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Multiple displacement amplification-based comprehensive
genome amplification in fresh-frozen and formalin-fixed/paraffin-embedded
clinical brain tumor samples
Markus Bredel, Claudia Bredel, Hannes Vogel, Griffith Harsh IV, Branimir
I. Sikic
Stanford University, Stanford, CA. E-mail:
mbredel@stanford.edu
Adequate quality and quantity of genomic DNA is a bottleneck in genetic analysis
of clinical tumor samples.
In brain tumor patients, most often only tiny amounts
of tissue are available from stereotactic needle biopsies.
The yield of genomic
template from these specimens by common methods of DNA isolation does frequently
not allow high-throughput genetic analysis in these patients, which however may
be important for treatment stratification in targeted therapy.
Much effort has
been invested in developing methods for whole genome amplification (WGA).
Substantial variation in the extent of amplification occurring between different
markers, incomplete coverage, and inadequate average DNA size has limited the
use of existing WGA methods, making them particularly unsuitable for diagnostic
testing.
Most recently, a novel technique for WGA has been described, termed
multiple displacement amplification (MDA), which provides a highly uniform
representation across the human genome.
MDA employs the Phi29 DNA polymerase and
random primers to generate high-molecular weight DNA.
The utility of MDA-based
WGA has been shown for plasmid and bacteriophage DNA as well as whole blood,
buccal swabs, buffy coats, and cultured cell lines.
Here we have extended the
use of MDA to the WGA of fresh-frozen (FF) as well as
formalin-fixed/paraffin-embedded (FP) clinical solid brain tumor samples.
Genomic DNA was isolated from snap-frozen glioblastoma specimens and
corresponding archival formalin-fixed/ paraffin-embedded specimens and varying
amounts of genomic DNA were subjected to overnight MDA.
We demonstrate that MDA
of nanograms of input template DNA generated microgram amounts of high-fidelity
output DNA.
Three nanograms of FF and FP input DNA resulted in 1610 ± 96-fold
and 1591 ± 72-fold WGA, respectively, demonstrating that the yield of genomic
DNA is comparable for the same amounts of input DNA from FF and FP samples.
When
the DNA input was increased, amplification efficiency as measured by the
fold-change of input to output DNA decreased steadily, suggesting saturation of
either the enzyme or the primers.
Fifty nanograms of DNA input generated only
110 ± 1-fold and 81 ± 7-fold amplification of DNA template from FF and FP
tissue, respectively.
Our results suggest that MDA-based WGA may be an
invaluable tool for DNA probe preparation for genotyping and DNA sequencing
analysis from limited patient DNA sources such as fresh-frozen stereotactic
biopsy material of brain tumors as well as archival
formalin-fixed/paraffin-embedded, and laser microdissected brain tumor tissues.
We will further report on the suitability of MDA-based WGA in quantitative
assessment of genome-wide gene dosages, utilizing comparative genomic
hybridization via 42,000-element cDNA microarrays (array-CGH) and real-time PCR.
Copyright © 2004 American Association for Cancer Research. All rights
reserved.
Source: http://aacr04.agora.com/planner/displayabstract.asp?presentationid=6795
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