USE WHOLE NUCLEIC ACID AMPLIFICATION FOR COMPLEX CLINICAL SAMPES AND GO

BACK TO GOOD (OLD) SOUTHERN RESOLUTION

Use author’s names as “personal communication” option in reference list

Brukner I 1, Damian L2, Dascal, A1. and Krajinovic, M.2

(IBRUKNER@JGH.MCGILL.CA)

ibrukner@gmail.com

+1 514 8038782 for further developments

1.Sir Mortimer B. Davis–Jewish General Hospital, 3755 Cote-Ste-Catherine, Montréal (QC), H3T 1E2, Canada.

2.Centre de Recherche, Hopital Sainte-Justine and Département de Pédiatrie, Université de Montréal, Montréal, QC, Canada.

Recently, Yilmas et al. (1) nicely illustrated a limited applicability of the whole genome amplification (WGA)
method (2) using Molecular Displacement Amplification (MDA) for/in complex microbial community samples.
They showed clear post-WGA bias in relative genome coverage of the community members and recommended that
until the source of this bias is understood and can be corrected, no quantitative inferences should be made from
comparison of MDA-amplified environmental nucleic acids. We agree that all tested commercial assays do not offer
adequate solution, yet already in 2005-2006 we developed, validated and published two WGA protocols eliminating
such bias (3,4 ). We wish to thank Yilmas et al., for raising these important methodological problems which can be
at least partly circumvented following our protocols (3,4).
The MDA bias during amplification of complex mixture of small genomes is largely due to the length heterogeneity
of these templates. For example, 300 human genome copies could be WGA-amplified without any evidence of bias,
but the same number of small plasmid DNA molecules – cannot be amplified at all. Amplification experiments with
the degraded samples and with the same samples previously ligated to create concatenated elongated templates (5 )
indicated that high quality WGA product could only be obtained with ligated templates. So, common sense strategy
was to apply ligation of degraded DNA or DNA composed of short fragments to increase the initial length of small
WGA templates (5 ). If subsequent applications do not require the preservation of the original linear order of
amplified sequence fragments, as in the case of SNP detection or short PCR diagnostics, than this procedure usually
helps. However, if the initial concentration of DNA fragments is bellow Kd of the DNA ligase (which may be the
case when genomic samples are of limited quantity) the ligation is very inefficient or does not occur at all ( ). In
such a case, ligation efficacy of the low concentration target DNA can be increased by addition of appropriate
concentration of a DNA linker consisting of polydA-dT homopolymer, inapproximate average length 8778 bp ( ).
This solution was shown to be surprisingly efficient; WGA was able to amplify 10-100 molecules of 3000bp long
linear plasmid DNA. In other words introducing high concentration of a long linker DNA renders ligation more
efficient leading to “splicing in” of the analyzed DNA fragments into a long template –thus rendering their
amplification by WGA protocol efficient and presumably independent on the DNA lengths in the sample, thanks to
the extraordinary processivity (80 000bp) of the Phi29 enzyme. We hope that here described solutions, will help to
extend the applicability of WGA protocols in research and molecular diagnostics.

The authors claim no commercial issues

1. Yilmaz, S., Allgaier, M & Hugenholtz, P. Nature Methods. 7, 944 (2010).
2. Lasken, R.S. Biochem. Soc. Trans. 37, 450–453 (2009).
3. Brukner, I., B. Paquin, M. Belouchi, D. Labuda, M. Krajinovic , Anal. Biochem. 339 345–347. (2005).
4.. Brukner, I., Labuda, D. and Krajinovic, M. Anal. Biochem., 354 154–156, (2006)
5. S. Panelli, G. Damiani, L. Espen, V. Sgaramella, BioTechniques 39 174–180. (2005)