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Sensitivity and specificity of immunoprecipitation of DNA containing 5-Methylcytosine.

Okitsu CY, Hsieh CL - BMC Res Notes (2015)

Bottom Line: More importantly, binding of DNA to these antibodies does not always indicate the presence of DNA methylation.It is clear that false positive and false negative findings can be easily reached even though it does not ify these convenient and simple methods completely.Great caution should be taken for the interpretation of IP results using these antibodies and rigorous confirmation by sodium bisulfite sequencing is essential.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology and Department of Biochemistry, University of Southern California, 1441 Eastlake Ave., Rm 5420, Norris Cancer Center, Los Angeles, CA, 90033, USA. cyen@usc.edu.

ABSTRACT

Background: Attempts to enrich or identify DNA with cytosine methylation have been commonly carried out using anti-5-methylcytosine or anti-MBD2 (methyl-CpG binding domain protein 2) antibody in immunoprecipitation (IP) assays. However, a careful and systematic control experiment to examine the sensitivity and specificity of this approach has not been reported. It is of critical importance to understand the potential pitfalls of this approach and to avoid potential misinterpretation of findings.

Findings: We found that increased concentration of antibody used in the assay increased the amount of overall DNA captured as expected. The increased number of methylated cytosines in/on the DNA fragment also increased the amount of DNA captured by the antibody. Importantly, the antibody can bind to some fully unmethylated DNA fragments, even when fully methylated DNA is present in the same experiment.

Conclusion: The sensitivity of anti-5-methylcytosine antibody and anti-MBD2 antibody/MBD2 binding varies with the number of methylated cytosines on the DNA target. The specificity of these antibodies can also vary for different DNA target sequences. DNA fragments with fewer CpG sites may not bind to these antibodies even when all are methylated while DNA fragments with more CpG sites may bind to the antibodies when only some of these sites are methylated. More importantly, binding of DNA to these antibodies does not always indicate the presence of DNA methylation. It is clear that false positive and false negative findings can be easily reached even though it does not ify these convenient and simple methods completely. Great caution should be taken for the interpretation of IP results using these antibodies and rigorous confirmation by sodium bisulfite sequencing is essential.

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Increase in anti-5-methyl-C antibody concentration increases the efficiency of IP. qPCR was used to quantitate various regions of the fully methylated target DNA to determine the percentage of pull down, calculated by dividing the amount of DNA after IP by the amount of input DNA, with different concentrations of the antibody for the IP. A) A schematic diagram of MseI restriction sites on pCLH22. MseI restriction sites are as marked by short lines on the circle and the nucleotide positions of DNA fragments examined are as numbered. The solid arrows indicate the locations of the qPCR amplicons with the number of CpG sites in the box under the name of each probe/primer set. The hollow arrows mark the regions examined by sodium bisulfite sequencing. B) Percentage of IP using different concentrations of antibody and no antibody with fully methylated pCLH22 as target. C) Percentage of IP using different concentrations of antibody and no antibody with entirely unmethylated pCLH22 as target.
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Fig1: Increase in anti-5-methyl-C antibody concentration increases the efficiency of IP. qPCR was used to quantitate various regions of the fully methylated target DNA to determine the percentage of pull down, calculated by dividing the amount of DNA after IP by the amount of input DNA, with different concentrations of the antibody for the IP. A) A schematic diagram of MseI restriction sites on pCLH22. MseI restriction sites are as marked by short lines on the circle and the nucleotide positions of DNA fragments examined are as numbered. The solid arrows indicate the locations of the qPCR amplicons with the number of CpG sites in the box under the name of each probe/primer set. The hollow arrows mark the regions examined by sodium bisulfite sequencing. B) Percentage of IP using different concentrations of antibody and no antibody with fully methylated pCLH22 as target. C) Percentage of IP using different concentrations of antibody and no antibody with entirely unmethylated pCLH22 as target.

