Multiplex amplifiable probe hybridization (MAPH)

Abstract

Changes in DNA copy number can result in a wide range of pathological conditions such as mental retardation, malformations, developmental delay, neurological disorders and congenital anomalies. These copy number changes may be large or small and they may include the entire or segments of a chromosome. Conventional chromosomal analysis (karyotyping) allows detection of chromosomal aberrations larger than 5 Mb, however smaller aberrations are not detectable with the existing G-banding analysis. Microdeletion syndromes, subtle deletions and complex rearrangements in the subtelomeric regions are typical examples of small abnormalities that cause clinical phenotypes but are not visible by routine cytogenetics. Such small imbalances, called "submicroscopic," can only be detected using other methods that provide high-resolution analysis. Several methodologies have been developed to detect these submicroscopic genetic imbalances. The introduction of fluorescence in situ hybridization (FISH) led to the identification of cryptic chromosomal imbalances that were undetectable with high-resolution G-banding chromosomal analysis. Examples include detection of most of the known microdeletion syndromes, microduplications, interstitial segment imbalances and subtelomeric rearrangements (1-5), using locus-specific or subtelomeric specific FISH probes. Although FISH methodology provided significant improvements in the field of cytogenetics, it does not offer an alternative approach to G-banded chromosomal analysis, as it is a targeted method, which does not provide genome-wide analysis. DNA-based methods such as quantitative PCR or real time PCR can also be applied to determine small copy number changes (6,7). These are highresolution methods but are also targeted to specific known loci. Simultaneous analysis of multiple loci is technically challenging and it is therefore difficult to screen large genomic regions and identify new genes. Until now, the only alternative method to chromosomal analysis, for genome wide screening was comparative genomic hybridization (CGH). Conventional CGH analysis uses metaphases and permits the detection ofchromosomal deletions or duplications as small as 3 Mb (8). The advancement from metaphase chromosome-based CGH to microarray based-CGH using BAC and PAC clones has increased the resolution to higher than 1 Mb (9,10). Microarray-based CGH has been performed successfully to screen total genome to identify subtle abnormalities in cancer samples (11), mental retardation (12). The sensitivity and specificity of array-CGH was further increased with recent improvements and modifications, such as tiling resolution BAC arrays covering the entire human genome with approximately one clone per 100 kb (13), hybridization of genomic representations to CGH arrays (14), arraying long oligonucleotide probes (15), Affymetrix SNP arrays (16,17) and other commercial solutions with extreme resolution (18). Array-CGH has been proven to be very sensitive and is rapidly becoming the method of choice for high-resolution genome-wide screening to detect known and unknown copynumber changes. A new DNA-based method for the reliable determination of locus copy number in complex genomes is multiplex amplifiable probe hybridization (MAPH) (19). This method relies on the fact that probes can be quantitatively recovered and amplified after hybridization to genomic DNA template. MAPH uses probes that can be specifically designed for any locus, telomere, chromosomal segment, whole chromosome or the total human genome at extremely high resolution, and enable the sensitive detection of loss or gain of genomic DNA sequences as small as 150 bp. However gel-based detection places a limit to the multiplicity of MAPH, as in most cases only 30-50 probes per assay are included. The latest advancement in MAPH methodology is the introduction of microarrays, resulting in the development of microarray-based MAPH (array- MAPH) (20,21). This new method combines the high specificity and sensitivity of MAPH with the high multiplicity of microarray format. Array-MAPH could be applied as a potential alternative to array-CGH, offering several advantages for the detection of copy number changes in the human genome. © 2008 Humana Press.