Nucleic Acid Isolation
DNA Applications on the 2100 Bioanalyzer
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Note: This is a review of the published article listed below. All information, quotes, figures, methods, and findings mentioned in this review are from that article, and are the property of its authors and/or the publication in which the article originally appeared.
Analyses that utilize DNA for biological characterizations typically depend upon labor-intensive techniques such as RFLP or those that offer only limited information such as PCR and electrophoresis. Agilent’s 2100 Bioanalyzer offers an alternative to traditional techniques for the quantification and high resolution analysis of DNA using a microfluidic chip. When used in conjunction with Agilent’s optimized DNA kits, the 2100 Bioanalyzer automates sizing and quantification of DNA fragments from 25 bp to 12 kb, offering a superior alternative to agarose gels, RFLP analysis, and multiplex techniques. Romulo Brena and others (2006) used the 2100 Bioanalyzer to quantify DNA methylation by combining the platform with bisulfite restriction analysis (termed “Bio-COBRA”). The group suggested that the main strength of this approach was that it allowed for rapid, accurate and cost-effective determination of DNA methylation percentages on a platform that enabled the comparison of those values across large sample sets. The data generated by this method was highly reproducible and, through use of an in vitro methylated DNA standard, experimental values could be converted to actual methylation values in a single step. Most importantly, no saturation of the system was observed within the dynamic range tested in the study (10–65 ng/µl). Results indicate that the combination of the COBRA assay with the 2100 Bioanalyzer offers a rapid, accurate and cost-efficient quantification of methylation patterns in any DNA sample.
Figure 1. DNA methylation standards for SALL3 (A) and TWIST2 (B).
Fragment sizes are indicated to the right of the gel. Methylation percentages for each lane are indicated at the top. The restriction map of the sequence is indicated at the bottom of the gel. BstUI sites are indicated with vertical lines on the restriction map. (C) Example of a SALL3 virtual gel generated by the Bioanalyzer software. (D) Fluorescence versus time data plot for lanes 8 and 9 from (C). From right to left, the fluorescence peaks correspond to the following digestion fragments: 208, 124, 36 and 26 bp. The 22 bp fragment overlaps with the front marker. As the methylation percent of the sample increases, there is a decrease in the fluorescence of the 208 bp peak and an increase in the fluorescence of the digested peaks (75% versus 87.5% plots). Plots likes the ones shown in this figure were used to calculate methylation percentages for all standards and samples tested.
Figure 2. Plots of observed versus expected DNA methylation values for SALL3, TWIST2 and C/EBPα methylation standards.
(A) SALL3, (B) TWIST2 and (C) C/EBPα results. Trend lines and r2 values are displayed for each plot. The non-linearity of the observed versus expected methylation values is most likely due to a PCR amplification bias.
Figure 3. Assessment of DNA methylation in clinical CLL samples and a human lung cancer cell line treated with 5-aza-2'dC.
(A) Methylation levels of TWIST2 in 19 primary CLL samples generated by Bio-COBRA and Southern blot. The correlation coefficient between the two data sets was 0.98. (B and E) Restriction digestions of SALL3 (B) and C/EBPα (E) in A549 cells treated with 5-aza-dC at six different concentrations for 72 h (concentrations are indicated at the top). (C and F) Bio-COBRA quantification of the restriction digestions shown in (B) and (E). As expected, low doses of the demethylating agent showed a pronounced effect in the DNA methylation status of the analyzed loci. (D and G) mRNA expression level of SALL3 (D) and C/EBPα (G). Three separate measurements were performed for each sample. For C/EBPα, the expression level measured in the untreated cell line was normalized to 1. For SALL3, the expression level detected at 0.10 µM was normalized to 1, since the untreated cell line shows no expression under the experimental conditions utilized in this study.
Original Research Paper:
Title: Accurate quantification of DNA methylation using combined bisulfite restriction analysis coupled with the Agilent 2100 Bioanalyzer platform.
Authors: Brena RM, Auer H, Kornacker K, Hackanson B, Raval A, Byrd JC, Plass C.
Journal: BMC Nucleic Acids Res. 2006 Feb 7;34(3):e17.
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