MLPA

What is the MLPA technique?

Changes in the copy number of specific chromosomal sequences, as well as the occurrence of deletions and duplications, demonstrate the presence of pathogenic mutations that contribute to a variety of genetic syndromes and diseases. There exist multiple methodologies to identify and diagnose these mutations and chromosomal disorders. One of the most widely utilized techniques is the MLPA molecular method. Multiplex ligation-dependent probe amplification (MLPA), is a molecular detection method proves to be both sensitive and effective in genetic diagnosis and pathobiology laboratories. Furthermore, it has proven valuable in research endeavors focused on the identification of gene deletions and duplications.

The MLPA method is a modified version of Multiplex PCR, wherein a maximum of 50 gene regions are amplified using a pair of primers. This allows for the identification and differentiation of sequences that differ by a single nucleotide. Subsequently, the results obtained from the MLPA technique are analyzed using specialized software programs.

The MLPA molecular method was initially introduced in 2002 by Jan Schuten in the scientific journal Nucleic Acid Research. It was employed to identify exon deletions in the human genes BRCA1, MSH2, and MLH1, which are known to cause hereditary breast and colon cancers. Presently, this method is widely used to identify various types of genetic and hereditary abnormalities that arise from deletion mutations in a broad spectrum of genes. Examples of such abnormalities include α-thalassemia, β-thalassemia, Duchenne muscular dystrophy (DMD), hereditary breast and colon cancers, phenylketonuria (PKU), Prader-Willi and Angelman syndrome, mental retardation, and more.

Principles of the MLPA method

In general, the MLPA method is a multiplex PCR technique that is utilized to assess the relative copy number of each DNA sequence from a set of 40 probes. Each of these probes is designed to target a distinct DNA sequence, primarily the exons of a specific gene. Each probe consisting of two half-probes, namely the 3′ and 5′ half-probes, which is responsible for determining the copy number of its corresponding DNA sequence. Furthermore, the probe includes a universal primer sequence that enables the amplification of all probes through multiplex PCR. Moreover, either one or both of the half-probes contain a stuffer sequence, which facilitates the differentiation of fragments during electrophoresis based on the probe’s length and consequently the size of the amplified product.

The MLPA reaction encompasses five discrete steps:

denaturation of the template DNA
hybridization of the probe with the template DNA in the specific region
DNA ligation
amplification of fragments via PCR
separation of PCR products through capillary electrophoresis
data analysis

The initial step involves denaturing the DNA and allowing the MLPA probes to bind to it. The two half-probes possess the ability to identify specific target sequences, which necessitate a precise match with the probes without any gaps. Following hybridization, PCR is executed. Notably, PCR is solely carried out using a pair of primers, one of which is tagged with a fluorescent label. Since only the bound probes undergo amplification during the subsequent PCR reaction, the count of probe ligation products is measured in relation to the number of target sequences in the sample. Eventually, the PCR products are separated based on their size utilizing the electrophoresis technique.

The height or area of the fluorescent peaks derived from PCR is quantified, and the value of the PCR product is normalized and compared with control DNA samples. This comparison effectively reveals the relative abundance of the target DNA sequence in the given DNA sample. The assessment of the reaction’s quality is accomplished by evaluating the presence of control peaks, which offer insights into the amplification efficiency and the appropriate amount of DNA used in the reaction. The fundamental aspect of the MLPA reaction lies in the fact that PCR does not amplify the target sequences directly, but rather the bound probes. Consequently, while a pair of PCR primers is utilized in conventional PCR, the amplification process in Multiplex PCR necessitates the use of specific primers.

Advantages

The simultaneous examination of multiple target sequences, approximately 40-50, within a single reaction
Ability to check point mutations, methylations and the copy number variation in a single analysis
utilizes a minimal amount of input DNA, ranging from 20-100 ng
does not require cell culture
requires a single pair of primer for amplification
cost-effective, rapid and straightforward method
An accurate method with high efficiency in genetic diagnosis tests
diagnosis of detections, microdeletions, duplications and small rearrangements.

Applications

point mutation analysis
examination of gene copy numbers within the human genome
Aneuploidy examination
Mental retardation study
sub chromosomal regions study
Examination of gene expression (RT-MLPA method)
DNA methylation test (MS-MLPA method)
Diagnosis of genetic abnormalities

Disadvantages

Time-consuming, complex and high cost of probe preparation
Contamination with healthy cells and failure to diagnose properly
Inability to diagnose mosaicism, tumor heterogeneity
Failure to recognize neutral copies in heterozygosity
Failure to distinguish diploidy from triploidy
Observing false-positive results because of alterations in the sequences of the probe or ligation location (specifically for the BRCA1 gene)
identification of false positive results during molecular diagnosis of Duchenne/Baker muscular dystrophy due to sequence change (the presence of different types of sequences in individuals of the same population) and as a result of probe hybridization sites