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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
Ligation-Mediated PCR and Concatamer-Mediated Multiple Displacement Amplification. (a) The principle of Ligation Mediated PCR (LM-PCR). 1-Ligation with excess of primers, 2-Polymerase chain reaction of individual fragments. In LM-PCR, each fragment is amplified independently so that due to intrinsic differences among individual fragments, some fragments are amplified less efficiently than others. This results in non-uniform representation of original genetic material in the resultant amplicon, which consequently leads to loss of genetic information and inaccurate results. (b) The principle of concatamer-mediated multiple displacement amplification. 1-Religation of DNA fragments with T4 DNA ligase, which leads to two types of products, 2.1-Linear Concatamers and 2.2-Circular Concatamers. 3.1 and 3.2-Annealing of random hexamer primers and addition of phi29-DNA polymerase leads to concatamers-mediated multiple displacement amplification from linear and circular concatamers respectively.
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Figure 1: Ligation-Mediated PCR and Concatamer-Mediated Multiple Displacement Amplification. (a) The principle of Ligation Mediated PCR (LM-PCR). 1-Ligation with excess of primers, 2-Polymerase chain reaction of individual fragments. In LM-PCR, each fragment is amplified independently so that due to intrinsic differences among individual fragments, some fragments are amplified less efficiently than others. This results in non-uniform representation of original genetic material in the resultant amplicon, which consequently leads to loss of genetic information and inaccurate results. (b) The principle of concatamer-mediated multiple displacement amplification. 1-Religation of DNA fragments with T4 DNA ligase, which leads to two types of products, 2.1-Linear Concatamers and 2.2-Circular Concatamers. 3.1 and 3.2-Annealing of random hexamer primers and addition of phi29-DNA polymerase leads to concatamers-mediated multiple displacement amplification from linear and circular concatamers respectively.

Mentions: Amplification of a complex mixture of relatively short DNA fragments, derived from mini scale experiments in chromatin immunoprecipitation (ChIP), degraded forensic material, cDNA synthesis, clinical diagnostics, stored tumor or other tissue samples is an area of genome research which has received scant attention. Abundant amounts and good quality of amplified material are required for high throughput analysis of this kind of complex genetic material. However, the currently available amplification techniques for complex mixtures of DNA fragments, often based upon PCR [e.g. Ligation-mediated PCR (LM-PCR)][1], do not amplify all fragments in equal proportion. In the later, every fragment is amplified independently and hence depending upon the presence or absence of GC-rich sequences and secondary (hairpin) structures in some DNA fragments, different elements of the mixture tend to amplify at different rates. This non-uniform amplification leads to relative loss of genetic material and ultimately can result in severe mis-representation of the fragments, producing inaccurate results and loss of important genetic information (fig. 1a).


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)

Ligation-Mediated PCR and Concatamer-Mediated Multiple Displacement Amplification. (a) The principle of Ligation Mediated PCR (LM-PCR). 1-Ligation with excess of primers, 2-Polymerase chain reaction of individual fragments. In LM-PCR, each fragment is amplified independently so that due to intrinsic differences among individual fragments, some fragments are amplified less efficiently than others. This results in non-uniform representation of original genetic material in the resultant amplicon, which consequently leads to loss of genetic information and inaccurate results. (b) The principle of concatamer-mediated multiple displacement amplification. 1-Religation of DNA fragments with T4 DNA ligase, which leads to two types of products, 2.1-Linear Concatamers and 2.2-Circular Concatamers. 3.1 and 3.2-Annealing of random hexamer primers and addition of phi29-DNA polymerase leads to concatamers-mediated multiple displacement amplification from linear and circular concatamers respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Ligation-Mediated PCR and Concatamer-Mediated Multiple Displacement Amplification. (a) The principle of Ligation Mediated PCR (LM-PCR). 1-Ligation with excess of primers, 2-Polymerase chain reaction of individual fragments. In LM-PCR, each fragment is amplified independently so that due to intrinsic differences among individual fragments, some fragments are amplified less efficiently than others. This results in non-uniform representation of original genetic material in the resultant amplicon, which consequently leads to loss of genetic information and inaccurate results. (b) The principle of concatamer-mediated multiple displacement amplification. 1-Religation of DNA fragments with T4 DNA ligase, which leads to two types of products, 2.1-Linear Concatamers and 2.2-Circular Concatamers. 3.1 and 3.2-Annealing of random hexamer primers and addition of phi29-DNA polymerase leads to concatamers-mediated multiple displacement amplification from linear and circular concatamers respectively.
Mentions: Amplification of a complex mixture of relatively short DNA fragments, derived from mini scale experiments in chromatin immunoprecipitation (ChIP), degraded forensic material, cDNA synthesis, clinical diagnostics, stored tumor or other tissue samples is an area of genome research which has received scant attention. Abundant amounts and good quality of amplified material are required for high throughput analysis of this kind of complex genetic material. However, the currently available amplification techniques for complex mixtures of DNA fragments, often based upon PCR [e.g. Ligation-mediated PCR (LM-PCR)][1], do not amplify all fragments in equal proportion. In the later, every fragment is amplified independently and hence depending upon the presence or absence of GC-rich sequences and secondary (hairpin) structures in some DNA fragments, different elements of the mixture tend to amplify at different rates. This non-uniform amplification leads to relative loss of genetic material and ultimately can result in severe mis-representation of the fragments, producing inaccurate results and loss of important genetic information (fig. 1a).

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