Multiplex Ligation-Dependent Probe Amplification (MLPA)
Multiplex ligation-dependent probe amplification (MLPA) is a method based on multiplex PCR that can amplify multiple target sequences with only one pair of primers. MLPA can detect copy number variations (CNVs) in the genome and analyze them with software. MLPA is an important diagnostic tool used in clinical pathology laboratories as well as in research fields such as genetic diseases and tumors. MLPA was invented to overcome the resolution and specificity limitations of traditional PCR methods in multiplex amplification. The principle of MLPA is to use two probes to hybridize to adjacent sites of the target sequence and then link the probes into a fragment that can be amplified by PCR. Each pair of probes has a specific length, which allows them to be separated by electrophoresis. By comparing the peak patterns of the sample and the reference sample, the relative quantity of the target sequence in the sample can be calculated. The advantages of MLPA are that it has high throughput, high sensitivity, high specificity, low cost, and easy operation. It can detect copy number changes from whole chromosomes to single exons. MLPA can also detect DNA methylation changes (MS-MLPA) and distinguish disease-related genes from highly similar pseudogenes.
Principles and steps of MLPA
The basic principle of MLPA is to use two probes to hybridize to adjacent sites of the target sequence and then link the probes into a fragment that can be amplified by PCR. Each pair of probes has a specific length, which allows them to be separated by electrophoresis. By comparing the peak patterns of the sample and the reference sample, the relative quantity of the target sequence in the sample can be calculated. MLPA can detect copy number changes from whole chromosomes to single exons, as well as DNA methylation changes, and distinguish disease-related genes from highly similar pseudogenes.
Fig.1 The principle of MLPA (Seidman, 2016)
The experimental operation steps of MLPA include sample preparation, reaction system, reaction conditions, electrophoresis detection, and data processing. Sample preparation mainly involves extracting and quantifying DNA and diluting DNA to an appropriate concentration. The reaction system is to mix DNA with MLPA probes and add a salt solution and water. The reaction conditions are divided into three parts: (a) DNA denaturation and probe hybridization; (b) ligation reaction; and © PCR amplification. Electrophoresis detection is to separate and quantify the PCR products by capillary electrophoresis and obtain peak patterns of different lengths. Data processing is done using Coffalyser. Net software for quality control and result analysis, and calculate the relative quantity of the target sequence in the sample.
Characteristics of MLPA
The main characteristics of MLPA are that it has high throughput, high sensitivity, high specificity, low cost, and easy operation. It can detect copy number changes from whole chromosomes to single exons, as well as DNA methylation changes, and distinguish disease-related genes from highly similar pseudogenes. MLPA and other similar methods, such as FISH, qPCR, Southern blotting, SNP array, and CGH array, have their own advantages and disadvantages and can be selected according to different purposes and conditions.
Table 1. Comparison of MLPA and other methods
| Method | Advantages | Disadvantages |
|---|---|---|
| MLPA | High throughput, high sensitivity, high specificity, low cost, easy operation, and the ability to detect various variations | Need to design specific probes, cannot detect unknown variations, cannot locate variation positions |
| FISH | The ability to directly observe cell karyotype, locate variation positions, and detect unknown variations | Low throughput, low sensitivity, high cost, complex operation, need for fluorescence microscope |
| qPCR | High sensitivity, high specificity, low cost, easy operation | Low throughput, need to design specific primers, inability to detect unknown variations or locate variation positions |
| Southern blotting | The ability to detect unknown variations | Low throughput, low sensitivity, high cost, complex operation, and large amount of DNA consumption |
| SNP array | High throughput, and the ability to detect copy number changes and single nucleotide polymorphisms | High cost, complex operation, cannot detect methylation changes and pseudogenes |
| CGH array | High throughput, and the ability to detect copy number changes and unknown variations | High cost, complex operation, cannot detect methylation changes and pseudogenes |
Applications of MLPA
MLPA has a wide range of applications in genetic disease diagnosis and can detect copy number variations that cause genetic diseases such as Duchenne muscular dystrophy, neurofibromatosis, Williams syndrome, Prader-Willi syndrome, Down syndrome, etc. MLPA can also detect DNA methylation changes, such as Prader-Willi syndrome, Angelman syndrome, Rett syndrome, etc. MLPA can also distinguish disease-related genes from highly similar pseudogenes, such as CFTR gene and the CFTR pseudogene.
MLPA also has an important application in tumor molecular typing and can detect copy number variations in tumors such as breast cancer, colon cancer, lung cancer, neuroblastoma, etc. MLPA can also detect microsatellite instability (MSI) in tumors, such as colorectal cancer, endometrial cancer, etc. MLPA can also detect chromosomal translocations in tumors, such as chronic myeloid leukemia (BCR-ABL1 translocation), follicular lymphoma (BCL2-IgH translocation), etc.
References
- Schouten JP, et al. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res. 2002 Jun 15;30(12):e57. 1
- Chaitanya KV. Diagnostics and Gene Therapy for Human Genetic Disorders. CRC Press; 2022. 2
- van Eijk R, et al. MLPAinter for MLPA interpretation: an integrated approach for the analysis, visualisation and data management of Multiplex Ligation-dependent Probe Amplification. BMC Bioinformatics. 2010 Jan 29;11:67. 10
- Eid OM, et al. Evaluation of MLPA as a comprehensive molecular cytogenetic tool to detect cytogenetic markers of chronic lymphocytic leukemia in Egyptian patients. J Genet Eng Biotechnol. 2021 Jun 28;19(1):98. 11
- El-Sayed ZA, et al. MLPA as a genetic assay for the prenatal diagnosis of aneuploidy in Egypt by its validation compared to the FISH technique. J Genet Eng Biotechnol. 2022 Jan 13;20(1):4. 12
- Stuppia L, et al. Use of the MLPA assay in the molecular diagnosis of gene copy number alterations in human genetic diseases. Int J Mol Sci. 2012 Mar 15;13(3):3245-76. 13
- Nygren AO, et al. Methylation-specific MLPA (MS-MLPA): simultaneous detection of CpG methylation and copy number changes of up to 40 sequences. Nucleic Acids Res. 2005 Aug 4;33(14):e128. 14
- Nygren AO, et al. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) for detection of promoter hypermethylation in well-defined DNA sequences in tumors: possibilities and pitfalls in diagnostics and follow-up of treatment in oncology. Expert Rev Mol Diagn. 2007 May;7(3):259-69. 15
- Seidman, Michael A., and Richard N. Mitchell. "Fundamental principles in cardiovascular genetics." Cardiovascular Pathology. Academic Press, 2016. 213-237.
