes 2, 4, 6, 8: minipreps digested with Bgl. II Lanes 3, 5, 7, 9: undigested minipreps • Different sizes of inserts suggests different GAPDH genes were cloned in this ligation 26 • Inserts can vary from 0. 5– 2. 5 kb depending on plant species

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

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

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. 29

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

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

i. Finch Landing Page 32

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

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

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

Finch. TV Sequence Chromatograms 36

Data Analysis Contiguous sequence • Need to examine all 4 sequences (2 forward, 2 reverse) • Determine overlap and align sequences 37 • Run the CAP 3 program to assemble the sequence fragments to a full-length “contig” or contiguous sequence

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

Example of an alignment Compare between groups before publishing data 39

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

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 41

Student Authors Great for a resume! 42

Try i. Finch http: //classroom 1. bio-rad. ifinch. com/Finch/ Username: BR_guest Password: guest 43 Tutorial movies available http: //www. geospiza. com/ifinch. Bio. Rad. html

Watch the Webinar Playback Planning Guide available for download 44

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 45