Arabidopsis Functional Genomics: Gene Tagging by Long Flanking Homology PCR
Arabidopsis is the most important model system for identifying plant genes and
characterizing their function. Although the entire Arabidopsis genome has been
sequenced, about 30% of all predicted gene products have not been assigned to any
functional category. Moreover, for a large proportion of the proteins that have been
annotated, the intracellular location and/or expression pattern information remain
unknown or unverified experimentally. Thus, a large gap remains in our understanding
of the function of a very significant portion of Arabidopsis gene products.
A first step towards determining the function of proteins is to learn their cellular
localization. Cold Spring Harbor Laboratory (CSHL) scientists have begun to
systematically analyze 4,000 Arabidopsis genes of unknown function by fluorescent
tagging of full-length protein products (FTFLP).
In this laboratory exercise, students will fluorescently tag Arabidopsis genes of unknown
function using a long flanking homology PCR technique. The tagged genes are then sent
to the laboratory of CSHL scientist, David Jackson, where they are transformed into
Arabidopsis plants. Images of the expressed proteins then are posted on our Plant
Genetics and Genomics website.
Generally, any specific subcellular localization pattern for a particular fluorescentlytagged protein will be interesting and will help shed light on a protein’s function. The
following examples illustrate interesting and biologically significant localization patterns
that may be discovered in these experiments.
Plasma membrane-specific targeting in the root epidermis and root hairs is important
because these parts of the plant constitute the first line of communication with the
rhizosphere (the zone surrounding the roots of plants in which complex relations exist
among the plant, the soil microorganisms and the soil itself), and very little is known
about particular proteins that may be deployed there. This localization pattern may have
special significance for our understanding of plant interactions with fungal and bacterial
pathogens.
For intercellular transport and communication the role of plasmodesmata is undisputed.
However, little is known about the identity of the proteins that comprise these
fascinating channels, which connect cells and regulate movement of molecules as large
as viral genomes. Identification of proteins that localize to plasmodesmata will greatly
help to understand function and composition of these channels.
Discovery of YFP-tagged proteins that target to the nuclear envelope (NE), nuclear
pores, or specific subnuclear sites will also have important implication on our knowledge
of cellular functions. Although many of the nuclear proteins with housekeeping
functions are conserved between animals and plants (e.g., histones and enzymes in DNA
and RNA metabolism), no homologs of the known animal NE proteins have been found
in the Arabidopsis genome. Identification of plant NE-associated proteins should reveal
novel structural components of this important compartment of the plant cell.
1
Gene Tagging by Long Flanking Homology (LFH) PCR.
To visualize localization and pattern of expression within individual cells and plant
tissues, a protein typically is tagged with a readily detected reporter. Due to their unique
autofluorescence and high quantum yields, GFP and its spectral variants, such as yellow
fluorescent protein (YFP), provide a sensitive and convenient tool to track biological
molecules in real time in animal and plant systems1.
The FTFLP strategy inserts a YFP tag into the final exon of genes of interest via a tripletemplate PCR technique. This brings the reporter under control of the gene’s promoter,
expressing fluoresence in a tissue- and developmentally specific manner. In the first step,
the gene of interest is amplified as two separate pieces, using two sets of primers (P1-4 in
figure below). Primers P2 and P3 incorporate sequences compementary to YFP. In the
second step, template YFP is added to the left and right (flanking) gene templates from
the first reaction, hence “triple templates.” Regions of overlap between the long flanking
homologies (LFH) and YFP create primers for PCR in both directions2. This method
illustrates how PCR has been adapted to create a fusion gene.
Insertion of YFP gene into Arabidopsis gene of unknown function.
Step 1. First PCR reaction using primers pairs P1/P2, and P3/P4
P2
P1
exon 1
exon 2
exon 3
P3
P4
exon 4
Firs t PCR products
Step 2. PCR products from Step 1 and P1 and P4 act as primers for second PCR reaction
P1
YFP
Final PCR product: Arabidopsis gene with YFP gene ins erted
P4
YFP
Indicates sequence complementary to YFP gene
2
Procedure I: Isolating DNA From Arabidopsis thaliana
Reagents
Equipment & Supplies
Edward's Extraction Buffer, 400 1.5 ml test tube, polypropylene
l
100-1000 µl micropipet and tips
Isopropanol, 400 l
Tris/EDTA (TE) Buffer
10 - 100 l micropipet and tips
Disposable pellet pestle
Shared Items
Microcentrifuge
Pre-lab Preparation

Plant Arabidopsis seeds and allow for 3-4 week growth period. For information
concerning growing Arabidopsis, refer to The Arabidopsis Information Resource
(TAIR) at WWW.arabidopsis.org.
