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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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Application of human genomic DNA with pUC19 as Stuffer DNA. (a) The schematics of the idea of Stuffer DNA. 1-In case of much diluted samples, relegation preferentially leads to self-circularization of fragments, which are then amplified as individual molecules. 2-The addition of Stuffer DNA derived from an unrelated source to the sample DNA favors intermolecular ligation and leads to the formation of long concatamers, both linear and circular. These long concatamers can then be reliably amplified using MDA technique. (b) Amplification of hgDNA with stuffer DNA using MDA technique. Two different amounts of hgDNA were taken (1:10 ng/μL and 2:1 ng/μL) and a fixed amount of pUC19 (100 ng) was used as stuffer DNA. The amplification products are indicated by asterisks. 0.8% agarose gel stained with SYBR Green was used for electrophoresis. (c) Re-digestions of samples [previously digested with AseI and MseI (both gives identical cohesive ends), religated and amplified], with the same enzyme (MseI) and a different enzyme (HpaII). Odd numbers represent the samples redigested with MseI, while even numbers represent HpaII digests (1,2: 10 ng and 3,4: 1 ng). Asterisks indicate HpaII fragments that do not contain internal MseI sites and thus are preserved after MseI digestion and religation. 10% polyacryamide gel stained with EB was used for electrophoresis. (d) q-PCR data of 10 ng and 1 ng hgDNA samples MDA amplified with stuffer DNA. 6 different primer pairs were used for quantitative analysis. As a template for q-PCR, native hgDNA (left graph), and the MDA-amplified DNA (10 ng – middle graph, 1 ng – right graph) were used. Shown are CT values calculated for each fragment according to Materials and Methods. (e) To see if the amplification preserves the proportions among different genomic regions, for each MDA experiment, ratios were calculated between CT values of different fragments, taking P1-hgDNA as reference for hgDNA control samples and P1-MDA for MDA processed samples. Data for P6-MDA in case of 1ng input is not shown, as it did not give the correct amplification product.
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Figure 4: Application of human genomic DNA with pUC19 as Stuffer DNA. (a) The schematics of the idea of Stuffer DNA. 1-In case of much diluted samples, relegation preferentially leads to self-circularization of fragments, which are then amplified as individual molecules. 2-The addition of Stuffer DNA derived from an unrelated source to the sample DNA favors intermolecular ligation and leads to the formation of long concatamers, both linear and circular. These long concatamers can then be reliably amplified using MDA technique. (b) Amplification of hgDNA with stuffer DNA using MDA technique. Two different amounts of hgDNA were taken (1:10 ng/μL and 2:1 ng/μL) and a fixed amount of pUC19 (100 ng) was used as stuffer DNA. The amplification products are indicated by asterisks. 0.8% agarose gel stained with SYBR Green was used for electrophoresis. (c) Re-digestions of samples [previously digested with AseI and MseI (both gives identical cohesive ends), religated and amplified], with the same enzyme (MseI) and a different enzyme (HpaII). Odd numbers represent the samples redigested with MseI, while even numbers represent HpaII digests (1,2: 10 ng and 3,4: 1 ng). Asterisks indicate HpaII fragments that do not contain internal MseI sites and thus are preserved after MseI digestion and religation. 10% polyacryamide gel stained with EB was used for electrophoresis. (d) q-PCR data of 10 ng and 1 ng hgDNA samples MDA amplified with stuffer DNA. 6 different primer pairs were used for quantitative analysis. As a template for q-PCR, native hgDNA (left graph), and the MDA-amplified DNA (10 ng – middle graph, 1 ng – right graph) were used. Shown are CT values calculated for each fragment according to Materials and Methods. (e) To see if the amplification preserves the proportions among different genomic regions, for each MDA experiment, ratios were calculated between CT values of different fragments, taking P1-hgDNA as reference for hgDNA control samples and P1-MDA for MDA processed samples. Data for P6-MDA in case of 1ng input is not shown, as it did not give the correct amplification product.

Mentions: As a model to test the usefulness of stuffer DNA, we used human genomic DNA (hgDNA) from HeLa cells (target DNA), while plasmid pUC19 was used as a stuffer DNA. hgDNA was digested with AseI, whereas pUC19 was digested with MseI. Both enzymes produce DNA fragments with compatible ends. Digestion of hgDNA with MseI gives very small fragments that are difficult to monitor with quantitative-PCR (q-PCR), we therefore used AseI instead. (The HpaII and HaeIII used in our preliminary experiments with pUC19 cannot be used for the analysis of genomic DNA, because because extensive methylation of human genomic DNA does not allow obtaining DNA fragments of sufficiently small size. Therefore, we had to choose different enzymes). After digestion of hgDNA, two dilutions were made, i.e. 10 ng/μL & 1 ng/μL. To each of these samples a fixed amount (100 ng) of pUC19, digested with MseI, was added, so that the final concentration of hgDNA + stuffer DNA became approximately 10 ng/μL. This concentration was previously shown to be sufficient for avoiding self-circularization (fig. 3). After religation, the samples were subjected to MDA. Fig. 4b shows the amplification products after MDA and amplification yield was estimated to be approximately 1000 fold. To control the quality of the amplification product, aliquots from the final amplified products were redigested with the original restriction enzyme (MseI), and a different enzyme (HpaII). Redigestion with MseI produced pattern identical to the original one, while the digest with HpaII consistently gave a smear (fig. 4c), confirming that the amplification preserved the original composition of the mixture, and resulted mostly from true religation of the DNA fragments. The HpaII redigests also yielded some bands superposed on a smear (fig. 4c; lanes 2 & 4). These bands represent the HpaII sites present between the MseI sites, therefore they remain preserved despite the scrambling of fragments during religation.


