Methods

The reagents used were of analytical grade purchased from Fluka and Sigma-Aldrich (Milwaukee, WI, USA). Modified oligonucleotides were purchased from Synthetic Genetics (San Diego, CA, USA). PCR primers were made in-house in a PE 8909 nucleic acid system. Experts in the field from different provinces of China collected and identified all TCM material used for this project.

1. Extraction of Genomic DNA

Plant DNA was extracted as described previously (19,20) with slight modifications. Briefly, about 0.2 g of fresh leaves from TCM was frozen in liquid nitrogen and ground into a fine powder using a mortar and pestle (Table 1). The powder was mixed with extraction buffer (25 mM Tris-HCl, pH 8.0, 50 mM EDTA, 0.5% SDS, 10 Ag/mL RNA A, 0.2% h-mercaptoethanol), and then incubated at 58°C for 1 hr, then centrifuged at 10,000 g for 10 min. The supernatant was recovered, mixed with equal volume of phenol:chloroform: isoamyl alcohol (1:24:1) by inversion, and centrifuged at 14,000 g for 10 min, 2 times. The supernatant was transferred to a new tube, mixed with chlo-roform:isoamyl alcohol, and centrifuged as above. The supernatant was recovered, mixed with 1/10 of 3M sodium acetate, pH 5.2 and 2.5 volumes of 95% ethanol, placed at -20°Cfor 1hr and centrifuged for 10minat 14,000 g. The DNA pellet was washed twice with 70% ethanol and resuspended in double-distilled autoclaved water.

2. Design and Fabrication of Probes

Species-Specific Oligonucleotide Probes. DNA-polymorphism-based analyses have been successfully used for the identification of herbal medicines (19,21,22). We chose the 5S-rRNA gene as our probe normally used as marker for phylogenetic analysis (23) because of its dual properties: it is widely conserved throughout prokaryotes and eukaryotes, but it is also highly variable at the internal transcribed spacer region within species (24). The 5S-rRNA gene from the TCM plants was amplified by PCR. The primers used for the amplification of the 5S- rRNA gene were: sense primer: 5' GGA TCC GTG CTT GGG CGA GAG TAG TA 3' and antisense: 5' GGA TCC TTA GTG CTG GTA TGA TCG CA 3' (19). PCR products were subcloned using the TA Cloning Kit (Invitrogen) and electroporated into Escherichia coli XL-1 blue. Plasmid DNA from positive clones was extracted by alkaline lysis, digested with restriction enzymes, analyzed by gel electrophoresis, and subjected to DNA sequencing using the AutoRead Sequencing Kit (Amersham Pharmacia Biotech). Oligonucleotide probes used for the microarrays described here were designed and fabricated based on the variable sequences obtained from the internal transcribed spacer (ITS) domain of the gene. The probes used for immobilization had a 5' disulfide modification, a carbon spacer (6-18 carbons), and 20-26 base sequences.

3. Development of Toxic TCM DNA Microarrays

Silicon surface modifications included a methodical cleaning procedure, a thermal oxidation process, and the self-assembly of thiol layers used as het-erofunctional linkers for immobilization of disulfide-modified probes. Initial cleaning, as well as thermal oxidation procedures, was performed in a class 1000 clean room using CMOS-compatible processing. Briefly, all n-type Si(100) wafers were immersed in a ''piranha'' bath (90% H2SO4, 10% H2O2; 125°C) for 10 min followed by 1-min treatment with 2% solution of hydrofluoric acid (HF). Then the wafers were rinsed 10 times with double-distilled water and blow-dried. A 1000-A layer of dry silicon dioxide was grown in an oxidation furnace at 1100 °C, followed by 4000 A of wet oxide (Fig. 1). The wafers were cut into 1-cm2 chips using a Disco automatic dicing saw DAD341.

Figure 1 Fabrication of TCM chip. Schematic diagram showing the steps followed during fabrication of the Si-based TCM chip.

