Biomedical Engineering Reference
In-Depth Information
The resolution of what is now called classical CGH is limited to a chromosomal band,
approximately 5-10Mb. This was overcome by the introduction of array comparative
genomic hybridization (aCGH) in 1997 [4, 5]. The method is essentially the same, but
now an array of genomic DNA clones or oligonucleotides serves as hybridization target,
rather than metaphase chromosomes. The resolution of aCGH is now defined by the choice
and/or the number of DNA clones and later oligonucleotides, and another advantage is that
it does not require karyotyping. At the present moment, aCGH using oligonucleotide or
single nucleotide polymorphism (SNP) arrays is most widely applied [6].
Multiple ligation-dependent probe amplification (MLPA), developed and first published
in 2002 by Schouten
et al
. [7], is an alternative DNA copy number analysis technique,
especially when specific genes or chromosomal regions are already known to be of interest.
MLPA requires only 20 ng of DNA, enough to allow the simultaneous quantification of up
to 50 different targets, which may be as small as 50 nucleotides. Another advantage of
MLPA lies in its reproducibility and specificity, allowing application in a routine diagnostic
setting while remaining time- and cost-efficient.
One increasingly important application is genomic profiling of FFPE samples. Across the
world, large collections of FFPE samples with clinical follow-up exist. However, the DNA
from FFPE samples shows varying levels of degradation depending largely on the length and
the method of fixation, and on age of the specimen. This chapter aims to describe in detail
the methods of oligonucleotide aCGH, SNP aCGH and MLPA, with special attention for
the use of DNA obtained from FFPE samples. These techniques have primarily been used
in cancer research; however, they are also suitable for the analysis of DNA copy number
aberrations in human genetic disorders.
1.2 Methods and approaches
1.2.1 Oligonucleotide aCGH
The first whole-genome microarray contained 2400 large-insert genomic clones, primarily
bacterial artificial chromosomes (BACs) [8]. With the total human genome covering about
3000Mb the resolution of this array is on average close to 1Mb, which is about one order of
magnitude higher than that obtained with classical CGH [1]. For a full coverage resolution,
about 30 000 BACs have been arrayed [9], increasing the resolution by another order of
magnitude. However, producing such large numbers of BACs for aCGH is expensive and
time consuming; and due to the large size of the BACs, the resolution limits for BAC aCGH
resolution were reached.
Several laboratories used cDNA arrays, initially designed for expression profiling, as
an alternative for measuring chromosomal copy number changes [10]. Even though this
approach has certainly yielded valuable information, it cannot compete with the oligonu-
cleotide platforms in terms of its maximal achievable resolution. Oligonucleotides allow a
sheer infinite resolution, great flexibility and are cost effective [6]. They also enable the
generation of microarrays for any organism for which the genome has been sequenced.
Using the same oligonucleotide array for CGH and expression profiling allows direct com-
parison of mRNA expression and DNA copy number ratios. In addition, oligonucleotide
arrays are being used, designed and accepted for expression profiling, and thus are widely
available.
