Cloning and Sequencing Explorer Series

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Cloning and Sequencing Explorer Series
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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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Chapter Task
1 Nucleic Acid Extraction Extract DNA
2 GAPDH PCR Initial PCR, Exo, Nested PCR
3 Electrophoresis Electrophorese PCR products
4 PCR Purification Purify PCR Products
5 Ligation Ligate PCR Product
6 Transformation 7 Plasmid Purification Transformation Mini-prep and restriction digest confirmation
8 Sequencing 9 Bioinformatics Prep and send sequencing reactions Analyze Sample Sequence
Day 1Day 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

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Chapter 1
1 Nucleic Acid Extraction
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 12 and set up PCR
reactions using freshly extracted genomic DNA.
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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

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Nucleic Acid ExtractionQuick Guide
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Chapter 2
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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Gene 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 dont know the exact DNA
• target sequence?
• How do you design primers?

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Variation in the Genetic Code
The GAPDH enzyme (protein) is highly conserved
but there are variations at the DNA level

• Introns are less conserved since they do
• not code for protein

Conservative substitution-does not change protein
properties
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Degenerate primers are used to account for
sequence variation
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Primers are designed using the consensus sequence
Plant Accession
Sequence GAPDH Number
Tobacco DQ682459 GATTTCGTTGTGGAATCCACTGG Car
rot AY491512 GAGTACATTGTGGAGTCCACTGG Blue
gem X78307 GAGTACGTCGTTGAGTCGACTGG Tomato
AB110609 GACTTCGTTGTTGAATCAACCGG Snapdragon
X59517 GAGTATATTGTGGAGTCCACTGG


Consensus sequence
GABTATGTTGTTGARTCTTCWGG
Primer set GA(GTC)TATGTTGTTGA(GA)TCTTC(AT)GG Yi
eld 12 primers Reverse primers are designed in
the same fashion
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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)GG
• GATTATGTTGTTGA(GA)TCTTC(AT)GG
• GACTATGTTGTTGA(GA)TCTTC(AT)GG
• GA(GTC)TATGTTGTTGAGTCTTC(AT)GG
• GA(GTC)TATGTTGTTGAATCTTC(AT)GG
• GA(GTC)TATGTTGTTGA(GA)TCTTCAGG
• GA(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
• Add 20 µl of blue mastermix with initial primers
  to each PCR tube
• Add 15 µl of sterile water to each tube
• Add 5 µl of DNA template to the appropriate tube
• Control Arabidopsis gDNA
• Control plasmid DNA
• Test gDNAs
• Negative control
• 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 Lambs ear gDNA samples
generated using Nucleic Acid Extraction module.
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2000 bp-
1500 bp-
1000 bp-
500 bp-
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
lambs 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
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Why is a Nested PCR reaction necessary?

• 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

INITIAL GACTATGTTGTTGAGTCTTCTGG FORWARD
PRIMER Arabidopsis GACTACGTTGTTGAGTCTACTGG
GAPC1AT3G04120
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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
• Add 1 µl of exonuclease 1 enzyme to the PCR
  reactions
• Incubate 15 min 37C
• Incubate 15 min 80C
• 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
• Mix 20 µl of diluted exo-treated template DNA
  with 20 µl of yellow mastermix with nested
  primers
• For controls, mix 20 µl of control pGAP plasmid
  and 20 µl of water with 20 µl of yellow mastermix
  with nested primers
• Amplify in thermal cycler (Annealing temp 46oC)

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PCR Products Initial vs. Nested Reactions
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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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Chapter 3
3 Electrophoresis
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Electrophoresis Quick Guide
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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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Laboratory Overview
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Chapter Task
1 Nucleic Acid Extraction Extract DNA
2 GAPDH PCR Initial PCR, Exo, Nested PCR
3 Electrophoresis Electrophorese PCR products
4 PCR Purification Purify PCR Products
5 Ligation Ligate PCR Product
6 Transformation 7 Plasmid Purification Transformation Mini-prep and restriction digest confirmation
8 Sequencing 9 Bioinformatics Prep and send sequencing reactions Analyze Sample Sequence
Day 1Day 2
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PCR Purification,Ligation, Transformation and
Plasmid Miniprep Purification

• Choose best PCR products for ligation
• Transformation and selection
• Plasmid Miniprep preparation

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Chapter 44 PCR Purification
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.
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Column Purification of PCR products
SEC is used to purify large PCR products from the
smaller primers, dNTPs and enzymes.
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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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LigationSticky End LigationIf the insert has
sticky ends then the vector should be cut with
the same enzyme to produce complementary
endsAllows for directional cloning
(cDNA)Blunt End LigationAll 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
lengthDesigned for blunt-end cloning MCS
(Multiple Cloning Site) has restriction enzyme
sites may be used for later manipulationHigh
copy numberb-lactamase gene for Amp
resistanceContains the eco47IR gene
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Positive SelectionThe 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 LigationProductsExpected
ResultsSelf-ligation of vectorLigation of
vector with multiple insertsLigation of vector
with primer-dimers or short DNA
fragmentsSelf-ligation of insertLigation of
one insert into vector
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Ligation Quick Guide
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Chapter 66 Transformation
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TransformationCompetent cellspGAP plasmid
control IPTGHeat Shock
Competent 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
b-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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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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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 [phone].
• Alternate options for sequencing would include
  local universities and other professional
  sequencing services.

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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 (dNTPs)
• 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
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

• Automated Sanger method of sequencing
• 4 fluorescent dyes- 1 for each base
• DNA fragments separated by CE
• Fragments separated in sequential order

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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