4. Hydroxylation of the Surface and Formation of Self-Assembled MPTS Layer

Organic contaminants were removed by immersing the 1-cm2 chips in a potassium permanganate-sulfuric acid solution (0.5 g KMNO4 in 15 mL H2SO4) for 30 sec, then rinsed with copious amounts of water. The cleaned chips were incubated for 35 min in 3.3% of ammonium hydroxide solution at 60°C to restore the OH groups in the surface, rinsed with isopropanol, and dried under nitrogen gas. The chips were placed in a heat-resistant container with a solution consisting of 100 mL isopropanol p.a., 800 ||L (3-mercapto-propyl)trimethoxysilane (MPTS), and 200 ||L H2O, then placed in the oven at 80°C for 35 min. The chips were then rinsed first with isopropanol, then with water, and blow-dried with N2 gas. Finally, the chips were heated at 115 ° C for 30 min to remove H2O traces.

5. Printing the Arrays

Species-specific thiol-modified oligonucleotide probes were diluted with sodium chloride/sodium citrate buffer (SSC; 3 M NaCl, 0.3 M Na citrate-2 H2O, pH 4.5) and 5% DMSO to a final concentration of 20 ||M and spotted (100-|im diameter) in 5 x 25 arrays onto the silicon surface to form disulfide bonds with the free thiol groups of the MPTS layer on the surface (Fig. 1). The microarrays were incubated overnight at room temperature in a humidified chamber, then washed in washing solution (2X SSC, 1 % SDS) for 10 min on a shaker and rinsed 3 times with water. They were blow-dried with nitrogen gas. The tip was rinsed 3 times with station washing buffer (10 mM Tris-HCl pH 7.0,0.5% Tween 20) between samples. DNA microarrays were printed using a Cartesian PixSys 7500 arrayer (Fig. 1).

6. Asymmetrical PCR Reactions, Hybridization and Detection

A 50-|aL total asymmetrical PCR reaction consisted of 20-100 ng DNA template, PCR buffer to a final concentration of 1.5 mM magnesium chloride, 2.5 units of Taq Polymerase (GIBCO/BRL), 0.4 |M dNTPs (GIBCO/BRL), 0.4 |aM antisense Texas Red-labeled primer, and 0.004 |M sense primer. The reactions were performed using the following profile: 94°C/4 min (94°C/1 min; 53°C/30 sec; 72°C/30 sec) x 45 cycles. Asymmetrical PCR products were speed-vacuumed for 2 hr and resuspended in 12 |L of hybridization buffer [6x SSC pH 7, 5x Denhardt's solution (1% BSA (bovine serum albumin), 2% Ficoll 400, 2% polyvinylpyrollidone (PVP)], 0.5 % sodium dodecylsulfate (SDS), sheared salmon sperm DNA (5 |ig/mL)). A schematic drawing of the hybridization procedure is shown in Figure 2. The arrays were covered with a cleaned microscope slide cover glass, filled with the PCR reaction by capillary force, and incubated in a hybridization

Figure 2 Schematic representation of hybridization assays used to identify regulated TCMs. The immobilized probe is 20-26 bases long and contains a 5'-thiol modification and an 18-carbon chain between the thiol group and the first base. This physical separation is needed for successful hybridization.

Figure 2 Schematic representation of hybridization assays used to identify regulated TCMs. The immobilized probe is 20-26 bases long and contains a 5'-thiol modification and an 18-carbon chain between the thiol group and the first base. This physical separation is needed for successful hybridization.

chamber at 53 °C overnight. Then the chips were washed 3 times with washing solution in a shaker for 10 min, then with water, and blow-dried with nitrogen gas. Hybridization was detected using a GSI Lumonics scanner 5000 and its software was used for detection and analysis of the data acquired from the microarrays.

Chinese Herbs

Chinese Herbs

Worried For Your Health More Than Ever? Want To PREVENT Rather Than CURE Your Illness? Then WE are the only ones who can answer YOUR concern of time by presenting the exclusive work piece, the explicit and special eBook on CHINESE HERBS- the call of time.

Get My Free Ebook


Post a comment