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Multiple displacement amplification for complex mixtures of DNA fragments.

Shoaib M, Baconnais S, Mechold U, Le Cam E, Lipinski M, Ogryzko V - BMC Genomics (2008)

Bottom Line: To circumvent this problem, an additional (stuffer) DNA was added during religation (religation concentration > 10 ng/microL), which helped in the formation of long concatamers and hence resulted in uniform amplification.To confirm its usefulness in research, DP1 bound chromatin was isolated through ChIP and presence of DHFR promoter was detected using q-PCR and compared with an irrelevant GAPDH promoter.The results clearly indicated that when ChIP material was religated in presence of stuffer DNA (improved MDA), it allowed to recover the original pattern, while standard MDA and MDA without stuffer DNA failed to do so.

View Article: PubMed Central - HTML - PubMed

Affiliation: Université Paris-Sud 11, CNRS UMR 8126 Interactions Moléculaires et Cancer, Institut de Cancérologie Gustave-Roussy, 94805 Villejuif Cedex, France. muhd_shoaib@yahoo.com

ABSTRACT

Background: A fundamental requirement for genomic studies is the availability of genetic material of good quality and quantity. The desired quantity and quality are often hard to obtain when target DNA is composed of complex mixtures of relatively short DNA fragments. Here, we sought to develop a method to representatively amplify such complex mixtures by converting them to long linear and circular concatamers, from minute amounts of starting material, followed by phi29-based multiple displacement amplification.

Results: We report here proportional amplification of DNA fragments that were first converted into concatamers starting from DNA amounts as low as 1 pg. Religations at low concentration (< 1 ng/microL) preferentially lead to fragment self-circularization, which are then amplified independently, and result in non-uniform amplification. To circumvent this problem, an additional (stuffer) DNA was added during religation (religation concentration > 10 ng/microL), which helped in the formation of long concatamers and hence resulted in uniform amplification. To confirm its usefulness in research, DP1 bound chromatin was isolated through ChIP and presence of DHFR promoter was detected using q-PCR and compared with an irrelevant GAPDH promoter. The results clearly indicated that when ChIP material was religated in presence of stuffer DNA (improved MDA), it allowed to recover the original pattern, while standard MDA and MDA without stuffer DNA failed to do so.

Conclusion: We believe that this method allows for generation of abundant amounts of good quality genetic material from a complex mixture of short DNA fragments, which can be further used in high throughput genetic analysis.

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Analysis of chromatin immunoprecipitation. (a) DP1 complex with DHFR and GAPDH promoters. NIH 3T3 cells were transiently transfected with pBBHN.DP1 vector and subjected to the chromatin immunoprecipitation procedure. Shown is the ratio between amounts of DFHR (lanes 1 and 2) and GAPDH DNA (lanes 3 and 4) pulled down from the DP1 transfected sample (lanes 2 and 4) and GFP transfected sample (lanes 1 and 3), considered as a nonspecific background (mean of 2 experiments). 500 μg of chromatin were used for the ChIP. 5% of the input chromatin for each sample was decrosslinked, processed and analyzed in the same way by q-PCR. For each sample the value of the signal is presented as a percent of input. (b) MDA without any religation. Immunoprecipitate from the same experiment as in fig. 5a was diluted 100 fold and subjected to MDA before q-PCR analysis. (c) MDA with religation. Immunoprecipitate from the same experiment as in fig. 5a was diluted 100 fold and then subjected to religation, MDA and q-PCR as above. (d) MDA with religation in the presence of stuffer DNA. Immunoprecipitate from the same experiment as in fig. 5a was diluted 100 fold and then subjected to religation in the presence of 100 ng of HaeIII digested pUC19 and then MDA and q-PCR as above.
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Figure 5: Analysis of chromatin immunoprecipitation. (a) DP1 complex with DHFR and GAPDH promoters. NIH 3T3 cells were transiently transfected with pBBHN.DP1 vector and subjected to the chromatin immunoprecipitation procedure. Shown is the ratio between amounts of DFHR (lanes 1 and 2) and GAPDH DNA (lanes 3 and 4) pulled down from the DP1 transfected sample (lanes 2 and 4) and GFP transfected sample (lanes 1 and 3), considered as a nonspecific background (mean of 2 experiments). 500 μg of chromatin were used for the ChIP. 5% of the input chromatin for each sample was decrosslinked, processed and analyzed in the same way by q-PCR. For each sample the value of the signal is presented as a percent of input. (b) MDA without any religation. Immunoprecipitate from the same experiment as in fig. 5a was diluted 100 fold and subjected to MDA before q-PCR analysis. (c) MDA with religation. Immunoprecipitate from the same experiment as in fig. 5a was diluted 100 fold and then subjected to religation, MDA and q-PCR as above. (d) MDA with religation in the presence of stuffer DNA. Immunoprecipitate from the same experiment as in fig. 5a was diluted 100 fold and then subjected to religation in the presence of 100 ng of HaeIII digested pUC19 and then MDA and q-PCR as above.

