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Cloning and Sequencing Explorer Series
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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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Day 1
Day 2
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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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Optional: Dependent on time constraints, analyze samples prior to the next steps. This may include agarose gel electrophoresis, fluorometry, or spectrometry.
Optional: If time permits proceed directly to Step 1–2 and set up PCR reactions using freshly extracted genomic DNA.
Chapter 1
1: Nucleic Acid Extraction
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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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Benefits of using plants
• Large number of species
• Lots of diversity
• Phylogenetic approaches
• Avoid ethical concerns associated with animals
• No pre-approval
Nucleic Acid ExtractionQuick Guide
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Chapter 2
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2: GAPDH PCR
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Optional: Although this protocol recommends analyzing the PCR products after both rounds of PCR have been completed, PCR results can be assessed using electrophoresis directly after this reaction is complete. Positive controls should yield visible bands. It is possible that some plant genomic DNA will not yield a visible band during the initial round of PCR and yet still be amplified after the second round of nested PCR. Note: If this is done — DO NOT add loading dye directly to the PCR reactions as loading dye may interfere with the subsequent round of PCR.
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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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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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Initial PCR may result in some amplicons that are non-specific
Because of the degenerate primer used to amplify the GAPDH of various plant species the initial PCR 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
PCR Animation
http://www.bio-rad.com/flash/07-0335/07-0335_PCR.html
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DNA sequence varies between species
Primers need to be designed to account for species variation
• What if you don’t know the exact DNA target sequence?
• How do you design primers?
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Variation in the Genetic Code
Introns are less conserved since they donot code for protein
Conservative substitution-does not change protein properties
The GAPDH enzyme (protein) is highly conserved but there are variations at the DNA level
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Degenerate primers are used to account for sequence variation
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Primers are designed using the consensus sequence Tobacco DQ682459
GATTTCGTTGTGGAATCCACTGGCarrot AY491512 GAGTACATTGTGGAGTCCACTGG
Blue gem X78307 GAGTACGTCGTTGAGTCGACTGG
Tomato AB110609 GACTTCGTTGTTGAATCAACCGG
Snapdragon X59517 GAGTATATTGTGGAGTCCACTGG
Consensus sequence GABTATGTTGTTGARTCTTCWGG
Primer set: GA(GTC)TATGTTGTTGA(GA)TCTTC(AT)GGYield: 12 primers
Reverse primers are designed in the same fashion
Plant Accession SequenceGAPDH Number
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Degenerate primers have optional bases in specified positions
To increase the probability that the primer will anneal to the target DNA, variable bases are designed into the primer.
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Multiple oligos comprise the forward primer
GAGTATGTTGTTGA(GA)TCTTC(AT)GGGATTATGTTGTTGA(GA)TCTTC(AT)GGGACTATGTTGTTGA(GA)TCTTC(AT)GG
GA(GTC)TATGTTGTTGAGTCTTC(AT)GGGA(GTC)TATGTTGTTGAATCTTC(AT)GG
GA(GTC)TATGTTGTTGA(GA)TCTTCAGGGA(GTC)TATGTTGTTGA(GA)TCTTCTGG
Position 3 has 3 bases
Position 15 has 2 bases
Position 21 has 2 bases
3 x 2 x 2 = 12
different oligonucleotides comprising the forward primer
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DNA Isolation and Amplification
To identify differences in GAPDH code we must isolate plant DNA and amplify the gene of interest using PCR first with degenerate primers (primers that account for variation in the DNA code)
A second PCR reaction (Nested PCR) is necessary to amplify the region which contains one of the GAPDH gene sequences (second set of primers are nested inside the initial PCR product sequence)
Biotechnology Explorer PCR primers are color-coded
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Setting up initial PCR Reactions
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Setting up Initial PCR Reactions Protocol
1. Add 20 µl of blue mastermix with initial primers to each PCR tube
2. Add 15 µl of sterile water to each tube
3. Add 5 µl of DNA template to the appropriate tube
• Control Arabidopsis gDNA
• Control plasmid DNA
• Test gDNAs
• Negative control
4. Amplify in thermal cycler (Annealing temp 52oC)
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Results of Initial PCR Reactions
1% agarose gel loaded with (20 µl) initial PCR samples.
Green bean and Lamb’s ear gDNA samples generated using Nucleic Acid Extraction module. Lane 1- 500 bp molecular weight ruler (10 µl),
Lane 2- PCR of control Arabidopsis gDNA with initial primers (20 µl)
Lane 3- PCR of green bean gDNA with initial primers (20 µl)
Lane 4- PCR of lamb’s ear gDNA with initial primers (20 µl)
Lane 5- PCR of pGAP plasmid control with initial primers (20 µl)
Sometimes no amplification is observed with initial PCR with some plants, or much fainter than this gel
500 bp-
1000 bp-1500 bp-2000 bp-
1 2 3 4 5
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• Use of degenerate primers may not give you an exact match to the target sequence
• Because there are multiple primers in the mix, the primer concentration for the matching primer is lower than normal (1/12th
concentration)
• Problems with initial PCR:– inefficient– non-specific
• Benefits of initial PCR:– cast a wide net– increase the pool of specific products
Why is a Nested PCR reaction necessary?
INITIAL GACTATGTTGTTGAGTCTTCTGGFORWARD PRIMER
Arabidopsis GACTACGTTGTTGAGTCTACTGGGAPC1AT3G04120
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Exonuclease 1 treatment is needed before the second round of PCR (nested PCR) is done
• The primers that were not incorporated into PCR product in the first reaction must be removed so that they do not amplify target DNA in the second round of PCR.
