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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.

Show MeSH
Religation at low concentration lead to non-proportional amplification. (a) Re-digestions of HpaII digested, religated and amplified samples. Religations were done at different concentrations. The concentrations go down in descending order from number 1 to 12. Six different concentrations were chosen i.e. 12 ng/μL, 10 ng/μL, 8 ng/μL, 4 ng/μL, 1 ng/μL, 100 pg/μL. Odd numbers represent the amplified samples redigested with the same enzyme (HpaII) while even numbers represent same samples redigested with a different enzyme (AflIII). (1,2: 12 ng/μL, 3,4: 10 ng/μL, 5,6: 8 ng/μL, 7,8: 4 ng/μL, 9,10: 1 ng/μL, 11,12: 100 pg/μL). The asterisks represent the fragments that are over amplified as compared to others. 10% polyacrylamide gels stained with EB were used for redigested samples. (b) Quantitative scanning of relative band intensities. The arrows shown in the scan of lane-11, corresponding to the asterisks in fig. 3a, represent DNA fragments that are relatively better amplified as compared to others in the same lane.
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Figure 3: Religation at low concentration lead to non-proportional amplification. (a) Re-digestions of HpaII digested, religated and amplified samples. Religations were done at different concentrations. The concentrations go down in descending order from number 1 to 12. Six different concentrations were chosen i.e. 12 ng/μL, 10 ng/μL, 8 ng/μL, 4 ng/μL, 1 ng/μL, 100 pg/μL. Odd numbers represent the amplified samples redigested with the same enzyme (HpaII) while even numbers represent same samples redigested with a different enzyme (AflIII). (1,2: 12 ng/μL, 3,4: 10 ng/μL, 5,6: 8 ng/μL, 7,8: 4 ng/μL, 9,10: 1 ng/μL, 11,12: 100 pg/μL). The asterisks represent the fragments that are over amplified as compared to others. 10% polyacrylamide gels stained with EB were used for redigested samples. (b) Quantitative scanning of relative band intensities. The arrows shown in the scan of lane-11, corresponding to the asterisks in fig. 3a, represent DNA fragments that are relatively better amplified as compared to others in the same lane.

Mentions: In the above experiments, we performed religation at relatively high DNA concentration (50 ng/μL) and then made several dilutions before MDA. However, in real-life experiments, it is expected that the starting amount of DNA will be much lower. In the next set of experiments, religations were carried out at different concentrations starting from 12 ng/μL down to 100 pg/μL. As shown in fig. 3a, redigestions of the amplified products with the same enzyme (HpaII) preserved the pattern, while a different enzyme (AflIII) produced the expected smeared product. However, going down in the religated DNA concentration, the pattern of HpaII redigestion started to deviate from the original one. Although, we used equal amounts of amplified material for redigestion and loaded equal amounts of redigested DNA in all lanes, it was observed that at certain DNA concentration, the amplification yield starts to decrease, generating less amplified material and hence observed low signal in the last lanes in fig. 3. We also observed that some fragments were completely lost while some were only diminished especially in lane-11. The asterisks and arrows in fig. 3a &3b respectively, represent the fragments that are relatively better amplified than others. The selective loss of most of the fragments in this experiment could have been due to self-circularization of these fragments, which, consequently amplify as individual molecules, instead of amplifying as part of concatamers. This leads to a significant bias in the relative efficiencies of the various DNA fragments during amplification. The relative intensities of various fragments were quantitatively measured using image processing software 'Image J' [5]. The scans shown in fig. 3b clearly indicates that as we go down in the DNA concentration during religation, we get non-uniform amplification evident by the aberrant peaks in the scans of lanes 9 & 11.


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)

Religation at low concentration lead to non-proportional amplification. (a) Re-digestions of HpaII digested, religated and amplified samples. Religations were done at different concentrations. The concentrations go down in descending order from number 1 to 12. Six different concentrations were chosen i.e. 12 ng/μL, 10 ng/μL, 8 ng/μL, 4 ng/μL, 1 ng/μL, 100 pg/μL. Odd numbers represent the amplified samples redigested with the same enzyme (HpaII) while even numbers represent same samples redigested with a different enzyme (AflIII). (1,2: 12 ng/μL, 3,4: 10 ng/μL, 5,6: 8 ng/μL, 7,8: 4 ng/μL, 9,10: 1 ng/μL, 11,12: 100 pg/μL). The asterisks represent the fragments that are over amplified as compared to others. 10% polyacrylamide gels stained with EB were used for redigested samples. (b) Quantitative scanning of relative band intensities. The arrows shown in the scan of lane-11, corresponding to the asterisks in fig. 3a, represent DNA fragments that are relatively better amplified as compared to others in the same lane.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553422&req=5

Figure 3: Religation at low concentration lead to non-proportional amplification. (a) Re-digestions of HpaII digested, religated and amplified samples. Religations were done at different concentrations. The concentrations go down in descending order from number 1 to 12. Six different concentrations were chosen i.e. 12 ng/μL, 10 ng/μL, 8 ng/μL, 4 ng/μL, 1 ng/μL, 100 pg/μL. Odd numbers represent the amplified samples redigested with the same enzyme (HpaII) while even numbers represent same samples redigested with a different enzyme (AflIII). (1,2: 12 ng/μL, 3,4: 10 ng/μL, 5,6: 8 ng/μL, 7,8: 4 ng/μL, 9,10: 1 ng/μL, 11,12: 100 pg/μL). The asterisks represent the fragments that are over amplified as compared to others. 10% polyacrylamide gels stained with EB were used for redigested samples. (b) Quantitative scanning of relative band intensities. The arrows shown in the scan of lane-11, corresponding to the asterisks in fig. 3a, represent DNA fragments that are relatively better amplified as compared to others in the same lane.
Mentions: In the above experiments, we performed religation at relatively high DNA concentration (50 ng/μL) and then made several dilutions before MDA. However, in real-life experiments, it is expected that the starting amount of DNA will be much lower. In the next set of experiments, religations were carried out at different concentrations starting from 12 ng/μL down to 100 pg/μL. As shown in fig. 3a, redigestions of the amplified products with the same enzyme (HpaII) preserved the pattern, while a different enzyme (AflIII) produced the expected smeared product. However, going down in the religated DNA concentration, the pattern of HpaII redigestion started to deviate from the original one. Although, we used equal amounts of amplified material for redigestion and loaded equal amounts of redigested DNA in all lanes, it was observed that at certain DNA concentration, the amplification yield starts to decrease, generating less amplified material and hence observed low signal in the last lanes in fig. 3. We also observed that some fragments were completely lost while some were only diminished especially in lane-11. The asterisks and arrows in fig. 3a &3b respectively, represent the fragments that are relatively better amplified than others. The selective loss of most of the fragments in this experiment could have been due to self-circularization of these fragments, which, consequently amplify as individual molecules, instead of amplifying as part of concatamers. This leads to a significant bias in the relative efficiencies of the various DNA fragments during amplification. The relative intensities of various fragments were quantitatively measured using image processing software 'Image J' [5]. The scans shown in fig. 3b clearly indicates that as we go down in the DNA concentration during religation, we get non-uniform amplification evident by the aberrant peaks in the scans of lanes 9 & 11.

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