Mentions: Three different concentrations of anti-5-methyl-C antibody, 2.5, 5, and 10 ng/ul, were used in each pull down experiment of 30 ul volume with 100 ng of MseI digested pCLH22 (Figure 1A) target DNA methylated at all CpG sites. Experiments using MseI digested fully unmethylated pCLH22 as target DNA were carried out as negative controls. Experiments with no antibody was done in parallel for all configurations as controls for binding of the target DNA to the protein G-sepharose beads. When 2.5 ng/ul concentration of antibody was used, from 0.3% (LTR1) to 3.5% (Hyg5) of the fully methylated pCLH22 DNA fragments were pulled down (Figure 1B). At this antibody concentration, approximately 0.1% to 0.5% of the unmethylated pCLH22 DNA fragments were pulled down from the six regions examined (Figure 1C). When 5 ng/ul concentration of antibody was used, the percent pull down was almost 1% for the LTR1 region and at 10.8% for Hyg5 region of the fully methylated pCLH22 (Figure 1B), while the unmethylated pCLH22 fragments were pulled down at the level of up to 0.7% (Figure 1C). Percent pull down increased to 1.9% for the LTR1 region and 24.8% for the Hyg5 region when antibody concentration was 10 ng/ul for fully methylated pCLH22 (Figure 1B), and was up to 2% for the unmethylated pCLH22 (Figure 1C). It is clear that the amount of DNA precipitated increases with increasing concentration of antibody for all DNA regions, except Luc2, examined for methylated DNA. While the same trend is also observed for unmemthylated DNA targets, the increases are much less notable and remain to be at what can be considered background noise level. Much more fully methylated DNA fragments with 37 and 143 CpG sites than totally unmethylated same DNA fragments (quantitated by qPCR of Luc1 and Hyg5 regions) were clearly and consistently pulled down in all the experiments. It is also noted that the DNA fragment harboring the Luc3 amplicon was pulled down by the beads along in the no antibody control at a higher rate when it is unmethylated (Figure 1C). These observation suggest that while more than seven methylated CpG sites has good potential to be distinguished from fully unmethylated DNA using the anti-5-methyl-C antibody, many more methylated CpG sites on the DNA fragment may be needed to be unequivocally identified all the time.Figure 1


Sensitivity and specificity of immunoprecipitation of DNA containing 5-Methylcytosine.

Okitsu CY, Hsieh CL - BMC Res Notes (2015)

