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Advantages of MLPA

Using MLPA for copy number detection offers many advantages over other techniques. First of all, methods which were primarily developed for detecting point mutations, such as sequencing and DHPLC, generally fail to detect copy numbers changes. Southern blot analysis, on the other hand, will reveal many aberrations but will not always detect small deletions and is not ideal as a routine technique. Although well-characterised deletions and amplifications can be detected by PCR, the exact breakpoint site of most deletions is unknown. Furthermore, when comparing MLPA to FISH, MLPA not only has the advantage of being a multiplex technique, but also one in which very small (50-70 nt) sequences are targeted, enabling MLPA to identify the frequent, single gene aberrations which are too small to be detected by FISH. Moreover, MLPA can be used on purified DNA. Finally, as compared to array CGH, MLPA is a low cost and technically uncomplicated method. Although MLPA is not suitable for genome-wide research screening, it is a good alternative to array-based techniques for many routine applications. The over 300 probe sets now commercially available are dedicated to applications ranging from the relatively common (Duchenne, DiGeorge syndrome, SMA) to the very rare (hereditary pancreatitis, Antithrombin deficiency, Birt-Hogg-Dube syndrome).

MLPA reaction

Typical for MLPA is that it is not target sequences that are amplified, but MLPA probes that hybridise to the target sequence. In contrast to a standard multiplex PCR, a single pair PCR primers is used for MLPA amplification. The resulting amplification products of a SALSA MLPA kits range between 130 and 480 nt in length and can be analysed by capillary electrophoresis. Comparing the peak pattern obtained to that of reference samples indicates which sequences show aberrant copy numbers.

The MLPA reaction can be divided in five major steps: 1) DNA denaturation and hybridisation of MLPA probes; 2) ligation reaction; 3) PCR reaction; 4) separation of amplification products by electrophoresis; and 5) data analysis (Figure 1). During the first step, the DNA is denatured and incubated overnight with a mixture of MLPA probes. MLPA probes consist of two separate oligonucleotides, each containing one of the PCR primer sequences. The two probe oligonucleotides hybridise to immediately adjacent target sequences (Figure 1 - step 1). Only when the two probe oligonucleotides are both hybridised to their adjacent targets can they be ligated during the ligation reaction (Figure 1 - step 2). Because only ligated probes will be exponentially amplified during the subsequent PCR reaction (Figure 1 - step 3), the number of probe ligation products is a measure for the number of target sequences in the sample. The amplification products are separated using capillary electrophoresis (Figure 1 - step 4). Probe oligonucleotides that are not ligated only contain one primer sequence. As a consequence, they cannot be amplified exponentially and will not generate a signal. The removal of unbound probes is therefore unnecessary in MLPA and makes the MLPA method easy to perform.

MLPA variations

A few variations on MLPA have been developed. RT-MLPA (Reverse Transcriptase MLPA) can be used for mRNA profiling (11). As the ligase enzyme cannot ligate probes which are bound to RNA, the RT-MLPA procedure starts with the reverse transcription of mRNA into cDNA. After this, RT-MLPA continuous as a normal MLPA reaction. Another variation is Methylation-Specific MLPA (MS-MLPA), which can be used for both copy number quantification and methylation profiling (12). MS-MLPA has proven to be a very useful method for the detection of imprinting diseases (13-15) and for the analysis of methylation aberrations in tumour samples (16, 17).

SALSA MLPA kits for a rapidly growing number of applications are available from MRC-Holland (Amsterdam, the Netherlands).

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