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Cloning and Sequencing Explorer Series

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Title: PowerPoint Presentation Author: ken shiokari Last modified by: Administrator Created Date: 6/29/2004 5:54:37 PM Document presentation format – PowerPoint PPT presentation

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Title: Cloning and Sequencing Explorer Series


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(No Transcript)
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Cloning and Sequencing Explorer Series
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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_at_bio-rad.com
  • Leigh Brown, M.A.
  • leigh_brown_at_bio-rad.com

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

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

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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

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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

13
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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Gene 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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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

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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
Initial PCR
Nested PCR
DNA template Genomic DNA
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

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PCR results1 agarose gel loaded with 20 µl
initial PCR samples and 5 µl nested PCR samples.
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Arabidopsis
Green bean
Lambs ear
pGAP
MW
I
N
I
N
I
N
I
N
2000 bp-
1500 bp-
1000 bp-
500 bp-
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Ligation, Transformation and Plasmid Minipreps
  • Choose best PCR products for ligation
  • Transformation and selection
  • Plasmid Miniprep preparation

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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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Analysis of plasmid digests
Bgl II Digest
GAPDH inserts
2.5 kb gt 2.0 kb gt 1.5 kb gt 1.0 kb gt 0.5
kb gt
pJet vector
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Example of a miniprep digestion with Bgl II
pJet 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
    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 0.52.5 kb depending on
    plant species

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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
GAPDH gene of interest
Always need to sequence reverse, complementary
strand
pJet cloning vector
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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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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

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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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iFinch Data Folders
Folder for each student group
sequences per folder (4)
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FinchTV Free application for viewing and
editing chromatograms
http//www.geospiza.com/finchtv.html
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FinchTV Sequence Chromatograms
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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

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

Contiguous sequence
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Example of an alignment
Compare between groups before publishing data
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BLAST Searches
Basic Local Alignment Search Tool, or BLAST
Searches a DNA/protein database for published
sequences that are similar to your sequence
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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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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
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