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)

Application of human genomic DNA with pUC19 as Stuffer DNA. (a) The schematics of the idea of Stuffer DNA. 1-In case of much diluted samples, relegation preferentially leads to self-circularization of fragments, which are then amplified as individual molecules. 2-The addition of Stuffer DNA derived from an unrelated source to the sample DNA favors intermolecular ligation and leads to the formation of long concatamers, both linear and circular. These long concatamers can then be reliably amplified using MDA technique. (b) Amplification of hgDNA with stuffer DNA using MDA technique. Two different amounts of hgDNA were taken (1:10 ng/μL and 2:1 ng/μL) and a fixed amount of pUC19 (100 ng) was used as stuffer DNA. The amplification products are indicated by asterisks. 0.8% agarose gel stained with SYBR Green was used for electrophoresis. (c) Re-digestions of samples [previously digested with AseI and MseI (both gives identical cohesive ends), religated and amplified], with the same enzyme (MseI) and a different enzyme (HpaII). Odd numbers represent the samples redigested with MseI, while even numbers represent HpaII digests (1,2: 10 ng and 3,4: 1 ng). Asterisks indicate HpaII fragments that do not contain internal MseI sites and thus are preserved after MseI digestion and religation. 10% polyacryamide gel stained with EB was used for electrophoresis. (d) q-PCR data of 10 ng and 1 ng hgDNA samples MDA amplified with stuffer DNA. 6 different primer pairs were used for quantitative analysis. As a template for q-PCR, native hgDNA (left graph), and the MDA-amplified DNA (10 ng – middle graph, 1 ng – right graph) were used. Shown are CT values calculated for each fragment according to Materials and Methods. (e) To see if the amplification preserves the proportions among different genomic regions, for each MDA experiment, ratios were calculated between CT values of different fragments, taking P1-hgDNA as reference for hgDNA control samples and P1-MDA for MDA processed samples. Data for P6-MDA in case of 1ng input is not shown, as it did not give the correct amplification product.
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Related In: Results  -  Collection

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Figure 4: Application of human genomic DNA with pUC19 as Stuffer DNA. (a) The schematics of the idea of Stuffer DNA. 1-In case of much diluted samples, relegation preferentially leads to self-circularization of fragments, which are then amplified as individual molecules. 2-The addition of Stuffer DNA derived from an unrelated source to the sample DNA favors intermolecular ligation and leads to the formation of long concatamers, both linear and circular. These long concatamers can then be reliably amplified using MDA technique. (b) Amplification of hgDNA with stuffer DNA using MDA technique. Two different amounts of hgDNA were taken (1:10 ng/μL and 2:1 ng/μL) and a fixed amount of pUC19 (100 ng) was used as stuffer DNA. The amplification products are indicated by asterisks. 0.8% agarose gel stained with SYBR Green was used for electrophoresis. (c) Re-digestions of samples [previously digested with AseI and MseI (both gives identical cohesive ends), religated and amplified], with the same enzyme (MseI) and a different enzyme (HpaII). Odd numbers represent the samples redigested with MseI, while even numbers represent HpaII digests (1,2: 10 ng and 3,4: 1 ng). Asterisks indicate HpaII fragments that do not contain internal MseI sites and thus are preserved after MseI digestion and religation. 10% polyacryamide gel stained with EB was used for electrophoresis. (d) q-PCR data of 10 ng and 1 ng hgDNA samples MDA amplified with stuffer DNA. 6 different primer pairs were used for quantitative analysis. As a template for q-PCR, native hgDNA (left graph), and the MDA-amplified DNA (10 ng – middle graph, 1 ng – right graph) were used. Shown are CT values calculated for each fragment according to Materials and Methods. (e) To see if the amplification preserves the proportions among different genomic regions, for each MDA experiment, ratios were calculated between CT values of different fragments, taking P1-hgDNA as reference for hgDNA control samples and P1-MDA for MDA processed samples. Data for P6-MDA in case of 1ng input is not shown, as it did not give the correct amplification product.
Mentions: As a model to test the usefulness of stuffer DNA, we used human genomic DNA (hgDNA) from HeLa cells (target DNA), while plasmid pUC19 was used as a stuffer DNA. hgDNA was digested with AseI, whereas pUC19 was digested with MseI. Both enzymes produce DNA fragments with compatible ends. Digestion of hgDNA with MseI gives very small fragments that are difficult to monitor with quantitative-PCR (q-PCR), we therefore used AseI instead. (The HpaII and HaeIII used in our preliminary experiments with pUC19 cannot be used for the analysis of genomic DNA, because because extensive methylation of human genomic DNA does not allow obtaining DNA fragments of sufficiently small size. Therefore, we had to choose different enzymes). After digestion of hgDNA, two dilutions were made, i.e. 10 ng/μL & 1 ng/μL. To each of these samples a fixed amount (100 ng) of pUC19, digested with MseI, was added, so that the final concentration of hgDNA + stuffer DNA became approximately 10 ng/μL. This concentration was previously shown to be sufficient for avoiding self-circularization (fig. 3). After religation, the samples were subjected to MDA. Fig. 4b shows the amplification products after MDA and amplification yield was estimated to be approximately 1000 fold. To control the quality of the amplification product, aliquots from the final amplified products were redigested with the original restriction enzyme (MseI), and a different enzyme (HpaII). Redigestion with MseI produced pattern identical to the original one, while the digest with HpaII consistently gave a smear (fig. 4c), confirming that the amplification preserved the original composition of the mixture, and resulted mostly from true religation of the DNA fragments. The HpaII redigests also yielded some bands superposed on a smear (fig. 4c; lanes 2 & 4). These bands represent the HpaII sites present between the MseI sites, therefore they remain preserved despite the scrambling of fragments during religation.

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