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Digoxigenin is derived from a plant steroid hormone digoxin found in the plants Digitalis sp. Digoxigenin has been used successfully in labeling nucleic acids. In this experiment we optimized minimum probe requirement for a nonradioactive digoxigenin-based gene detection system in the model plant Arabidopsis thaliana. We showed that 1 μL of labeled probe was sufficient to hybridize onto 1–10 μg of target plasmid DNA. We also examined the sensitivity of labeled probe and showed that 2 μL of labeled probe was not able to hybridize with 1 μg of target DNA, although 2 μL of labeled probe was able to detect target DNA ranging from 2 to 10 μg. To test the efficacy of our optimization protocol, we used 1 μL of labeled plasmid DNA pU16893 harboring an Arabidopsis housekeeping gene elongation factor-1 and showed that the elongation factor-1 gene could be detected in Arabidopsis genome under various environmental conditions. This paper describes a nonradioactive in situ hybridization technique to detect nucleic acids in plants.
When comparing radioactive and nonradioactive in situ hybridization, nonradioactive methods have shown to have many advantages, including shorter development time, higher histological resolution, and higher detection sensitivity.1 Digoxigenin (DIG)-labeled probes are not as expensive as radioisotope-labeled probes, they last longer, and there is no environmental hazard caused by radiation.1 DIG-labeled probes and chemiluminescent substrates have proved to be a popular method for nucleic acid detection because of susceptibility, quicker results, and dependability.1 DIG polymerase chain reaction (PCR)-labeled probes were used to detect transgenes in barley, rice, and Nicotiana benthamiana.2 DIG-labeled probes have also been used successfully in detecting transgenes in other transgenic plants, such as rice, potato, sugarbeet, maize, and wheat.3 The genome of the primitive land plant Selaginella kraussiana was analyzed using a DIG-based Southern blot4 method to identify nine xyloglucan endotransglycosylase/hydrolase genes.5 DIG-based dot blot hybridization has been reported to provide a dependable, easy, and cost-efficient tool for plant molecular breeders, in which a large number of single nucleotide polymorphisms can be analyzed for breeding purposes.6 A DIG-based Southern blot4 was used to hybridize large Arabidopsis thaliana DNA fragments. This is a simple nonradioactive labeling technique that can be used in number of universal labeling practices.7 To avoid using radioactive gene labeling, nonradioactive-labeled probes can be used for single-copy sequence detection and decrease result time from days to a few hours.8 A nonradioactive DIG-based mapping protocol was also developed to screen bacterial artificial chromosome library of the plant Actinidia chinensis.9
This article describes a nonradioactive DIG-based gene detection protocol applicable in plant molecular biology and plant biotechnology. We optimized minimum amount of probe required for successful gene detection. We also tested various amounts of target plant genomic DNA for successful in situ hybridization. A DIG-labeled (DIG-dUTP) dot blot method was optimized for detection of a housekeeping gene known as elf-1 (elongation factor gene) found in Arabidopsis thaliana. The elf-1 gene is found in both plants10 and animals.11 It was chosen for our research because this gene was reported to be most stably expressed in plants compared with other housekeeping genes under various experimental conditions.10 This protocol will encourage plant molecular biologists and biotechnologists to use and optimize a safer alternative (compared with radioactivity based) approach for nucleic acid detection and hybridization in transgenic and nontransgenic plants.
For the entire experiment the following two kits were mainly used and protocols followed from the manufacturer’s instructions with modifications. The buffers and reagents were also diluted following the manufacturer’s instructions.
Two types of plasmid DNAs were used:
Test DNA pU16893 was labeled according to the DIG High Prime DNA Labeling and Detection Starter Kit I protocol. For complete DNA denaturation, 1 μg of pU16893 was added to 15 μL of sterile deionized water and denatured at 99°C for 10 min, then chilled on ice. The denatured pU16893 was labeled with the addition of 4 μL of DIG-High Prime and contents were briefly centrifuged. In order to increase DIG-labeled DNA yield, the sample was incubated overnight at 37°C in an MJ Mini Thermal Cycler (Bio-Rad, Hercules, CA). After overnight incubation, the reaction was stopped with the addition of 2 μL of 0.2M EDTA and heated for 10 min at 65°C.
