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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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Multiple displacement amplification of concatamers. (a) Digestions and religations of 50 ng pUC19 with HpaII and HaeIII. Lanes 1,2 & 3 correspond to 50 ng each of pUC19 undigested, digested with HpaII, digested with HpaII and religated, respectively; while lanes 4,5 and 6 correspond to the same amounts of pUC19 undigested, digested with HaeIII, digested with HaeIII and religated, respectively. 2% agarose gel stained with EB was used for electrophoresis. (b) Frequency distribution of circular and linear concatamers. The horizontal axis shows the increasing size of concatamers in nanometers while the vertical axis shows the percentages of both linear and circular concatamers with respect to their size in nanometers. An example of electron micrograph showing the presence of both circular and linear concatamers as shown in the insert. (c) & (d) MDA of HpaII and HaeIII digested and religated samples respectively. Five dilutions (in ascending order, 1:1 pg, 2:10 pg, 3:100 pg, 4:1 ng, 5:10 ng) were made after religation in both cases along with untreated pUC19 (1 ng) taken as positive control. The amplification products are indicated by asterisks. 0.8% agarose gels stained with SYBR Green were used for electrophoretic analyses. (e) Re-digestions of HpaII digested, religated and amplified samples. Odd numbers represent the samples redigested with the same enzyme (HpaII) while even numbers represent samples redigested with a different enzyme (AseI). (1,2:1 pg, 3,4:10 pg, 5,6:100 pg, 7,8:1 ng, 9,10:10 ng). 10% polyacryamide gels stained with EB were used.
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Figure 2: Multiple displacement amplification of concatamers. (a) Digestions and religations of 50 ng pUC19 with HpaII and HaeIII. Lanes 1,2 & 3 correspond to 50 ng each of pUC19 undigested, digested with HpaII, digested with HpaII and religated, respectively; while lanes 4,5 and 6 correspond to the same amounts of pUC19 undigested, digested with HaeIII, digested with HaeIII and religated, respectively. 2% agarose gel stained with EB was used for electrophoresis. (b) Frequency distribution of circular and linear concatamers. The horizontal axis shows the increasing size of concatamers in nanometers while the vertical axis shows the percentages of both linear and circular concatamers with respect to their size in nanometers. An example of electron micrograph showing the presence of both circular and linear concatamers as shown in the insert. (c) & (d) MDA of HpaII and HaeIII digested and religated samples respectively. Five dilutions (in ascending order, 1:1 pg, 2:10 pg, 3:100 pg, 4:1 ng, 5:10 ng) were made after religation in both cases along with untreated pUC19 (1 ng) taken as positive control. The amplification products are indicated by asterisks. 0.8% agarose gels stained with SYBR Green were used for electrophoretic analyses. (e) Re-digestions of HpaII digested, religated and amplified samples. Odd numbers represent the samples redigested with the same enzyme (HpaII) while even numbers represent samples redigested with a different enzyme (AseI). (1,2:1 pg, 3,4:10 pg, 5,6:100 pg, 7,8:1 ng, 9,10:10 ng). 10% polyacryamide gels stained with EB were used.

Mentions: In the first set of experiments, we tested whether formation of concatamers, before isothermal amplification, of small amounts of starting material allows preservation of initial composition of the complex mixture of DNA fragments. We used pUC19 DNA digested with different restriction enzymes as a source of DNA fragments for religation and amplification. The digestion of pUC19 by almost any enzyme produces well recognizable and reproducible pattern, which can be compared later with that of the amplified DNA. Accordingly, pUC19 DNA was first digested either with HpaII (producing cohesive ends) or HaeIII (producing blunt ends), and the resulting fragments subjected to religation with T4 DNA ligase. Religations were done at relatively high DNA concentration (50 ng/μL) to ensure the formation of linear and circular concatamers, which are a prerequisite for optimal multiple displacement amplification (MDA). As seen in fig 2a, HpaII and HaeIII digests yields distinct patterns of fragments ranging from 500 and 800 bp, respectively, to as low as 75 bp (lanes 2 & 5). The religation reaction converted these distinct patterns to long smears ranging from 0.6–12 Kb (lanes 3 & 6), representing long linear and circular concatamers. In most of our experiments, HpaII fragments gave longer religation products compared with those relegated from HaeIII products. This was expected, as blunt ends, generated by HaeIII, are ligated less efficiently.


