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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@bio-rad.com
Leigh Brown, M.A.
leigh_brown@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 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
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
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Cloning
Direct PCR cloning
Transformation
Ligation
Student use the
following
techniques
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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
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
Genomic DNA Extraction
DNA Precipitation
DNA Quantitation
GAPDH
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
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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
gene
duplication
Endosymbiotic
event
GAPA
gene
duplication
GAPC/CP
GAPA/B
Host Cell
GAPC
Most algae
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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
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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)
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• 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
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•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
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Using Nested
PCR to
increase your
final PCR
product
Initial PCR
DNA template:
Genomic DNA
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
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• Results: intense, bold band on agarose gel
PCR results
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MW
2
Arabidopsis
I
1% agarose gel loaded
with 20 µl initial PCR
samples and 5 µl
nested PCR samples.
2000 bp1500 bp1000 bp500 bp-
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3
N
4
5
Green bean
I
N
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7
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Lamb’s ear
I
N
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pGAP
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 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
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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)
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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
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Example of a
miniprep
digestion with
Bgl II
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2
3
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5
6
7
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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
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• 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
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Setting up
Sequencing
Reactions
GAPDH gene of interest
Always need to
sequence
reverse,
complementary
strand
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pJet 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.
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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
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• Screens for GAPDH & vector sequences
iFinch Data
Folders
Folder for each
student group
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# sequences per folder (4)
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
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• Run the CAP3 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
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Contiguous sequence
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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Try iFinch
http://classroom1.bio-rad.ifinch.com/Finch/
Username: BR_guest
Password: guest
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Tutorial movies available
http://www.geospiza.com/ifinchBioRad.html
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
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