Cloning and Sequencing Explorer Series

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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

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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

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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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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
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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.com?Support?Webinars