In order to determine the efficiency of the labeled probe pU16893 (described above), a dot blot was performed. Labeled control plasmid DNA pBR328 (linearized with BamH1) was supplied with the DNA labeling kit. pBR328 (5 ng/μL) was diluted to 1 ng/μL. This dilution was used to make a series of dilutions which included 1000 pg/μL, 10 pg/μL, 1 pg/μL, 0.1 pg/μL, and 0.01 pg/μL. To test the labeling efficiency, the labeled probe (pU16893, test DNA) was diluted from 100 ng/μL (approximate theoretical yield by assumption) to 1000 pg/μL, 10 pg/μL, 1 pg/μL, 0.1 pg/μL, and 0.01 pg/μL. After serial dilutions were complete, the samples were spotted in 1-μL aliquots on a positively charged nylon membrane (Roche Diagnostics GmbH, Manheim, Germany, Cat. No. 1 417 240) in the order described above and shown in Figure 1: pBR328 (top row) and pU16893 (bottom row). The DIG-labeled control DNA (pBR328) and the labeled test DNA (pU16893) were fixed to the membrane with a CL-1000 Ultra Violet Cross Linker (UV strength 1200 × 100 μJ/cm2) (UVP, Upland, CA).
Washing the membrane required a number of incubation steps. At a temperature of 25°C, membrane washing was carried out in the following manner: the membrane was incubated for 2 min in a glass container with 20-mL of 1X maleic acid buffer, then transferred to 10 mL of 1X blocking solution and incubated for 30 min. Afterwards, the membrane was transferred to 10 mL of antibody solution and incubated for 30 min. The membrane was then washed twice for 15 min with 10 mL of 1X washing buffer solution and then incubated in 10 mL of 1X detection buffer for 5 min. All incubation steps were performed on a rotary shaker at 100 rpm in 25°C. In a lightproof container, 40 μL of immunodetection color substrate solution NBT/BCIP (nitroblue tetrazolium chloride, 5-bromo-4-chloro-3-indolyl-phosphate in 67% DMSO) was added to 2 mL of 1X detection buffer. The membrane was then added to this solution and allowed to set for 10 h in a stationary condition. Finally, the reaction was stopped by washing the membrane in sterile deionized water for 5 min, and intensities of spots were compared (Fig. 1).
Different aliquots of denatured test DNA pU16893 were spotted on a positively charged nylon membrane in the following order (Fig. 2): 10 μg, 8 μg, 6 μg, 4 μg, 2 μg, 1 μg and sterile deionized water (as negative control). Afterwards, the membrane was placed on a Whatman 3MM-paper (Whatman International Ltd., Kent, UK, Cat. No. 3030-861) soaked with 10X sodium citrate/sodium chloride (SSC), and the denatured DNA was fixed to the membrane by UV cross-linking as described above. The membrane was then washed with sterile deionized water and allowed to dry. Afterwards, the membrane was incubated in 10 mL of heated (37°C) DIG Easy Hyb solution in an incubator at 37°C for 30 min.
Two microliters of the denatured labeled probe pU16893 (following the protocols described above in “Labeling Test DNA pU16893 Harboring Elf-1 Gene with DIG” section) was added to a hybridization bottle containing preheated (42°C) hybridization solution along with the membrane containing target DNA pU16893 (described above) and then hybridized overnight in a hybridization chamber with constant rotation. A standard hybridization glass bottle was used for the hybridization experiment. After hybridization, the membrane was given two post-hybridization stringency washes as follows: 10 mL of 2X SSC, 0.1% SDS for 5 min at 25°C and prewarmed (65°C) 10 mL of 0.2X SSC, 0.1% SDS for 15 min. Then denatured target pU16893 (spotted on the membrane) was detected using the Roche kit procedure for immunological detection at 25°C under constant agitation. The nylon membrane was washed for 5 min in washing buffer. Then the membrane was incubated for 30 min in 100 mL of blocking solution, 30 min in 20 mL of antibody solution, washed 2 times at 15 min each in 100 mL of washing buffer and 5 min in 20 mL of detection buffer. The membrane was then placed in a lightproof container that contained 200 μL of NBT/BCIP immunodetection solution and 10 mL of detection buffer. The solution containing the membrane was placed in a dark area for 10 h in a stationary condition. After a 10-h reaction period, NBT/BCIP reaction was stopped with a 5-min sterile deionized water wash of the membrane (Fig. 2A).
