What is Multiplex Ligation-Dependent Probe Amplification (MLPA)?

Multiplex Ligation-Dependent Probe Amplification (MLPA) is a widely used molecular biology technique for detecting the copy number of various DNA sequences in human genetic disease research. This technique involves the ligation of probe oligonucleotides followed by PCR amplification, allowing the analysis of up to 50 multiplex probe pairs designed to hybridize with specific target loci.

Each probe pair is designed to generate amplification products of a specific length. By incorporating universal sequences at their termini, all ligated probes can be amplified in a single PCR reaction using one primer pair. The forward PCR primer is labeled with an Applied Biosystems™ 6-FAM™ fluorescent tag, enabling detection and quantification based on the molecular sizes of the probes, as determined through automated capillary electrophoresis (CE).

Multiplex Ligation-Dependent Probe Amplification (MLPA) process visualized in three steps: 1) Denaturation & Hybridization, 2) Ligation, 3) Amplification with fluorescent PCR primers to detect copy number variations.

This method has been successfully used in the study of diseases caused by exon deletions and duplications, such as Duchenne Muscular Dystrophy (DMD) and BRCA1/BRCA2 gene mutations. Additionally, advancements in the MLPA technique now enable its application in quantitative methylation analysis of various genomic sequences.

Why Choose MLPA?

MLPA is preferred for its precise, high-throughput analysis, even with limited DNA samples. Unlike traditional PCR, MLPA can test multiple genetic loci at once, making it a cost-effective and reliable solution for detecting genetic abnormalities. It is also more sensitive than other multiplex PCR methods, offering better detection of copy number variations (CNVs).

MLPA combines DNA probe hybridization with PCR technology, offering the following advantages:

• High Efficiency: A single reaction can detect copy number variations in up to 50 target sequences.
• Rapid Turnaround: Complete experiments can be performed within 24 hours.
• Operational Simplicity: Different reagent kits adhere to nearly identical procedures, rendering the technique easy to learn and master.

Technical Comparison: MLPA and Alternative Genetic Analysis Methods

Applications of MLPA

MLPA is a trusted method for detecting genetic variations with high sensitivity, and it finds application across various research and clinical fields. It enables researchers to analyze structural variations in the genome, providing insights into genetic disorders and disease mechanisms. This versatile technology supports a wide range of scientific inquiries, including but not limited to:

• Detection of Small-Scale Gene Rearrangements:

  Validated genes include BRCA1, BRCA2, MSH2, MLH1, DMD, APC, SMA, NF1, NF2, VHL, TSC1/2 etc.

• Detection of Large-Scale Genomic Rearrangements:

  Validated disorders include Williams syndrome, Prader-Willi/Angelman syndrome, DiGeorge syndrome, Cri du Chat syndrome, Pelizaeus-Merzbacher disease, CMT1A, and HNPP.

• Sub telomeric Copy Number Variations
• Chromosomal Aneuploidy Testing

• Tumor Diagnosis Research:

  DNA copy number analysis in malignancies such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), oligodendrogliomas, melanomas, and neuroblastomas.

• Quantitative Methylation Analysis:

  Applications include Prader-Willi/Angelman syndrome, Beckwith-Wiedemann syndrome, MGMT, MLH1, Fragile X syndrome, and tumor suppressor gene inactivation.

• mRNA Expression Analysis:

  Focusing on genes involved in apoptosis and inflammatory response.

Multiplex Ligation Dependent Probe Amplification Assay Service Workflow

At N2 Jenomics Lab Pvt. Ltd. , we provide a streamlined, end-to-end MLPA service to ensure consistent, high-quality results. Our standardized workflow is designed to support reproducibility and accelerate discovery across genomic studies.

1. Sample Submission

Submit your DNA samples, including tissue, blood, or cell lines. Our team ensures that they meet quality standards for analysis.

2. DNA Extraction and QC

If needed, we offer DNA extraction services and perform quality checks to ensure optimal DNA quality for MLPA.

3. Probe Hybridization and Ligation

Probes are hybridized to your DNA and ligated to target regions for CNV analysis.

4. PCR Amplification

Multiple DNA regions are amplified simultaneously using fluorescent primers, enabling efficient CNV detection.

5. Capillary Electrophoresis

The amplified products are analyzed by capillary electrophoresis for accurate CNV quantification.

6. Data Analysis and Reporting

We provide detailed statistical and annotation reports, along with graphical results for easy interpretation.

Sample Requirements for MLPA

Tips:

• Ship samples on blue ice or dry ice to preserve integrity.
• DNA extraction services available upon request.
• For special sample types or low-input scenarios, contact us for a customized plan.

