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Cloning and Sequencing Explorer Series

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Stan HitomiCoordinator – Math & SciencePrincipal – Alamo SchoolSan Ramon Valley Unified School DistrictDanville, CA

Kirk BrownLead Instructor, Edward Teller Education CenterScience 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

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Partnering with Bio-Rad

Geospiza Inc.Seattle, Wa

Bellarmine University Louisville, Kentucky Prof. Dave RobinsonDr. Joann Lau

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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

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Appropriate courses

• Molecular Biology

• Recombinant DNA Techniques

• Biotechnology

• Molecular Evolution

• Bioinformatics

• Advanced Cell Biology

• Advanced Genetics

• Advanced Plant Biology

• Independent Research

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Laboratory Overview

Microbial CulturingAntibiotic SelectionSterile Technique

Genomic DNA ExtractionDNA PrecipitationDNA Quantitation

GAPDH PCRNested PCRDegenerate primersExonuclease

Gel ElectrophoresisDNA Gel InterpretationBand IdentificationStandard Curve Use

CloningDirect PCR cloningTransformationLigation

PCR PurificationSize Exclusion Chromatography

Plasmid MiniprepRestriction Enzyme DigestionGel Electrophoresis

SequencingAutomated sequencing

BioinformaticsSequence Data EditingContig AssemblyIntron-Exon Prediction

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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

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Output of the lab is a tangible productPublication in Genbank database

Students as Authors of Sequence Data

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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 ExtractionDNA PrecipitationDNA Quantitation

GAPDH PCRNested PCRDegenerate primersExonuclease

Gel ElectrophoresisDNA Gel InterpretationBand IdentificationStandard Curve Use

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Benefits of using plants

• Large number of species

• Lots of diversity

• Phylogenetic approaches

• Avoid ethical concerns associated with animals

• No pre-approval

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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

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Why use GAPDH?

Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH)

• Enzyme of glycolysis

• Structure and reaction mechanism well-studied

• Multitude of sequences

• Highly conserved

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Host Cell

Endosymbiotic event

Most algae

gene duplication

Land plants

gene duplication

Mesostigma(small group of green algae)

GAPC

GAPA GAPA/B

GAPC/CPGene Families

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DNA Extraction

• Use young, fresh plant-tissue

• DNA extraction at room temperature

• Time requirement ~30 minutes

• Does not require DNA quantification

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PCR Reactions

Initial

Nested

• Color-coded PCR primers (hallmark of Bio-Rad PCR kits)

• Two positive controls• Arabidopsis• pGAP (plasmid DNA)

• One negative control

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•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

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Nested PCR amplifies only regions within the GAPDH gene

Nested PCR is more specific

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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 PCR Nested PCR

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PCR results

1% agarose gel loaded with 20 µl initial PCR samples and 5 µl nested PCR samples.

Arabidopsis Green bean Lamb’s ear pGAPMW

I N I N I N I N

1 2 3 4 5 6 7 8 9

500 bp-

1000 bp-

1500 bp-2000 bp-

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• Choose best PCR products for ligation

• Transformation and selection

• Plasmid Miniprep preparation

Microbial CulturingAntibiotic SelectionSterile Technique Cloning

Direct PCR cloningTransformationLigation

PCR PurificationSize Exclusion Chromatography

Plasmid MiniprepRestriction Enzyme DigestionGel Electrophoresis

Ligation, Transformation and Plasmid Minipreps

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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)

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Picking colonies for plasmid minipreps

Each colony is a clonal growth (clones)from one transformed bacteria

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Plasmid minipreps

• Isolate plasmid DNA (40 minutes)

• Restriction digest (1 hour)

• Electrophorese to confirm inserts (20 minutes)

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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

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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

1 2 3 4 5 6 7 8 9

Digested and undigested DNA were electrophoresed on a 1% TAE agarose gel

pJet vector

GAPDH inserts

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Setting up Sequencing Reactions

• Add sequencing primers to DNA (10 minutes)

• Load 96-well plate

• Send sealed plate off to sequencing facility for sequencing

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Setting up Sequencing Reactions

Always need to sequence reverse, complementary strand

GAPDH gene of interest

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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.

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• 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

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Bioinformatics

• Two month subscription to genetic analysis software from Geospiza

• Data is stored on iFinch server

• Data can be accessed 24/7

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iFinch Landing Page

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What does iFinch do?

• Upload and store DNA sequence data

• Examine the quality of the sequences

• Screens for GAPDH & vector sequences

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Folder for each student group

iFinch Data Folders

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http://www.geospiza.com/finchtv.html

FinchTV

Free application for viewing and editing chromatograms

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FinchTV

Sequence Chromatograms

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DataAnalysis

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

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• Students compare 3 sequences

• What is the accurate sequence?

• Usually requires going back to chromatograms

Assembling the

full-length contig

Contiguous sequence

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Example of an alignment

Compare between groups before publishing data

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Searches a DNA/protein database for published sequences that are similar to your sequence

Basic Local Alignment Search Tool,

or BLAST

BLAST Searches

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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

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Student Authors

Great for a resume!

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Try iFinch

http://www.geospiza.com/ifinchBioRad.html

http://classroom1.bio-rad.ifinch.com/Finch/

Username: BR_guest

Password: guest

Tutorial movies available

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Watch the Webinar Playback

Planning Guide available for download

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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.comSupportWebinars