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 fluorescently-tagged 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.
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 triple-template 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.
Procedure I: Isolating DNA From Arabidopsis thaliana
Edward's Extraction Buffer, 400 ml / 1.5 ml test tube, polypropylene / Microcentrifuge
Isopropanol, 400 ml / 100-1000 µl micropipet and tips
Tris/EDTA (TE) Buffer / 10 - 100 ml micropipet and tips
Disposable pellet pestle
Pre-lab Preparation
q 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 ml 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 ml of supernatant to a fresh tube.
7. Add 350 ml 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 ml of TE Buffer.
10. Template DNA can be used immediately or stored at -20°C.
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.
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 mix10X 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).
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.
Denaturing 94oC 3 min
35 Cycles of Denaturing 94oC 30 sec
Annealing 62oC 15 sec
Extending 68oC 6 min
Final extending 68oC 10 min
Holding 4oC 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 / Size of fragment (in base pairs)Arabidopsis gene AT2g22170 P1 and P2 / 2100
Arabidopsis gene AT2g22170 P3 and P4 / 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. To a fresh tube, add 50 ul of distilled water.
2. Add 25 ul cresol red loading dye.
3. Add 25 ul of 1 kb DNA ladder stock.
4. 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 / Sample loaded / Amount1 / 1 kb ladder / 5 ul
2 / Product P1/P2 + cresol red loading dye / 20 ul
3 / Product P3/P4 + cresol red loading dye / 20 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.
6. Visualize DNA.
7. 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.
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 / For each reaction / Master mix10X PFU Buffer / 1.0 ul / 11.0 ul
PFU DNA Polymerase / 0.5 ul / 5.5 ul
10X dNTPs / 1.0 ul / 11.0 ul
Purified P1/P2 / 2.0 ul
Purified P3/P4 / 2.0 ul
Purified YFP product* / 1.0 ul / 11.0 ul
Distilled H20 / 2.5 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.
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.
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 / For each reaction / Master mix10X ExTaq Buffer / 2.0 ul / 22.0 ul
ExTaq DNA Polymerase / 0.5 ul / 5.5 ul
10X dNTPs / 2.5 ul / 27.5 ul
‘Polished PCR’ products / 5.0 ul
Primer P1 / 1.0 ul / 11.0 ul
Primer P4 / 1.0 ul / 11.0 ul
Distilled H20 / 8.0 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.
Denaturing 94oC 3 min
30 Cycles of Denaturing 94oC 30 sec
Annealing 62oC 15 sec
Extending 68oC 8 min
Final extending 68oC 10 min
Holding 4oC 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.