Why Choose N2 Jenomics Lab Pvt. Ltd. for MLPA Service?

N2 Jenomics Lab Pvt. Ltd. offers a comprehensive MLPA service with expert support, fast results, and reliable data. Our team ensures high-quality testing using the latest technology, with quick turnaround times and global support. We follow strict quality control to provide accurate and dependable results for your research needs.

• Expert Team: Our team of geneticists and molecular biologists ensures high-quality results with every test.
• State-of-the-art Technology: We use the latest MLPA platforms to guarantee accurate and reliable data.
• Fast Turnaround: Receive your comprehensive MLPA report in a timely manner, ensuring that your research or clinical decision-making isn't delayed.
• Global Reach: Serving clients across the globe with customer support in multiple time zones.
• Quality Assurance: We follow rigorous quality control protocols, ensuring that our tests meet the highest standards of accuracy and reliability.

References:

1. de Boer, S., White, S.J. Genotyping multiallelic copy number variation with multiplex ligation-dependent probe amplification (MLPA). In: Genotyping. Springer, New York, NY, 147–153 (2017). https://doi.org/10.1007/978-1-4939-6442-0_9
2. Cuevas, D., Velasco, A. et al. Intratumor heterogeneity in endometrial serous carcinoma assessed by targeted sequencing and multiplex ligation-dependent probe amplification (MLPA): a descriptive study. Histopathology 75, 724–733 (2019). DOI: 10.1111/his.14001
3. Fu, X., Shi, Y., Ma, J. et al. Advances of multiplex ligation-dependent probe amplification technology in molecular diagnostics. BioTechniques 73, 205–213 (2022). DOI: 10.2144/btn-2022-0017

Demo Results

Figure 1. MLPA electropherogram comparison showing reference control peaks and reduced peaks in the test sample, indicating copy number deletions in specific gene loci.

Figure 2. Infographic MLPA result showing comparative bar peaks between control and test sample across multiple gene loci, with highlighted copy number variation.

Figure 3. MLPA result visualization in software UI style, displaying reference vs sample plots with highlighted copy number variation signals.

MLPA Assay FAQs

1. What types of samples can be used for MLPA?

You can use genomic DNA extracted from various sources such as blood, saliva, or tissue samples.

2. Can MLPA detect point mutations?

MLPA is primarily used for detecting copy number variations (deletions, duplications), but it can be adapted for mutation detection in specific cases.

3. What is the cost of MLPA testing?

The cost of MLPA testing varies depending on the number of targets analyzed. Please contact us for a quote.

MLPA Assay Case Studies

Reference

Xia Y., Feng Y., Xu L., Chen X., Gao F., Mao S. (2021). Case Report: Whole-Exome Sequencing With MLPA Revealed Variants in Two Genes in a Patient With Combined Manifestations of Spinal Muscular Atrophy and Duchenne Muscular Dystrophy. Frontiers in Genetics, Volume 12, Article 605611. DOI:10.3389/fgene.2021.605611.

1. Background

This case involves an 11-month-old male patient presenting with poor motor development and progressive muscle weakness. Clinical features resembled both Spinal Muscular Atrophy (SMA) and Duchenne Muscular Dystrophy (DMD), which are distinct neuromuscular genetic disorders. Accurate genetic diagnosis was crucial given overlapping symptoms and implications for treatment.

2. Methods

To pinpoint the genetic cause(s), clinicians used a two-tier genetic testing approach:

• Whole-Exome Sequencing (WES) to screen for sequence variants across all coding regions.
• Multiplex Ligation-Dependent Probe Amplification (MLPA) to specifically detect copy number variations (CNVs) such as deletions in target genes.

MLPA is a well-established method for exon-level CNV detection, making it suitable for DMD and SMA loci.

3. Results

The combined analysis identified:

• A homozygous deletion of exons 7 and 8 in the SMN1 gene, consistent with SMA.
• A deletion in exon 50 of the DMD gene, diagnostic of DMD.

These findings were confirmed by MLPA following WES predictions.

MLPA genetic test reports showing zero copy of SMN1 exons 7 and 8 and a homozygous deletion of DMD exon 50 in the patient sample, compared to reference controls.

4. Conclusions

The integration of MLPA and WES enhanced diagnostic accuracy in this complex case involving dual neuromuscular disorders. This approach highlights the value of combining broad variant detection (WES) with targeted CNV profiling (MLPA) for comprehensive genetic diagnosis in clinically overlapping phenotypes.