Mentions: To test whether our methodology was useful for a research application, we performed analysis of chromatin immunoprecipitation samples. We used binding of DHFR promoter with transcription factor DP1 as a model in our experiments. NIH3T3 cells were transiently transfected with a vector expressing in vivo biotinylated DP1, and the DP1 bound chromatin was isolated as described previously [6]. The presence of DHFR promoter was detected with q-PCR and compared with an irrelevant GAPDH promoter. Chromatin from NIH3T3 cells, transfected with GFP was used as a negative control. As seen on the fig. 5a, the DP1 bound chromatin contains significant amount of DHFR promoter, compared to the control chromatin immunoprecipitate, and no difference between the two samples is seen in the case of q-PCR analysis of GAPDH promoter. To test our improved MDA method, we diluted the samples 100 fold and performed three different amplifications: A. Direct MDA amplification without religation, B. With a sample subjected to religation before MDA, C. With a sample subjected to religation in the presence of stuffer DNA before MDA. The amplified material was analyzed by q-PCR for the presence of DHFR and GAPDH promoter containing fragments. As seen from the fig. 5b–d, MDA of the low amounts of the immunoprecipitated material does not allow to recover the original pattern presented on the fig. 5a, unless it was first religated in the presence of stuffer DNA. We conclude that the use of MDA for the uniform amplification of DNA mixtures obtained from the chromatin Immunoprecipitation experiments benefits from religation in the presence of stuffer DNA.


Multiple displacement amplification for complex mixtures of DNA fragments.

Shoaib M, Baconnais S, Mechold U, Le Cam E, Lipinski M, Ogryzko V - BMC Genomics (2008)

Analysis of chromatin immunoprecipitation. (a) DP1 complex with DHFR and GAPDH promoters. NIH 3T3 cells were transiently transfected with pBBHN.DP1 vector and subjected to the chromatin immunoprecipitation procedure. Shown is the ratio between amounts of DFHR (lanes 1 and 2) and GAPDH DNA (lanes 3 and 4) pulled down from the DP1 transfected sample (lanes 2 and 4) and GFP transfected sample (lanes 1 and 3), considered as a nonspecific background (mean of 2 experiments). 500 μg of chromatin were used for the ChIP. 5% of the input chromatin for each sample was decrosslinked, processed and analyzed in the same way by q-PCR. For each sample the value of the signal is presented as a percent of input. (b) MDA without any religation. Immunoprecipitate from the same experiment as in fig. 5a was diluted 100 fold and subjected to MDA before q-PCR analysis. (c) MDA with religation. Immunoprecipitate from the same experiment as in fig. 5a was diluted 100 fold and then subjected to religation, MDA and q-PCR as above. (d) MDA with religation in the presence of stuffer DNA. Immunoprecipitate from the same experiment as in fig. 5a was diluted 100 fold and then subjected to religation in the presence of 100 ng of HaeIII digested pUC19 and then MDA and q-PCR as above.
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Related In: Results  -  Collection