Procedure
1. Grind tissue in a microfuge with plastic pestle for 1 minute. Note: Whole plants,
single rosette leaves, single cauline leaves, whole and partial inflorescences have all
worked. However, best results are obtained using 1-2 whole leaves.
2. Add 400 l of Edward's Extraction Buffer.
3. Grind briefly (to remove tissue from pestle).
4. Vortex 5 seconds; leave at room temperature for 5 minutes.
5. Microfuge for 2 minutes.
6. Transfer 350 l of supernatant to a fresh tube.
7. Add 350 l of isopropanol, mix, leave at room temperature for 3 minutes.
8. Microfuge for 5 minutes, decant, air dry pellet for 10-15 minutes.
9. Resuspend DNA pellet in 100 l of TE Buffer.
10. Template DNA can be used immediately or stored at -20C.
3
Procedure II: Amplifying Arabidopsis Genes by PCR: First Round PCR
The amplification reactions will be performed in 2 consecutive stages. The first round of
PCR, comprised of two PCR reactions, will amplify the selected gene in two fragments.
In most genes, the YFP tag will be inserted 30 base pairs upstream of the STOP codon.
This location will minimize disturbance of the contiguous plant protein sequence, and it
will avoid N-terminal fusions that are known to destroy native targeting patterns.
Importantly, avoiding fusion directly to the C-terminus will ensure the activity of
membrane anchoring signals (i.e., farnesylation and myristylation sequences), typically
found within 10 C-terminal amino acid residues.
The first round of PCR requires two pairs of gene-specific primers. The first pair (P1
and P2) will amplify the 5’ end of the gene; the second pair (P3 and P4) will amplify the
3’ end of the gene. Primers P2 and P3 each have a 26 nucleotide extension that is
complementary to the 5’ and 3’ YFP linker sequences, respectively.
Step 1. First PCR reaction using primers pairs P1/P2, and P3/P4
P2
P1
exon 1
exon 2
exon 3
P3
P4
exon 4
Firs t PCR products
ExTaq DNA Polymerase
Best results are obtained using Ex Taq DNA polymerase from PanVera (catalog number
RR001A). This enzyme combines the robust activity of Taq polymerase with an
efficient 3’to 5’ exonuclease activity for increased fidelity. The polymerase is supplied
with 10X buffer and a 10X dNTP mix (2.5 mM each).
Procedure – Tagging Arabidopsis gene AT2g22170
The following is the reaction mix for a single 20 ul reaction. To minimize the number of
pipetting steps, a “master mix” comprising all reagents except primers and DNA should
be made. A master mix for 11 reactions is listed below. This will provide for 10 PCR
reactions.
Reagent
For each reaction
Master mix
10X ExTaq Buffer
2.0 ul
22.0 ul
ExTaq DNA Polymerase
0.5 ul
5.5 ul
10X dNTPs
2.0 ul
22.0 ul
Primer 1* (10 uM stock)
2.0 ul
Primer 2* (10 uM stock)
2.0 ul
Arabidopsis genomic DNA
1.0 ul
Distilled H20
10.5 ul
115.5 ul
*The primer used will differ depending on the reaction (P1 and P2 or P3 and P4).
4
1. Vortex the “master mix” and aliquot 15 ul per 0.2 ul PCR tube.
2. For each gene, 2 PCR reactions are necessary. One tube will be labeled
‘P1/P2,’and the other ‘P3/P4’.
3. Use a micropipet with a fresh tip to add 4 ul of primer mix P1/P2 to the
appropriate reaction tube.
4. Use a micropipet with a fresh tip to add 4 ul of primer mix P3/P4 to the
appropriate reaction tube.
5. Use a micropipet with a fresh tip to add 1 ul of Arabidopsis genomic DNA to
each tube. Change tips between tubes.