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)

Multiple displacement amplification of concatamers. (a) Digestions and religations of 50 ng pUC19 with HpaII and HaeIII. Lanes 1,2 & 3 correspond to 50 ng each of pUC19 undigested, digested with HpaII, digested with HpaII and religated, respectively; while lanes 4,5 and 6 correspond to the same amounts of pUC19 undigested, digested with HaeIII, digested with HaeIII and religated, respectively. 2% agarose gel stained with EB was used for electrophoresis. (b) Frequency distribution of circular and linear concatamers. The horizontal axis shows the increasing size of concatamers in nanometers while the vertical axis shows the percentages of both linear and circular concatamers with respect to their size in nanometers. An example of electron micrograph showing the presence of both circular and linear concatamers as shown in the insert. (c) & (d) MDA of HpaII and HaeIII digested and religated samples respectively. Five dilutions (in ascending order, 1:1 pg, 2:10 pg, 3:100 pg, 4:1 ng, 5:10 ng) were made after religation in both cases along with untreated pUC19 (1 ng) taken as positive control. The amplification products are indicated by asterisks. 0.8% agarose gels stained with SYBR Green were used for electrophoretic analyses. (e) Re-digestions of HpaII digested, religated and amplified samples. Odd numbers represent the samples redigested with the same enzyme (HpaII) while even numbers represent samples redigested with a different enzyme (AseI). (1,2:1 pg, 3,4:10 pg, 5,6:100 pg, 7,8:1 ng, 9,10:10 ng). 10% polyacryamide gels stained with EB were used.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Multiple displacement amplification of concatamers. (a) Digestions and religations of 50 ng pUC19 with HpaII and HaeIII. Lanes 1,2 & 3 correspond to 50 ng each of pUC19 undigested, digested with HpaII, digested with HpaII and religated, respectively; while lanes 4,5 and 6 correspond to the same amounts of pUC19 undigested, digested with HaeIII, digested with HaeIII and religated, respectively. 2% agarose gel stained with EB was used for electrophoresis. (b) Frequency distribution of circular and linear concatamers. The horizontal axis shows the increasing size of concatamers in nanometers while the vertical axis shows the percentages of both linear and circular concatamers with respect to their size in nanometers. An example of electron micrograph showing the presence of both circular and linear concatamers as shown in the insert. (c) & (d) MDA of HpaII and HaeIII digested and religated samples respectively. Five dilutions (in ascending order, 1:1 pg, 2:10 pg, 3:100 pg, 4:1 ng, 5:10 ng) were made after religation in both cases along with untreated pUC19 (1 ng) taken as positive control. The amplification products are indicated by asterisks. 0.8% agarose gels stained with SYBR Green were used for electrophoretic analyses. (e) Re-digestions of HpaII digested, religated and amplified samples. Odd numbers represent the samples redigested with the same enzyme (HpaII) while even numbers represent samples redigested with a different enzyme (AseI). (1,2:1 pg, 3,4:10 pg, 5,6:100 pg, 7,8:1 ng, 9,10:10 ng). 10% polyacryamide gels stained with EB were used.
Mentions: In the first set of experiments, we tested whether formation of concatamers, before isothermal amplification, of small amounts of starting material allows preservation of initial composition of the complex mixture of DNA fragments. We used pUC19 DNA digested with different restriction enzymes as a source of DNA fragments for religation and amplification. The digestion of pUC19 by almost any enzyme produces well recognizable and reproducible pattern, which can be compared later with that of the amplified DNA. Accordingly, pUC19 DNA was first digested either with HpaII (producing cohesive ends) or HaeIII (producing blunt ends), and the resulting fragments subjected to religation with T4 DNA ligase. Religations were done at relatively high DNA concentration (50 ng/μL) to ensure the formation of linear and circular concatamers, which are a prerequisite for optimal multiple displacement amplification (MDA). As seen in fig 2a, HpaII and HaeIII digests yields distinct patterns of fragments ranging from 500 and 800 bp, respectively, to as low as 75 bp (lanes 2 & 5). The religation reaction converted these distinct patterns to long smears ranging from 0.6–12 Kb (lanes 3 & 6), representing long linear and circular concatamers. In most of our experiments, HpaII fragments gave longer religation products compared with those relegated from HaeIII products. This was expected, as blunt ends, generated by HaeIII, are ligated less efficiently.

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