• Exonuclease I will be added to the PCR products
• The enzyme must be inactivated before proceeding to the nested PCR
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Exo treatment
Protocol
1. Add 1 µl of exonuclease 1 enzyme to the PCR reactions
2. Incubate 15 min 37°C
3. Incubate 15 min 80°C
4. Dilute 2 µl Exo-treated PCR product in 98 µl water
Note: Thermal cycler can be programmed for exo incubations
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Nested PCR amplifies only regions within the GAPDH gene
Nested PCR is more specific
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Setting up Nested PCR Reactions Protocol
1. Mix 20 µl of diluted exo-treated template DNA with 20 µl of yellow mastermix with nested primers
2. For controls, mix 20 µl of control pGAP plasmid and 20 µl of water with 20 µl of yellow mastermix with nested primers
3. Amplify in thermal cycler (Annealing temp 46oC)
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GAPC
GAP-C2
GAPCP-1
GAPCP-2
Nested GAPDH PCR product
PCR Products
Initial vs. Nested Reactions
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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
Chapter 3
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3: Electrophoresis
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Electrophoresis Quick Guide
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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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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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Day 1
Day 2
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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
PCR Purification,Ligation, Transformation and Plasmid Miniprep Purification
Chapter 4
4: PCR Purification
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Additional tasks to perform prior to next stage:
Starter cultures must be inoculated one day prior to the transformation with a starter colony from the HB101 LB agar starter plate. Incubate cultures with shaking overnight at 37oC.
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Optional: Electrophorese 5 µl of the purified sample along with 5 µl of the unpurified sample on an agarose gel.
Column Purification of PCR products
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SEC is used to purify large PCR products from the smaller primers, dNTPs and enzymes.
PCR Purification Quick Guide
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Ligation and Transformation
• Blunt-end PCR product & ligate to pJet vector
• Preparation of competent bacteria cells • Efficient heat-shock transformation
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Chapter 5
5: Ligation
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Ligation
Sticky End Ligation:If the insert has sticky ends then the vector should be cut with the same enzyme to produce complementary ends
Allows for directional cloning (cDNA)
Blunt End Ligation:All DNA ends are compatible, not necessary to cut vector and insert with the same restriction enzymes
Treating the PCR product with a proofreading DNA polymerase removes 3’-A added by Taq DNA polymerase in PCR, leaving blunt ends ready for ligation.
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pJet1.2 Blunted Vector
2974 bp in length
Designed for blunt-end cloning MCS (Multiple Cloning Site) has restriction enzyme sites may be used for later manipulation
High copy number
-lactamase gene for Amp resistance
Contains the eco47IR gene
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Positive Selection
The eco471R gene codes for the Eco47I restriction enzyme which is toxic to E.coli.
When the eco471R gene is disrupted by the insertion of DNA into the cloning site, the gene will no longer be expressed and the transformed cells will grow on selective media.
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Possible LigationProducts
Expected Results:
Self-ligation of vector
Ligation of vector with multiple inserts
Ligation of vector with primer-dimers or short DNA fragments
Self-ligation of insert
Ligation of one insert into vector
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Ligation Quick Guide
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Chapter 6
6: Transformation
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Transformation
Competent cells
pGAP plasmid control
IPTG
Heat ShockCompetent Cells are very delicate: keep on ice
Bacterial transformation with ligation mixtures is very inefficient (less efficient than transformation with plasmid DNA). Transform with pGAP plasmid as control
Isopropyl -D-1-thiogalactopyranoside (IPTG) is added to the selective medium to artificially increase the expression of the ampr gene and increase transformation efficiency
Heat Shock: plating bacteria from ice to agar plates at 370C
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Transformation Quick Guide
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Chapter 77: Plasmid Purification
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Plasmid Minipreps
• Isolate plasmid DNA
• Restriction digest
• Electrophorese to confirm inserts
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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 Purification and Restriction Digest AnalysisQuick Guide
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Optional: It is recommended that 5 µl of undigested DNA also be run next to your digested samples. Prepare these samples by combining 5 µl of miniprep DNA with 5 µl of sterile water.
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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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Chapter 8
8: Sequencing
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Sequencing• Primers, control plasmid and barcoded 96-well
plate are included. Actual sequencing service is not included with the purchase of the series.
• Bio-Rad Laboratories has been working with Eurofins MWG/Operon which is a company that offers sequencing services around the world. We have been able to partner with Eurofins MWG/Operon so that they can offer a highly discounted rate. The benefit of being able to utilize the discounted services of Eurofins MWG/Operon is that their turnaround time is 1-2 days and as an ISO9001 company they adhere to specific quality standards. To contact Eurofins MWG/Operon please visit www.operon.com/bio-rad or call (800)688-2248.
• Alternate options for sequencing would include local universities and other professional sequencing services.
Sequencing Methods • Maxam-Gilbert: Chemical Degradation
– 5’-end of DNA labeled with a radioactive tag– Labled DNA is divided into 4 test tubes with
chemicals which cleave after a particular base– PAGE to seperate cleaved fragments– X-ray film (autoradiography) – Derive DNA sequence from X-ray film image– Limited to about 100 bases
• Sanger: Chain Termination– Single-stranded template of DNA– Template DNA is divided into 4 test tubes with
• Sequencing primers• DNA polymerase• Labled nucleotides (dNTP’s)• Modified nucleotide, different dideoxynucleotide (ddNTP)
to each reaction tube– Allow for DNA synthesis, when ddNTP is incorporated
polymerisation will stop– PAGE and autoradiography can be used to separate
and visualize fragment sizes and read sequence
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Setting up Sequencing Reactions
• Add sequencing primers to DNA • Load 96-well plate
• Send sealed plate off to chosen 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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• Automated Sanger method of sequencing
• 4 fluorescent dyes- 1 for each base
• DNA fragments separated by CE
• Fragments separated in sequential order
Sequencing
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Chapter 9
9: Bioinformatics
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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