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Rapid detection of genomic,,,duplications and deletions using,,,the Agilent 2100 bioanalyzer

Duplications and deletions are known to cause a wide variety of human diseases. Many different methods are used for detecting such rearrangements, most based on either FISH or the quantitative analysis of PCR products. FISH is popular and accurate, but very labour intensive and expensive, while quantitative PCR methods are technically demanding and difficult to design. Recently two related methods (MAPH and MLPA) have been developed and applied to different disorders. Several methods for the analysis of the products were used, using a labeled primer and either gel or capillary electrophoresis. This Application Note describes the Agilent 2100 bioanalyzer in combination with the DNA 500 chip for the detection of copy number changes within the genome using MAPH and MLPA. The speed, ease of data analysis and potential for automation make this an attractive alternative for conventional methods in a diagnostic setting.
Alterations in genomic DNA can be broadly divided into 3 main classes: (i) qualitative changes, where the DNA sequence is altered, (ii) changes of order e.g. translocations and inversions, and (iii) quantitative changes, involving the deletion or duplication of a stretch of DNA. Sequencing has long been the gold standard for qualitative changes, and fluorescent in situ hybridization (FISH) and pulsed-field gel electrophoresis (PFGE) are the most widely applied methods for detecting the reordering of genomic segments. Until recently, the 3 methods predominantly used for quantitative analysis were Southern blotting, quantitative or breakpoint-specific PCR, and FISH. Southern blotting is time-consuming and laborious, and it is especially difficult to detect duplications. Quantitative PCR allows the multiplexing of up to 15 products in a single reaction,
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