Introduction to MLPA
MLPA is an abbreviation for "multiplex ligation-dependent probe amplification", which is a high-throughput technique for qualitative and quantitative analysis of target sequences in nucleic acids to be tested. The basic principle is to use a simple probe to hybridize with the target sequence DNA, followed by ligation, PCR amplification, product separation by capillary electrophoresis and data collection, and analysis software to analyze the collected data and draw conclusions. It can detect copy number variations of up to 50 nucleotide sequences simultaneously and can detect fragments smaller than 60bp. It can be used to detect deletions or amplifications of some disease-related genes or chromosomes.
The history of the MLPA method dates back to 2002, when the Dutch scholar Dr. Schouten proposed this multiplex ligation-dependent probe amplification technique. He described the principle and application of this method in a paper.The MLPA method was developed and commercialized by MRC-Holland, which offers a variety of MLPA kits and analysis software.Since its introduction, the MLPA method has been popular with many laboratories because it can detect copy number changes and methylation status of genes quickly, sensitively and accurately.The MLPA method is also used to detect deletion or amplification of some genes or chromosomes associated with diseases, such as hereditary breast cancer, colon cancer, hereditary gastric cancer, neuroblastoma, etc.
MLPA is mainly used to detect copy number variations (CNV) in DNA, which is the increase or decrease of DNA fragments larger than 1 kb (some literature suggests larger than 50 bp) on chromosomes, mainly in the form of deletions and duplications at the submicroscopic level. CNV can result from genomic rearrangements (e.g. chromosomal deletions, duplications, insertions and translocations) or non-allelic recombination (mispairing and swapping between homologous sequences).CNV can affect gene expression and function and is associated with many genetic diseases and cancers.MLPA works by using a pair of specific probes to identify the target DNA sequence. The adjacent probes are then ligated with a ligase to form a molecule that can be amplified by PCR. The length of each probe pair is different so that PCR products can be separated by electrophoresis and detected by fluorescence. By comparing the peak patterns of the sample and the reference sample, the relative amount of the target sequence in the sample DNA can be calculated.MLPA can be used to detect CNVs associated with diseases such as hereditary breast cancer, colon cancer, Duchenne muscular dystrophy, etc. MLPA can also be used to detect CNVs in tumors to optimize tumor staging.
-Step 1: Hybridization of probe and target sequence DNA. Sample DNA is mixed with the specific probe, hybridization buffer is added, and the hybridization reaction is performed in a thermostat or water bath so that the probe and target sequence DNA are fully complementary to each other.
-Step 2: Ligation of the probe. Transfer the hybridization reaction system to a new PCR tube, add ligating buffer and ligase, and place in a thermostat or water bath to perform the ligation reaction so that adjacent probes are joined into a single molecule that can be amplified by PCR.
-Step 3: Probe amplification. Transfer the ligation reaction system to a new PCR tube, add PCR buffer, dNTPs, universal primers and PCR enzymes, and place it in a PCR instrument to perform the amplification reaction so that the connected probe molecules are amplified exponentially.
-Step 4: Electrophoretic analysis of amplification products. The amplification reaction system is mixed with molecular weight internal standard and formamide and placed in capillary electrophoresis apparatus for electrophoretic separation and fluorescence detection to obtain peak patterns of probe molecules of different lengths.
-Step 5: Data analysis and result interpretation. Use professional software to perform peak detection, normalization, quality control and result evaluation of electrophoresis data to determine whether there is copy number variation in sample DNA.
-The length of the probe should be between 40-70 nucleotides, preferably between 50-60 nucleotides.
-The Tm value of the probe should be between 55-65°C, preferably around 60°C, and the Tm values of the two probes should not differ by more than 5°C.
-The GC content of the probes should be between 30-70%, preferably between 40-60%, and the GC content of the two probes should differ by no more than 10%.
-G or C should be avoided at the end of the probes to prevent the formation of secondary structures or non-specific hybridization.
