Novel genetic and epigenetic alterations in ... - Ous-research.no

Discussionserve as positive controls. Also, in the case of NF1, the exon probes were divided in twoseparate probe mixes (exon 1, 3, 5 etc. is in one probe mix and exon 2, 4, 6 etc. is in theother). In order to analyze the whole gene, the use of both probe sets will make thequantification of each exon independent from its adjacent exon. In that way there are twoexperiments that will confirm a whole gene- or a multi exon loss/gain, hence, the two probesets function as internal controls.Any factors that may interfere in probe binding will lower MLPA success. Point mutationsor indels at, or close to, the ligation site significantly causes reduced probe-sequence affinity.This may cause reduced levels of ligated and amplified product, and the probe to be scoredas deleted[159]. In our case this problem was surpassed as we performed mutation analysesin parallel, ensuring that all probes scored as deleted did not contain such aberrations in theproximity to the primers. Striving to have as equal hybridization and amplification efficiencyas possible is important as a more effective amplification of one probe may cause it to bemistakenly scored as a gain. Size is also an issue with MLPA as the fragments are separatedby this. Theoretically, as smaller fragments are amplified more efficiently than larger, it mightseem like a bad choice to use in a multiplex setup. However, if this was a general problemone would assume that the longest probes would be reported as lost in the majority of thecases. In the analysis of NF1, we did not see this effect as longer fragments had similarquantities as the smaller ones. As with all quantitative PCR assays, it is important to measurethe product while it is in the linear phase, in which the amount of the products areproportional to the copy number of the sequences. Optimal probe design, PCR-setup as wellas control regions are all taken care of when using a commercially available kit.The advantage of MLPA compared to methods such as fluorescent in situ hybridization orarray-CGH is the fact that it is far less expensive, less labor intensive, and therefore quicker.Overall, MLPA has proved to be a sensitive, time- and cost-efficient method to detect copynumber changes for a wide variety of genes[160-162].53