Multiplex Ligation-Dependent Probe Amplification

Abstract

Genetic aberrations in tumors are predictive for chemosensitivity and survival. A test is needed that allows simultaneous detection of multiple changes and that is widely applicable in a routine diagnostic setting. Multiplex ligation-dependent probe amplification (MLPA) allows detection of DNA copy number changes of up to 45 loci in one relatively simple, semiquantitative polymerase chain reaction-based assay. To assess the applicability of MLPA, we performed MLPA analysis to detect relevant genetic markers in a spectrum of 88 gliomas. The vast majority of these tumors (n ‫؍‬ 79) were previously characterized by comparative genomic hybridization. With MLPA kit P088 (78 cases), complete and partial loss of 1p and 19q were reliably identified, even in samples containing only 50% tumor DNA. Distinct 1p deletions exist with different clinically prognostic consequences, and in contrast to the commonly used diagnostic strategies (loss of heterozygosity or fluorescent in situ hybridization 1p36), P088 allows detection of such distinct 1p losses. Combining P088 with P105 will further increase the accurate prediction of clinical behavior because this kit identified markers (EGFR , PTEN , and CDKN2A) of high-grade malignancy in 41 cases analyzed. We conclude that MLPA is a reliable diagnostic tool for simultaneous identification of different region-specific genetic aberrations of tumors.

Diagnostyka rearanżacji genu SHOX u kobiet 46,XX z idiopatyczną niskorosłością

“…Tests that enable the identification of selected chromosome aberrations by other methods are increasingly common: FISH (fluorescence in situ hybridization), QF-PCR (quantitative fluorescence polymerase chain reaction), and MLPA (multiplex ligation-dependent probe amplification) [20][21][22][23][24][25][26][27][28][29][30][31][32]. These methods, having comparable efficacy and diagnostic reliability, making it possible to diagnose SHOX gene rearrangements [33].…”

“…Studies employing comparative genomic hybridisation (CGH) are expensive but also are more often used, allowing for simultaneous evaluation of tens of thousands of sequences. In our conditions, with the results of cytogenetic study, excluding the presence of structural aberrations on the Xp arm, the diagnosis of which could be possible at the level of a single chromosome band, we decided to apply the MLPA technique [20][21][22].…”

New applications and developments in the use of multiplex ligation‐dependent probe amplification

“…It has been shown that normal DNA contamination reduces the amplitude of cancer-specific copy number variations [58] and thus makes their detection more difficult. A titration experiment showed that 50% contamination with normal cells is the limit at which loss of one allele can be still reliably detected by MLPA [64].…”

“…Although a successful MLPA analysis of paraffin DNA specimens has been reported, difficulties are also recognized [16,49,64]. Success may depend on the amount of DNA that is extracted from the paraffin, fixative used and period of fixation, the age of the block, the degree of contamination with normal cells, and other factors.…”

Satisfying your neuro-oncologist: a fast approach to routine molecular glioma diagnostics

“…Typically, 125 ng DNA were used for MLPA 3 performed on a robotic thermocycler system (RoboAmp 4200, ALS Jena) using oil seals (LCS, Ventana). MLPA reaction products were examined by capillary gel electrophoresis (ABI GA 3500) and quantified by Coffalyser (MRC Holland).…”