Multiplex Ligation-Dependent Probe Amplification (MLPA)

Multiplex Ligation-Dependent Probe Amplification (MLPA)

CD Genomics is one of the professional sequencing companies with extensive experience in detecting gene mutations. We can provide high-quality Multiplex Ligation-Dependent Probe Amplification (MLPA) services for detecting copy number changes and methylation quantification of genomic DNA, or for mRNA analysis.

MLPA is a simple, easy-to-execute, high-throughput technology that can detect DNA mutations of up to 40 sequences in a single reaction. The technology can detect mental retardation and congenital disorders such as microdeletion syndrome and down syndrome; neuromuscular diseases, such as CMT; diseases of secretion and metabolism, such as congenital adrenal cortical hyperplasia; blood diseases, such as hemophilia; tumor characterization, such as leukemia, breast cancer, colon cancer, stomach cancer, etc.

The principle and process of MLPA

The MLPA technology designs corresponding oligonucleotide hybridization probe pairs according to the sequence of the target gene, and hybridizes to adjacent positions on the DNA, respectively. After the hybridization probe and genomic DNA are fully hybridized, they are ligated under the action of ligase to form a complete hybridization probe for amplification. In this way, the copy number of the target gene is converted into an equal number of hybridization probes for amplification. PCR products with different lengths after amplification by universal primers can be analyzed by capillary electrophoresis. Resulting chromatograms show size-separated fragments ranging from 130 to 490 bp. The target gene copy number can be judged by the relative area ratio of different product peaks on the map. Comparing the electrophoresis pattern of the test sample with the electrophoresis pattern of the control sample, the deletion or duplication of the genomic region of interest can be detected.

The principle of MLPAFigure 1. The principle of MLPA. (Sun et al, 2020)

Advantages of MLPA service

  • Highly sensitive, powerful and high throughput.
  • Can be used to detect deletions, insertions, CNV and SNPs. It can distinguish between point mutations and gene copy / deletion, and easily determine the relative copy number of all exons in a gene. Unlike FISH, MLPA can detect small genetic changes.
  • Results can be obtained within 24 hours, and due to multiple reactions, information can be collected quickly and efficiently.
  • One PCR reaction can detect 40-50 genomic DNA sequences.
  • Identify sequences that differ by only 1 base.
  • A variety of applications can be implemented with minor changes to the MLPA protocol. For example, MLPA can also be used to detect methylation patterns in DNA (MS-MLPA) by adding additional digestion steps.

Workflow of MLPA service

Workflow of MLPA service

Sample requirements

  • Tissue samples: if the material provided is biological material such as fresh tissue or blood cells, please provide enough material to extract more than 2ug of genetic DNA. Stored in low-temperature refrigerator at -20℃ or -80℃, transported with liquid nitrogen or dry ice; local or short-distance transport can be ice pack transport.
  • Cultured cells in vitro (≥ 106 cells).
  • DNA samples: DNA concentration ≥ 20ng / ul, total DNA ≥ 1ug, OD260/280 between 1.7 and 2.0, without PCR inhibitor. Samples must be shipped in a cold ice pack. FFPE samples can also be received.
  • Blood samples: it is required to be stored in anticoagulant tubes or cryopreservation tubes with a volume greater than 2ml. Please transport them in dry ice to ensure that DNA is not degraded.

Want to know about other Non-NGS gene mutation detection methods?

References:

  1. Sun J, Xu J, Liang P, et al. Molecular identification of Clonorchis sinensis and discrimination with other opisthorchid liver fluke species using multiple Ligation-depended Probe Amplification (MLPA). Parasites & vectors, 2011, 4(1): 98.
  2. de Boer S, White S J. Genotyping Multiallelic Copy Number Variation with Multiplex Ligation-Dependent Probe Amplification (MLPA)//Genotyping. Humana Press, New York, NY, 2017: 147-153.
  3. 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, 2019.
* For Research Use Only. Not for use in diagnostic procedures.

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