-Probes should avoid the presence of repetitive, homologous or complementary sequences to prevent the formation of secondary structures or non-specific hybridization.
-Probes should avoid the presence of SNP sites to prevent affecting hybridization efficiency or specificity.
-Sample quality: The quality and quantity of sample DNA has a great influence on the results of MLPA reactions, so it is necessary to use a suitable DNA extraction method, avoid DNA degradation and contamination, use purified water or TE buffer to dilute DNA, and use nanospectroscopy or fluorometric methods to determine DNA concentration.
-reaction conditions: optimization of reaction conditions is also important for the efficiency and specificity of MLPA reactions, so it is necessary to prepare the reaction system accurately according to the instructions of the kit, avoid primer dimerization and non-specific amplification, use a thermostat or water bath for hybridization and ligation reactions, and use high-fidelity (or called long fragment) PCR enzymes for amplification reactions.
-Electrophoresis analysis: The quality control of electrophoresis analysis mainly includes fluorescence calibration, molecular weight internal standard, peak detection and normalization steps, which require the use of fluorescence calibration standards matching the probe markers for spectral calibration to accurately detect the dye markers on the primers. Sample peak size and correction for injection error (usually ROX), peak detection and normalization using specialized (e.g. Coffalyser) software is required to eliminate variation between samples.
-Results assessment: Quality control of results assessment includes analysis and interpretation of data from reference, QC and assay samples, using appropriate (e.g., normal human) reference samples as a baseline for normal copy number (usually 2) and QC samples as controls (e.g., synthetic or known patients) for known copy number variations (e.g., 1 or 3). Professional (e.g., Coffalyser) software is required for data analysis and interpretation of results (e.g., calculating copy number ratios and setting thresholds for abnormalities) to determine whether a copy number variation exists in the test sample.
-Step 1: Peak detection. Peak detection is performed on the electrophoresis data to obtain the peak height or peak area value for each probe for each sample.
-Step 2: Normalization. The peak values of each sample are normalized (usually by dividing by the subscript peak and multiplying by a factor) to eliminate variation between samples (e.g., injection volume, PCR efficiency, etc.) to obtain a normalized value for each probe for each sample.
-Step 3: Quality control. Assess the quality of each sample (e.g., calculate CV values), check for abnormalities (e.g., missing, decreased, or increased) or low signal-to-noise ratios (SNR), and exclude samples or probes that do not pass (e.g., below 0.85 or above 1.15).
-Step 4: Evaluation of results. The normalized values for each probe for each sample are compared (usually by dividing by the reference sample mean and multiplying by 2), and the copy number ratio (ratio) for each probe for each sample is calculated to determine whether there is copy number variation (e.g., missing, duplicate, or normal).
-Step 5: Interpretation of results. Depending on the purpose of the test and the kit, the copy number ratio per probe for each sample is interpreted and combined with the data from the reference and quality control samples to draw a final conclusion and report (e.g., whether a microdeletion syndrome or other genetic disorder is present).
-Detection of hereditary breast and ovarian cancers The MLPA method can detect copy number changes in the BRCA1 and BRCA2 genes, such as deletions or duplications, that are associated with the development of hereditary breast and ovarian cancers .
The MLPA method detects copy number changes, such as deletions or duplications, in the MSH2, MLH1, PMS2 and MSH6 genes, which are associated with the development of hereditary non-polyposis colon cancer (HNPCC) .
-The MLPA method can detect copy number changes in the MYCN gene, such as amplification or normal, which are associated with the prognosis and grading of neuroblastoma .
-The MLPA method can detect copy number changes in multiple genes or segments on chromosome 21, such as trisomy or normal, which are associated with the diagnosis of Down syndrome.
The MLPA method detects DNA methylation status of specific genes or loci, such as methylated or non-methylated, which are associated with regulation of gene expression and epigenetics .
