Resolution: BAC vs. Oligo Arrays
The first set of variables encountered in array CGH platforms concerns the selection, design, and production of the array platform. Broadly speaking, these are all a function of the specific information required from the test. One of the first considerations in aCGH is whether or not the mutation is in a known mutation or not and, if so, what size of aberration it represents.
“Choosing the DNA source and microarray type depends on the chromosomal changes and resolution that are to be detected by the assay,” said Todd Martinsky, executive vp, TeleChem International(www.arrayit.com). Telechem provides a variety of technical solutions to developing custom array-based clinical tests. “Our NanoPrint microarrayer, SpotLight scanner, surface chemistries, and other sample preparation products play a key role in any microarray experimental lifecycle.”
Early low-resolution approaches using fixed chromosomes have been superseded by BAC (Bacterial Artificial Chromosome)-based arrays, which perform a low-resolution interrogation of the test sample and are typically deployed to detect large-scale gross genomic rearrangements, deletions, insertions, or amplifications. A further refinement of the technique, oligonucleotide-based arrays, can detect smaller mutations in the test sample and offer the highest resolution in aCGH.
“With oligonucleotides, it is possible to tile across a gene at specific lengths at a defined resolution,” said Martinsky. This type of custom design affords a greater degree of resolution than can be obtained by spotting larger, less well-defined sequences of DNA.
“Oligonucleotide arrays are typically used when you don’t see gross deletions with BAC arrays or FISH,” said Henry Sadowski, Ph.D., senior scientist and director of product applications, Enzo Life Sciences (www.enzo.com).
Besides the resolution factor, another distinct advantage of aCGH is the speed at which custom chips can be designed, manufactured, and delivered. “CombiMatrix offers a range of custom array design and manufacturing services,” said Dr. Mohammed. “In some cases the turnaround on these arrays can be overnight.”
The type of resolution needed in the test dictates many of the technical parameters required for the success of the application. “If the assay is designed to discover new chromosomal disease associations, higher density microarrays are required,” said Martinsky. “These require more expensive microarray detection systems because of the required resolution.”
Conversely, microarray platforms designed to detect specific known mutations are typically of lower density and require less sophisticated detection systems. “With fewer than 2,000 BACs spotted into a microarray, it is possible to detect more than 70 recognized microdeletion or microduplication syndromes,” Martinsky pointed out.
The enormous logistical advantages that aCGH provides over traditional cytogenetic diagnostics can be appreciated by considering the situation that occurs when infants present at birth or in early childhood development with defined mental or physical defects, which physicians use as the basis for the tentative diagnosis of a specific syndrome.
In order to make a specific diagnosis they typically order a series of genetic tests. Traditionally, these would have taken the form of serial tests using some form of in situ hybridization in order to identify or rule out a known large-scale genetic change.
“In practical terms this can mean that using FISH, a clinician may have to spend thousands and thousands of dollars and many weeks to iteratively look at a small number of disorders and at the end may or may not have identified an abnormality,” said Peter J. Welch, Ph.D., director of gene-expression profiling R&D at Invitrogen (www.invitrogen.com). The advent of array technology, however, greatly increases the options open to clinicians seeking a diagnosis.
“Arrays offer two dramatic improvements over FISH-based CGH analysis,” said Dr. Welch. “Much finer resolution interrogation of the genome is possible with the use of specific probes on the arrays targeted to smaller regions of the genome.” The second area of improvement, Dr. Welch pointed out, is the ability of an array CGH platform to interrogate multiple regions of the genome at a time. “Array CGH converts a serial methodology to a parallel methodology,” Dr. Mohammed observed.