Increase in anti-5-methyl-C antibody concentration increases the efficiency of IP. qPCR was used to quantitate various regions of the fully methylated target DNA to determine the percentage of pull down, calculated by dividing the amount of DNA after IP by the amount of input DNA, with different concentrations of the antibody for the IP. A) A schematic diagram of MseI restriction sites on pCLH22. MseI restriction sites are as marked by short lines on the circle and the nucleotide positions of DNA fragments examined are as numbered. The solid arrows indicate the locations of the qPCR amplicons with the number of CpG sites in the box under the name of each probe/primer set. The hollow arrows mark the regions examined by sodium bisulfite sequencing. B) Percentage of IP using different concentrations of antibody and no antibody with fully methylated pCLH22 as target. C) Percentage of IP using different concentrations of antibody and no antibody with entirely unmethylated pCLH22 as target.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig1: Increase in anti-5-methyl-C antibody concentration increases the efficiency of IP. qPCR was used to quantitate various regions of the fully methylated target DNA to determine the percentage of pull down, calculated by dividing the amount of DNA after IP by the amount of input DNA, with different concentrations of the antibody for the IP. A) A schematic diagram of MseI restriction sites on pCLH22. MseI restriction sites are as marked by short lines on the circle and the nucleotide positions of DNA fragments examined are as numbered. The solid arrows indicate the locations of the qPCR amplicons with the number of CpG sites in the box under the name of each probe/primer set. The hollow arrows mark the regions examined by sodium bisulfite sequencing. B) Percentage of IP using different concentrations of antibody and no antibody with fully methylated pCLH22 as target. C) Percentage of IP using different concentrations of antibody and no antibody with entirely unmethylated pCLH22 as target.
Mentions: Three different concentrations of anti-5-methyl-C antibody, 2.5, 5, and 10 ng/ul, were used in each pull down experiment of 30 ul volume with 100 ng of MseI digested pCLH22 (Figure 1A) target DNA methylated at all CpG sites. Experiments using MseI digested fully unmethylated pCLH22 as target DNA were carried out as negative controls. Experiments with no antibody was done in parallel for all configurations as controls for binding of the target DNA to the protein G-sepharose beads. When 2.5 ng/ul concentration of antibody was used, from 0.3% (LTR1) to 3.5% (Hyg5) of the fully methylated pCLH22 DNA fragments were pulled down (Figure 1B). At this antibody concentration, approximately 0.1% to 0.5% of the unmethylated pCLH22 DNA fragments were pulled down from the six regions examined (Figure 1C). When 5 ng/ul concentration of antibody was used, the percent pull down was almost 1% for the LTR1 region and at 10.8% for Hyg5 region of the fully methylated pCLH22 (Figure 1B), while the unmethylated pCLH22 fragments were pulled down at the level of up to 0.7% (Figure 1C). Percent pull down increased to 1.9% for the LTR1 region and 24.8% for the Hyg5 region when antibody concentration was 10 ng/ul for fully methylated pCLH22 (Figure 1B), and was up to 2% for the unmethylated pCLH22 (Figure 1C). It is clear that the amount of DNA precipitated increases with increasing concentration of antibody for all DNA regions, except Luc2, examined for methylated DNA. While the same trend is also observed for unmemthylated DNA targets, the increases are much less notable and remain to be at what can be considered background noise level. Much more fully methylated DNA fragments with 37 and 143 CpG sites than totally unmethylated same DNA fragments (quantitated by qPCR of Luc1 and Hyg5 regions) were clearly and consistently pulled down in all the experiments. It is also noted that the DNA fragment harboring the Luc3 amplicon was pulled down by the beads along in the no antibody control at a higher rate when it is unmethylated (Figure 1C). These observation suggest that while more than seven methylated CpG sites has good potential to be distinguished from fully unmethylated DNA using the anti-5-methyl-C antibody, many more methylated CpG sites on the DNA fragment may be needed to be unequivocally identified all the time.Figure 1

Bottom Line: More importantly, binding of DNA to these antibodies does not always indicate the presence of DNA methylation.It is clear that false positive and false negative findings can be easily reached even though it does not ify these convenient and simple methods completely.Great caution should be taken for the interpretation of IP results using these antibodies and rigorous confirmation by sodium bisulfite sequencing is essential.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology and Department of Biochemistry, University of Southern California, 1441 Eastlake Ave., Rm 5420, Norris Cancer Center, Los Angeles, CA, 90033, USA. cyen@usc.edu.

ABSTRACT

Background: Attempts to enrich or identify DNA with cytosine methylation have been commonly carried out using anti-5-methylcytosine or anti-MBD2 (methyl-CpG binding domain protein 2) antibody in immunoprecipitation (IP) assays. However, a careful and systematic control experiment to examine the sensitivity and specificity of this approach has not been reported. It is of critical importance to understand the potential pitfalls of this approach and to avoid potential misinterpretation of findings.

Findings: We found that increased concentration of antibody used in the assay increased the amount of overall DNA captured as expected. The increased number of methylated cytosines in/on the DNA fragment also increased the amount of DNA captured by the antibody. Importantly, the antibody can bind to some fully unmethylated DNA fragments, even when fully methylated DNA is present in the same experiment.

Conclusion: The sensitivity of anti-5-methylcytosine antibody and anti-MBD2 antibody/MBD2 binding varies with the number of methylated cytosines on the DNA target. The specificity of these antibodies can also vary for different DNA target sequences. DNA fragments with fewer CpG sites may not bind to these antibodies even when all are methylated while DNA fragments with more CpG sites may bind to the antibodies when only some of these sites are methylated. More importantly, binding of DNA to these antibodies does not always indicate the presence of DNA methylation. It is clear that false positive and false negative findings can be easily reached even though it does not ify these convenient and simple methods completely. Great caution should be taken for the interpretation of IP results using these antibodies and rigorous confirmation by sodium bisulfite sequencing is essential.

Show MeSH
Related in: MedlinePlus