title: Multiple displacement amplification for complex mixtures of DNA fragments.
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.
  
  
  CONCLUSIONS
  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.
authors.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
authors.forename: Muhammad
authors.initials: M
authors.lastname: Shoaib
authors.forename: Sonia
authors.initials: S
authors.lastname: Baconnais
authors.forename: Undine
authors.initials: U
authors.lastname: Mechold
authors.forename: Eric
authors.initials: E
authors.lastname: Le Cam
authors.forename: Marc
authors.initials: M
authors.lastname: Lipinski
authors.forename: Vasily
authors.initials: V
authors.lastname: Ogryzko
mesh_terms: D047369:Chromatin Immunoprecipitation; D004247:DNA / Q000032:analysis* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*
chemical_list: D004247:DNA
references.citation: J Biol Chem. 1992 Feb 5;267(4):2594-9
references.pmid: 1733957
references.citation: Nat Genet. 1998 Jul;19(3):225-32
references.pmid: 9662393
references.citation: Genomics. 2002 Dec;80(6):691-8
references.pmid: 12523365
references.citation: Gene. 1985;33(1):103-19
references.pmid: 2985470
references.citation: Nucleic Acids Res. 2004 May 21;32(9):e76
references.pmid: 15155823
references.citation: J Mol Diagn. 2004 Aug;6(3):236-42
references.pmid: 15269301
references.citation: Nat Biotechnol. 2002 Sep;20(9):936-9
references.pmid: 12161758
references.citation: Genome Res. 1996 Oct;6(10):986-94
references.pmid: 8908518
references.citation: Proc Natl Acad Sci U S A. 2002 Apr 16;99(8):5261-6
references.pmid: 11959976
references.citation: J Biol Chem. 1993 Feb 5;268(4):2719-26
references.pmid: 8428945
references.citation: Genome Res. 2001 Jun;11(6):1095-9
references.pmid: 11381035
references.citation: Proc Natl Acad Sci U S A. 1989 Sep;86(17):6686-90
references.pmid: 2771952
references.citation: Proc Natl Acad Sci U S A. 2005 Nov 29;102(48):17332-6
references.pmid: 16286637
references.citation: Am J Pathol. 2004 Jan;164(1):23-33
references.pmid: 14695315
references.citation: Nucleic Acids Res. 2004 May 18;32(9):e71
references.pmid: 15150323
references.citation: Anal Biochem. 2004 Feb 1;325(1):68-76
references.pmid: 14715286
references.citation: J Biol Chem. 1989 May 25;264(15):8935-40
references.pmid: 2498321
references.citation: Genome Res. 2003 May;13(5):954-64
references.pmid: 12695328
references.citation: Nat Genet. 2005 Jun;37 Suppl:S11-7
references.pmid: 15920524
references.citation: Proc Natl Acad Sci U S A. 1984 Sep;81(17):5325-9
references.pmid: 6433348
references.citation: Genome Res. 2004 Nov;14(11):2357-66
references.pmid: 15520297
references.citation: Proc Natl Acad Sci U S A. 1999 Apr 13;96(8):4494-9
references.pmid: 10200290
references.citation: Am J Hum Genet. 2005 Jul;77(1):78-88
references.pmid: 15918152
references.citation: J Biol Chem. 2004 Apr 23;279(17):16895-8
references.pmid: 14988392
references.citation: Hum Mutat. 2006 Jul;27(7):603-14
references.pmid: 16786504
references.citation: Genome Res. 2003 Feb;13(2):294-307
references.pmid: 12566408