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Bioorganic and Medicinal Chemistry Letters
Volumn 21, Issue 22, 2011, Pages 6905-6908
The detection of a transgenic soybean biochip using gold label silver stain technology
Author keywords

Biochip; Biotin; Gold label silver stain; Gold nanoparticles; Streptavidin; Transgenics
Indexed keywords

BIOTIN; DEOXYURIDINE TRIPHOSPHATE DERIVATIVE; GOLD NANOPARTICLE; STREPTAVIDIN;
EID: 80054750751     PISSN: 0960894X     EISSN: 14643405     Source Type: Journal    
DOI: 10.1016/j.bmcl.2011.07.037     Document Type: Article
Times cited : (12)
References (43)
  • 25
    • 84857501021 scopus 로고    scopus 로고
    • After mixing the biotin-11-dUTP with the primers listed in Table 1, single and dual PCR amplification were performed using either DNA from the green soy bean plant for the negative control or DNA from a transgenic soybean plant. The reaction volume was 20 μL. This symmetrical PCR mixture contained both forward and reverse primers, each with a final concentration of 0.6 μM, 100-500 ng of genomic DNA, a 1× PCR buffer solution and 1 U of Taq enzyme. The PCR amplification conditions were as follows: an initial denaturation step at 94 °C for 5 min; 45 cycles consisting of a denaturation step at 94 °C for 30 sec, an annealing step at 54 °C for 40 s, and an extension step at 72 °C for 1 min; and a final extension step at 72 °C for 7 min. The resulting PCR products were detected using gel electrophoresis
    • After mixing the biotin-11-dUTP with the primers listed in Table 1, single and dual PCR amplification were performed using either DNA from the green soy bean plant for the negative control or DNA from a transgenic soybean plant. The reaction volume was 20 μL. This symmetrical PCR mixture contained both forward and reverse primers, each with a final concentration of 0.6 μM, 100-500 ng of genomic DNA, a 1× PCR buffer solution and 1 U of Taq enzyme. The PCR amplification conditions were as follows: an initial denaturation step at 94 °C for 5 min; 45 cycles consisting of a denaturation step at 94 °C for 30 sec, an annealing step at 54 °C for 40 s, and an extension step at 72 °C for 1 min; and a final extension step at 72 °C for 7 min. The resulting PCR products were detected using gel electrophoresis.
  • 26
    • 80054732425 scopus 로고    scopus 로고
    • The pH of a 1-ml solution containing GNPs (colloidal gold) was adjusted to 6.5. A total of 1 ml of streptavidin (10 mg/L) was added to the GNP solution while it was being agitated. The solution was then incubated at room temperature for 30 min
    • The pH of a 1-ml solution containing GNPs (colloidal gold) was adjusted to 6.5. A total of 1 ml of streptavidin (10 mg/L) was added to the GNP solution while it was being agitated. The solution was then incubated at room temperature for 30 min.
  • 27
    • 84857503020 scopus 로고    scopus 로고
    • The sheet glass (chip substrate) was soaked in a solution containing methanol and HCl (1:1) for 30 min. After washing with distilled water, the chip was then soaked in a H2SO4 solution for 30 min. The chip was first washed with distilled water and then with 95% alcohol. The chip was dried using N2. Next, the chip was soaked in a silanization solution comprising 95% alcohol and silane (49:1) for 10 min. After a thorough wash with distilled water, the chip was dried with N2. Finally, the chip was baked in a drying oven at 110 °C for 30 min min to complete the silanization process. The silanized chip was soaked in 95% alcohol for 5 min, washed thoroughly with distilled water and dried. A 25% solution of glutaraldehyde was diluted to 5% with 0.01 MPB solution, and the chip was then soaked in this diluted solution for 2 h. The chip was washed with 0.01 MPB solution to remove any residual glutaraldehyde. After washing with distilled water, the chip was air-dried before use