6. Program the thermal cycler for 35 cycles according to the following cycle profile.
94oC
Denaturing
3 min
94oC 30 sec
62oC 15 sec
68oC 6 min
35 Cycles of Denaturing
Annealing
Extending
68oC
4oC
Final extending
Holding
5
10 min
indefinitely
Procedure III: Analysis and Purification of Amplified DNA
Agarose gel electrophoresis should be carried out to determine that PCR products of the
appropriate lengths have been generated. Sizes of PCR products are listed below.
Primer pairs
Arabidopsis gene AT2g22170 P1 and P2
Arabidopsis gene AT2g22170 P3 and P4
Size of fragment (in base pairs)
2100
1100
Run a 1.0 % agarose gel for separation of PCR products. A DNA size marker should also
be loaded. A 1 kb ladder from New England BioLabs (N3232S) will be used for this
course. The ladder is supplied at a concentration of 0.5 ug/ul and should be kept frozen.
A small amount of the stock can be diluted as follows:
1.
2.
3.
4.
To a fresh tube, add 50 ul of distilled water.
Add 25 ul cresol red loading dye.
Add 25 ul of 1 kb DNA ladder stock.
Vortex.
Procedure
1. Obtain one tube containing the 1 kb DNA ladder and another tube containing
cresol red loading dye.
2. Transfer 5 ul of cresol red loading dye to tubes P1/P2 and P3/P4.
3. Load the gel as follows:
Lane
1
2
3
Sample loaded
1 kb ladder
Product P1/P2 + cresol red loading dye
Product P3/P4 + cresol red loading dye
Amount
5 ul
20 ul
20 ul
4.
5.
6.
7.
Connect gel to a power supply set to 110V and run for 40 minutes.
The gel may be stained with either ethidium bromide or methylene blue.
Visualize DNA.
Use a disposable scalpel to excise the appropriately-sized bands. Minimize the
size of the gel slice by removing extra agarose.
8. Extract DNA using QIAquick Gel Extraction Kit (Qiagen, catalog # 28704).
9. Elute DNA in 30 ul elution buffer.
10. Run gel to determine success of DNA extraction.
M
P1/ P3/ YFP
P2 P4
Figure 1. 1% agarose gel of purified P1/P2, P3/P4 and YFP fragments.
6
Procedure IV: ‘Polishing’ ends of PCR products
Taq polymerase has a nontemplate-dependent activity that adds a single deoxyadenosine
(A) nucleotide to the 3' end of PCR products. This extra nucleotide would disrupt the
reading frame and could result in the production of a truncated protein. To remove this
extra nucleotide, it is necessary to incubate the PCR products with a DNA polymerase
that possesses exonuclease activity, and does not produce this overhang. Treated DNA
will have blunt ends.
PFU DNA Polymerase from Stratagene (catalog number 600153) possesses both DNA
polymerase and exonuclease activity and is supplied with a 10X buffer.
Procedure
The following is the reaction mix for a single 10 ul reaction. To minimize the number of
pipetting steps, a “master mix” comprising all reagents except PCR products can be
made immediately prior to setting up the reaction. A master mix for 11 reactions is
shown below. This will provide for 10 reactions.
Reagent
10X PFU Buffer
PFU DNA Polymerase
10X dNTPs
Purified P1/P2
Purified P3/P4
Purified YFP product*
Distilled H20
For each reaction
Master mix
1.0 ul
0.5 ul
1.0 ul
2.0 ul
2.0 ul
1.0 ul
2.5 ul
11.0 ul
5.5 ul
11.0 ul
11.0 ul
27.5 ul
*Purified YFP fragment will be supplied. The YFP gene serves as the third template in
Procedure V. Please see end of protocol for synthesis of YFP PCR product.
1. Vortex the “master mix” and aliquot 6 ul per 0.2 ul PCR tube
2. Label tube “P” for polishing step.
3. Use a micropipet with a fresh tip to add 2 ul of purified P1/P2 to tube “P”.
4. Use a micropipet with a fresh tip to add 2 ul purified P3/P4 to the same tube.
5. Program the thermal cycler to ramp to 72oC for 30 minutes and then to hold at
4oC indefinitely.