The same procedure was used to test the 1 μL denatured labeled probe pU16893 (Fig. 2B).
Total Arabidopsis genomic plant DNA was extracted from plant leaves of whole plants that were in 25°C (normal greenhouse environment), incubated at 4°C overnight (cold stressed), and incubated at 40°C overnight (heat stressed). DNA was extracted using UltraClean Plant DNA Isolation Kit (Ca. No. G-3196–250, MoBio, Carlsbad, CA) following the manufacturer’s protocol. Plant genomic DNAs from these various conditions were denatured at 99°C for 10 min. One microgram of genomic Arabidopsis DNA from these three environmental conditions was spotted on a positively charged nylon membrane along with 1 μg of pU16893 as a positive control and water as a negative control (Fig. 3). Afterwards, the membrane was placed on a Whatman 3MM paper soaked with 10X SSC and DNA was fixed to the membrane by UV light crosslinking as described above. The membrane was then washed with sterile deionized water and allowed to air dry. Then the membrane was incubated in 10 mL of preheated (37°C) DIG Easy Hyb solution in an incubator at 37°C for 30 min. One microliter of labeled DNA probe (pU16893) was denatured at 99°C for 10 min and added to 3 mL of heated (42°C) DIG Easy Hyb solution in a hybridization bottle. Then the spotted plant DNA-bound membrane was added to the hybridization solution containing 1 μL of DNA probe (pU16893) and hybridized overnight at 42°C using a hybridization chamber, as previously described. After hybridization, the membrane was given two post-hybridization stringency washes and underwent immunological detection procedure, ending with NBT/BCIP incubation, as described in the previous experiments.
All blots were scanned and adjusted to grayscale for publication quality images.
The theoretical yield of a labeled probe is approximately 100 ng/μL using the Roche kit. With this assumption we diluted our test labeled pU16893 probe from 100 ng/μL into 1000 pg/μL, 10 pg/μL, 1 pg/μL, 0.1 pg/μL, and 0.01 pg/μL. The control labeled probe pBR328 (supplied with the Roche kit) was spotted with the same dilution series and compared side by side (Fig. 1). When spot intensities were compared, the labeled probe pU16893 matched very closely with control probe pBR328 (Fig. 1). From this observation we can conclude that our probe yield was approximately 100 ng/μL.
From our experiment it is evident that 2 μL of the labeled test probe pU16893 proved to be excessive to hybridize with 1 μg of the denatured DNA pU16893 (spot 6, Fig. 2A). Although, 1 μL of the labeled probe pU16893 proved to be a sufficient amount of probe to hybridize with 1 μg of denatured pU16893 (spot 6, Fig. 2B). At the time of gene detection by dot blot or Southern blot,4 it is important to use optimal amount of probe otherwise the blot may generate too much background or poor or no hybridization at all.
The purpose of this experiment was to detect a housekeeping gene in Arabidopsis under various environmental conditions using our optimized in situ hybridization technique. We showed that 1 μL of labeled pU16893 harboring elf-1 gene was enough to hybridize onto Arabidopsis genomic DNA. The housekeeping gene elf-1 was detected under various environmental conditions as expected (Fig. 3).
This experiment aimed to demonstrate the efficacy of a nonradioactive gene detection technique. We showed that 1 μL of labeled probe was enough to detect 1 μg of target DNA. It also became evident that the amount of labeled probe is critical for a successful hybridization. We showed that 2 μL of labeled probe was not able to detect 1 μg of target DNA. This gene detection technique was sensitive enough to detect a wide range of target DNA from 10 μg to 1 μg. Radioactive probes are hazardous and difficult to discard. Nonradioactive probes are sensitive and a safer alternative. This type of DIG-based detection process does not require the blots to be exposed to X-ray films at all as required by traditional Southern blot4 experiments. We hope this report will generate more interest among plant biologists and plant biotechnologists to use DIG-based in situ gene detection system.
The authors sincerely acknowledge the assistance from members of Dr. Basu’s laboratory including Sam Zwenger and Silvia Retana Ramirez. This project was partially funded by a Research Enrichment and Development Initiative fellowship, University of Northern Colorado, awarded to Dr. Chhandak Basu. This paper was written for academic and educational purposes. The authors declare no financial interests on any of these protocols. The kits, reagents, and DNA referred to in this paper are developed by respective entities and are/may be patented/trademarked.