    • The sheet glass (chip substrate) was soaked in a solution containing methanol and HCl (1:1) for 30 min. After washing with distilled water, the chip was then soaked in a H2SO4 solution for 30 min. The chip was first washed with distilled water and then with 95% alcohol. The chip was dried using N2. Next, the chip was soaked in a silanization solution comprising 95% alcohol and silane (49:1) for 10 min. After a thorough wash with distilled water, the chip was dried with N2. Finally, the chip was baked in a drying oven at 110 °C for 30 min min to complete the silanization process. The silanized chip was soaked in 95% alcohol for 5 min, washed thoroughly with distilled water and dried. A 25% solution of glutaraldehyde was diluted to 5% with 0.01 MPB solution, and the chip was then soaked in this diluted solution for 2 h. The chip was washed with 0.01 MPB solution to remove any residual glutaraldehyde. After washing with distilled water, the chip was air-dried before use.
  • 29
    • 84857503021 scopus 로고    scopus 로고
    • The probes were diluted to 100 pmol/μL in carbonic acid buffer. A total of 0.15 μL of the probe solution for each point was manually spotted on the prepared chip. The chip was then sealed in watch glass and allowed to sit at room temperature overnight or longer. After being incubated in a 37 °C water bath for 1 h, the chip was removed from the watch glass, washed in 0.1% SDS for 5 min, washed five times with distilled water and then blown dry. The chip was soaked in a sodium borohydride reagent for 30 min to reduce the surface-bound aldehyde groups to hydroxyl groups (Fig. 1)
    • The probes were diluted to 100 pmol/μL in carbonic acid buffer. A total of 0.15 μL of the probe solution for each point was manually spotted on the prepared chip. The chip was then sealed in watch glass and allowed to sit at room temperature overnight or longer. After being incubated in a 37 °C water bath for 1 h, the chip was removed from the watch glass, washed in 0.1% SDS for 5 min, washed five times with distilled water and then blown dry. The chip was soaked in a sodium borohydride reagent for 30 min to reduce the surface-bound aldehyde groups to hydroxyl groups (Fig. 1).
  • 32
    • 84857505588 scopus 로고    scopus 로고
    • The PCR products were denatured at 95 °C for 10 min and then placed on ice for 3 min. The PCR products were diluted in a ratio of 1:3 with the hybridization solution. To ensure hybridization with every probe on the chip, 0.15 μL of that diluted solution was added to the chip, followed by incubation at 44 °C for 1 h. After hybridization, the chip was sequentially washed in a 2× SSC/0.1% SDS solution for 10 min, a 0.1× SSC/0.1% SDS solution for 10 min, and a 2× PBN (0.3 M NaNO3, 10 mM PB, pH 7) solution for 2 min. Following these washes, the chip was soaked in a solution containing the streptavidin-modified GNPs for 5 min. The chip was again sequentially washed in 2× SSC/0.1% SDS for 10 min, 0.1× SSC/0.1% SDS for 10 min, and then 2× PBN for 2 min
    • The PCR products were denatured at 95 °C for 10 min and then placed on ice for 3 min. The PCR products were diluted in a ratio of 1:3 with the hybridization solution. To ensure hybridization with every probe on the chip, 0.15 μL of that diluted solution was added to the chip, followed by incubation at 44 °C for 1 h. After hybridization, the chip was sequentially washed in a 2× SSC/0.1% SDS solution for 10 min, a 0.1× SSC/0.1% SDS solution for 10 min, and a 2× PBN (0.3 M NaNO3, 10 mM PB, pH 7) solution for 2 min. Following these washes, the chip was soaked in a solution containing the streptavidin-modified GNPs for 5 min. The chip was again sequentially washed in 2× SSC/0.1% SDS for 10 min, 0.1× SSC/0.1% SDS for 10 min, and then 2× PBN for 2 min.
  • 33
    • 84857501019 scopus 로고    scopus 로고
    • 2O, with the prior three mixtures combined together at the same time) at room temperature for several min. The chip was then washed and blown dry
    • 2O, with the prior three mixtures combined together at the same time) at room temperature for several min. The chip was then washed and blown dry.

* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.