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times are sufficient to ensure practically complete hybridization of target sequences and their corresponding probe oligonucleotides. Only probes hybridized to a perfectly matched target DNA are ligated and then exponentially amplified in the following PCR (Fig. 3). MLPA probes that do not find a target sequence cannot be ligated and consequently will not be amplified in the PCR. Probes that are hybridized to a target but have a mismatch close to the ligation point are not ligated because of the high sensitivity of the thermostable Ligase-65. This allows for the discrimination of closely related sequences such as the SMN1 and SMN2 genes both involved in spinal muscular atrophy. Because the relative peak area of each amplification product reflects the relative copy number of the target sequence in the DNA sample, comparison of gel patterns with patterns obtained on a control sample allows absolute copy number quantification. To obtain reproducible results, MLPA currently needs at least 20 ng human DNA, corresponding to approximately 3000 cells or 6000 single-copy target sequences.

Specificity of the Probes

When 40 probes, each consisting of two oligonucleo-tides, are present in one tube with genomic DNA, not 40 but 1600 different target sequences are theoretically able to hybridize with two oligonucleotides and make them a substrate for ligation. However, the specificity of MLPA is very high and no nonspecific products are generated during the PCR.[8] Control experiments showed that no product was generated when the short synthetic oligonucleotides were omitted or replaced by an identical oligonucleotide with a mismatch at the 3' end.[5] Several factors contribute to exquisite specificity of MLPA: 1) the need for the two oligonucleo-tides to anneal in juxtamposition on a target nucleic acid;

Fig. 2 Detection of exon deletions in the BRCA1 gene with MLPA. One hundred nanograms of DNAs from individuals from diagnosed breast- and/or ovarian-cancer-prone families were analyzed by MLPA using probe mix P002. The P002 mix contains one probe for each of the 24 BRCA1 exons (1-24), one extra each for the exon 1 and the large exon 11 as well as several controls probes (C) specific for different genes on different chromosomes were also designed and used in a single MPLA assay together with BRCA1 probes. A detailed description of the P002 probe mix can be obtained at www.mrc-holland.com. (a) A CE pattern obtained from a control DNA sample containing two copies of the normal BRCA1 gene. (b) A CE pattern obtained from DNA with a heterozygous BRCA1 exon 13 deletion. (c) A CE pattern obtained from DNA with a heterozygous BRCA1 exon 22 deletion. (View this art in color at www.dekker.com.)

Fig. 2 Detection of exon deletions in the BRCA1 gene with MLPA. One hundred nanograms of DNAs from individuals from diagnosed breast- and/or ovarian-cancer-prone families were analyzed by MLPA using probe mix P002. The P002 mix contains one probe for each of the 24 BRCA1 exons (1-24), one extra each for the exon 1 and the large exon 11 as well as several controls probes (C) specific for different genes on different chromosomes were also designed and used in a single MPLA assay together with BRCA1 probes. A detailed description of the P002 probe mix can be obtained at www.mrc-holland.com. (a) A CE pattern obtained from a control DNA sample containing two copies of the normal BRCA1 gene. (b) A CE pattern obtained from DNA with a heterozygous BRCA1 exon 13 deletion. (c) A CE pattern obtained from DNA with a heterozygous BRCA1 exon 22 deletion. (View this art in color at www.dekker.com.)

2) hybridization and ligation are performed at 54-60°C, which reduces unspecific binding and increases specificity;

3) the use of thermophilic ligases requiring NAD (e.g., Ligase-65) that are very sensitive to mismatches next to the ligation site.

Probe Signal and Reproducibility

Twenty-eight probes specific for sequences on chromosomes X, 21, 18, and 13 generated, on average, a 1.44-fold higher signal on the respective trisomy DNA samples as compared with control DNA.[8] This is close to the theoretical 1.5-fold increase. Reproducibility of results in MLPA depends on several factors: 1) the purity of DNA samples, the method of PCR product analysis with capillary electrophoresis being superior to fluorescent detection on slab gels; 2) the software used for peak detection and the method used for normalization of data. Whereas DNA degradation hardly influences the results, the presence of PCR inhibitors, e.g., traces of phenol, does. In general, the standard deviation of results obtained in MLPA varies between 4% and 10% for each probe.

In MLPA the relative signal strength depends on the relative amount of the target sequences present in the sample. In model experiments, it was shown that an almost linear increase of the signal could be obtained when up to fivefold increase of synthetic targets was added to a DNA sample.[8] Addition of higher amounts of synthetic target resulted in an additional nonlinear increase in signal of that probe.

Probe design

Synthetic oligonucleotide 1»

M13 derived oligonucleotide

Primer X Target specific sequence, Stuffer sequence Primer Y

Multiplex hybridization and ligation

No ligation of mismatched

Target 1

Target 1

Target 2

PCR with universal primers X and Y

^ No exponential amplification of nonligated probes

Fragment Analysis

Fig. 3 Outline of MLPA. 1. Probe design: Each MLPA probe consists of two oligonucleotides, one short synthetic and one long M13 derived. The short synthetic oligonucleotide consists of a 5' universal primer sequence X and a target-specific 3' sequence. The M13-derived oligonucleotide consists of a 5' target-specific sequence designed to hybridize in juxtaposition to the synthetic probe, a 3' universal primer sequence Y and a stuffer sequence that has a different length for each probe. 2. Multiplex hybridization and ligation: One femtomole of each probe oligonucleotide is hybridized to the denatured target DNA. Only perfectly matched probes oligonucleotides are ligated by the thermostable Ligase-65. 3. PCR: Only ligated probes are exponentially amplified by the single primer pair X and Y. 4. Fragment analysis: The amplified fragments are separated and analyzed by capillary electrophoresis.

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