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Figure 5: Analysis of chromatin immunoprecipitation. (a) DP1 complex with DHFR and GAPDH promoters. NIH 3T3 cells were transiently transfected with pBBHN.DP1 vector and subjected to the chromatin immunoprecipitation procedure. Shown is the ratio between amounts of DFHR (lanes 1 and 2) and GAPDH DNA (lanes 3 and 4) pulled down from the DP1 transfected sample (lanes 2 and 4) and GFP transfected sample (lanes 1 and 3), considered as a nonspecific background (mean of 2 experiments). 500 μg of chromatin were used for the ChIP. 5% of the input chromatin for each sample was decrosslinked, processed and analyzed in the same way by q-PCR. For each sample the value of the signal is presented as a percent of input. (b) MDA without any religation. Immunoprecipitate from the same experiment as in fig. 5a was diluted 100 fold and subjected to MDA before q-PCR analysis. (c) MDA with religation. Immunoprecipitate from the same experiment as in fig. 5a was diluted 100 fold and then subjected to religation, MDA and q-PCR as above. (d) MDA with religation in the presence of stuffer DNA. Immunoprecipitate from the same experiment as in fig. 5a was diluted 100 fold and then subjected to religation in the presence of 100 ng of HaeIII digested pUC19 and then MDA and q-PCR as above.
Mentions: To test whether our methodology was useful for a research application, we performed analysis of chromatin immunoprecipitation samples. We used binding of DHFR promoter with transcription factor DP1 as a model in our experiments. NIH3T3 cells were transiently transfected with a vector expressing in vivo biotinylated DP1, and the DP1 bound chromatin was isolated as described previously [6]. The presence of DHFR promoter was detected with q-PCR and compared with an irrelevant GAPDH promoter. Chromatin from NIH3T3 cells, transfected with GFP was used as a negative control. As seen on the fig. 5a, the DP1 bound chromatin contains significant amount of DHFR promoter, compared to the control chromatin immunoprecipitate, and no difference between the two samples is seen in the case of q-PCR analysis of GAPDH promoter. To test our improved MDA method, we diluted the samples 100 fold and performed three different amplifications: A. Direct MDA amplification without religation, B. With a sample subjected to religation before MDA, C. With a sample subjected to religation in the presence of stuffer DNA before MDA. The amplified material was analyzed by q-PCR for the presence of DHFR and GAPDH promoter containing fragments. As seen from the fig. 5b–d, MDA of the low amounts of the immunoprecipitated material does not allow to recover the original pattern presented on the fig. 5a, unless it was first religated in the presence of stuffer DNA. We conclude that the use of MDA for the uniform amplification of DNA mixtures obtained from the chromatin Immunoprecipitation experiments benefits from religation in the presence of stuffer DNA.

Bottom Line: To circumvent this problem, an additional (stuffer) DNA was added during religation (religation concentration > 10 ng/microL), which helped in the formation of long concatamers and hence resulted in uniform amplification.To confirm its usefulness in research, DP1 bound chromatin was isolated through ChIP and presence of DHFR promoter was detected using q-PCR and compared with an irrelevant GAPDH promoter.The results clearly indicated that when ChIP material was religated in presence of stuffer DNA (improved MDA), it allowed to recover the original pattern, while standard MDA and MDA without stuffer DNA failed to do so.

View Article: PubMed Central - HTML - PubMed

Affiliation: Université Paris-Sud 11, CNRS UMR 8126 Interactions Moléculaires et Cancer, Institut de Cancérologie Gustave-Roussy, 94805 Villejuif Cedex, France. muhd_shoaib@yahoo.com

ABSTRACT

Background: A fundamental requirement for genomic studies is the availability of genetic material of good quality and quantity. The desired quantity and quality are often hard to obtain when target DNA is composed of complex mixtures of relatively short DNA fragments. Here, we sought to develop a method to representatively amplify such complex mixtures by converting them to long linear and circular concatamers, from minute amounts of starting material, followed by phi29-based multiple displacement amplification.

Results: We report here proportional amplification of DNA fragments that were first converted into concatamers starting from DNA amounts as low as 1 pg. Religations at low concentration (< 1 ng/microL) preferentially lead to fragment self-circularization, which are then amplified independently, and result in non-uniform amplification. To circumvent this problem, an additional (stuffer) DNA was added during religation (religation concentration > 10 ng/microL), which helped in the formation of long concatamers and hence resulted in uniform amplification. To confirm its usefulness in research, DP1 bound chromatin was isolated through ChIP and presence of DHFR promoter was detected using q-PCR and compared with an irrelevant GAPDH promoter. The results clearly indicated that when ChIP material was religated in presence of stuffer DNA (improved MDA), it allowed to recover the original pattern, while standard MDA and MDA without stuffer DNA failed to do so.

Conclusion: We believe that this method allows for generation of abundant amounts of good quality genetic material from a complex mixture of short DNA fragments, which can be further used in high throughput genetic analysis.

Show MeSH