Multiplex ligation-dependent probe amplification

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MLPA in GeneMarker

Multiplex Ligation-dependent Probe Amplification (MLPA) is a molecular biology technique for the detection of genetic variations, such as copy number variations (CNVs), mutations, and single nucleotide polymorphisms (SNPs) within genes. Developed in the early 2000s, MLPA has become a vital tool in genetic testing and research, offering a relatively quick and cost-effective method for analyzing multiple targets in a single reaction.

Overview

MLPA utilizes a set of two oligonucleotide probes for each target sequence. Each probe consists of two parts: one that is complementary to the target sequence and another that contains a universal primer sequence. When these probes hybridize to their respective target sequences in a sample DNA, they are ligated together by a ligase enzyme. This ligation event only occurs if the probes are perfectly annealed next to each other on the target DNA, ensuring specificity. The ligated probes are then amplified using PCR with fluorescently labeled universal primers. The resulting amplicons are analyzed by capillary electrophoresis, allowing for the quantification of the target sequences.

Applications

MLPA is widely used in clinical diagnostics and research for a variety of applications, including:

• Detection of gene deletions or duplications, common in genetic disorders such as Duchenne Muscular Dystrophy and Charcot-Marie-Tooth disease.
• Analysis of gene copy number in cancer genetics, to identify gains or losses of oncogenes and tumor suppressor genes.
• Screening for known mutations in a panel of genes associated with a particular disease.
• Methylation analysis for the study of epigenetics and imprinting disorders.

Advantages and Limitations

The main advantage of MLPA over other techniques, such as quantitative PCR (qPCR) or microarray analysis, is its ability to multiplex, or simultaneously analyze, up to 50 different DNA sequences in a single reaction, making it highly efficient and cost-effective. Additionally, MLPA does not require large amounts of DNA and can be performed on fixed tissues, including formalin-fixed, paraffin-embedded (FFPE) samples.

However, MLPA has some limitations. It cannot detect point mutations or small insertions/deletions that do not alter the probe binding sites. The technique also requires careful design and validation of probes for each target sequence, which can be time-consuming.

Procedure

The MLPA procedure involves several key steps: 1. DNA extraction and quantification: Sample DNA is extracted from the source material and quantified. 2. Probe hybridization: MLPA probes are mixed with the DNA sample and hybridized under specific conditions. 3. Ligation: If probes are correctly hybridized next to each other on the target DNA, they are ligated together. 4. PCR amplification: The ligated probes are amplified using PCR. 5. Capillary electrophoresis: The amplified products are separated and quantified using capillary electrophoresis.

Conclusion

MLPA is a powerful tool for genetic analysis, offering a unique combination of multiplexing capability, sensitivity, and specificity. Its applications in clinical diagnostics and research continue to expand as new probes and protocols are developed. Despite its limitations, MLPA fills a critical niche in the genetic testing landscape, providing valuable information for the diagnosis, prognosis, and study of genetic disorders and diseases.

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