7
Procedure V: Triple Template PCR. Inserting YFP gene into Arabidopsis genes
The P1/P2 and P3/P4 products of the first PCR reaction serve as long primers in a Long
Flanking Homology PCR using the YFP fragment and each other as overlapping
templates (i.e. triple-template reaction). The resulting full-length product is further
amplified in the same reaction using P1 and P4 primers.
Step 2. PCR products from Step 1 and P1 and P4 act as primers for second PCR reaction
P1
YFP
Final PCR product: Arabidopsis gene with YFP gene inserted
P4
YFP
Indicates s equence complementary to YFP gene
Procedure
The following is the reaction mix for a single 20 ul reaction. To minimize the number of
pipetting steps, a “master mix” comprising all reagents except ‘polished’ PCR products
should be made immediately prior to use. A master mix for 11 reactions is listed below.
This will provide for 10 PCR reactions.
Reagent
10X ExTaq Buffer
ExTaq DNA Polymerase
10X dNTPs
‘Polished PCR’ products
Primer P1
Primer P4
Distilled H20
For each reaction
Master mix
2.0 ul
0.5 ul
2.5 ul
5.0 ul
1.0 ul
1.0 ul
8.0 ul
22.0 ul
5.5 ul
27.5 ul
11.0 ul
11.0 ul
88.0 ul
1. Vortex the “master mix” and aliquot 15 ul per 0.2 ul PCR tube.
2. Label the tube TT (for triple template).
3. Use a micropipet with a fresh tip to add 5 ul ‘polished’ PCR products to the TT
reaction tube.
4. Program the thermal cycler for 30 cycles according to the following cycle profile.
8
94oC
Denaturing
3 min
94oC 30 sec
62oC 15 sec
68oC 8 min
30 Cycles of Denaturing
Annealing
Extending
68oC
4oC
Final extending
Holding
10 min
indefinitely
Procedure V: Analyzing Amplified DNA by Gel Electrophoresis
Agarose gel electrophoresis should be carried out to determine that a PCR product of the
appropriate length has been generated. YFP tagged AT2g22170 should be 3.9 Kb in
length.
Run a 1.0 % agarose gel to analyze PCR products. A DNA size marker should also be
loaded. A 1 kb ladder from New England BioLabs (N3232S) will be used for this course.
Procedure
1. Obtain a tube containing the 1 kb DNA ladder and a tube containing 6 ul cresol
red loading dye.
2. Transfer 10 ul TT PCR product to the tube containing cresol red loading dye.
3. Load the gel as follows:
Lane
Sample loaded
Amount
1
1 kb ladder
5 ul
2
TT PCR product + cresol red loading dye 15 ul
4. Connect gel to a power supply set to 110V and run for 40 minutes.
5. The gel may be stained with either ethidium bromide or methylene blue.
Figure 2. YFP-tagged AT2g22170
9
References
1.
Hanson, M.R., and Kohler, R.H. (2001). GFP imaging: methodology and application to
investigate cellular compartmentation in plants. J. Exp. Bot. 52, 529-539.
2.
Wach, A. (1996). PCR-synthesis of marker cassettes with long flanking homology
regions for gene disruptions in S. cerevisiae. Yeast 12: 259-265.
Supplemental Information
Purified YFP PCR product is supplied. It was synthesized using the following
procedure.
YFP Left
GGC CGG CCT GGA GGT GGA GGT GGA GCT GTG AGC A
YFP Right
GGC CCC AGC GGC CGC AGC AGC ACC AGC AGG ATC
The following is the reaction mix for a single 50 ul reaction.
Reagent
10X ExTaq Buffer
ExTaq DNA Polymerase
10X dNTPs
YFP plasmid DNA (50 ng total)
Left Primer (5’) (10X stock)
Right Primer (3’) (10X stock)
Distilled H20
For each reaction
5.0 ul
0.5 ul
5.0 ul
0.5 ul
5.0 ul
5.0 ul
29.0 ul
Recommended Agarose Percentage for Size Separation
% Agarose
0.5
0.7
1.0
1.2
1.5
3-4 (sieving
agarose)
Size Fragments Separated
(bp)
1,000 to 30,000
800 to 12,000
500 to 10,000
400 to 7,000
200 to 3,000
10 to 1000
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