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Multiplex Ligation-dependent Probe Amplification shortly called as MLPA is an important method for the detection of variation in the Copy Number of DNA or RNA sequence. Using this technique separation of sequences differing in one nucleotide is possible. A copy number of up to 45 nucleic acid sequence can be set up in a single reaction (Shakthivel Murugan et al, 2010) and as many as 96 samples can be handled at once. The specialty of MLPA is that the amplification takes place to the probes that hybridize the specific sequence of interest or the target.
Principle and Working
Multiplex Ligation-dependent Probe Amplification consists of 5 steps namely denaturation of the DNA and probes hybridization, the ligation step followed by the polymerase chain reaction, then the electrophoretic separation of product that was amplified and finally the analysis of the data.(www.mlpa.com)
After the DNA is denatured the addition of the probe takes place. The probe consists of two parts, a short synthetic and a long probe oligonucleotide usually derived from M-13 phage. The short part of the probe consists of a 3' end that is made up of 21 to 30 nucleotide which is target specific and at the 5' end it contains a 19 nucleotide sequence identical to labeled PCR primer. And for the long part of the probe cloning of the specific or target sequence is done in M-13 derived SALSA vector having a length of 25 to 43 nucleotides(Schouten.et.al.2002).
Then the hybridization of the short and the long probe take place to target sequence that is present immediately. It is noted that ligation step takes place only when both the short and the long probe hybridize to their adjacent target sequence and the reason behind this is that during the PCR reaction amplification occurs only for the probes that have been ligated. There is a proportional relationship between the ligated probe product and the target sequence that is present in the sample. Then capillary electrophoresis is run in order to separate the products obtained. The probes that are not bound do not amplify or generate a signal since they contain only a single probe and thus making MLPA a simple method. .(www.mlpa.com)
The data analysis is an important step in the MLPA technique. Consider a gene that contains a large mutation. Here the general positioning or location of the probes would be in the exons while the control probes are obtained from regions where there is a low probability of affecting a genome. A comparison is done between reference sample ratio to that of the normalized probe signals against control probe's average or total signal. A bar chart is plotted with the ratios of probes signal intensity and a drop or increase in signal intensity is a proof for deletion or duplication taking into accounts the consecutive probes. Validation is done by running different probe sets.
Fig 1: Step 1 to 4 indicate the Denaturation & hybridization, Ligation, Amplification and Analysis. Taken from www.mlpa.com.
MLPA has a number of applications from detection of copy number variation in DNA which includes analyzing the deletion or insertion, change in the number of chromosomes and can also be extended to sub-chromosomal variations.
In case of epigenetic aberrations like Angelman's or prader-willi syndrome, Methylation Specific MLPA ,a modified MLPA assay has been used for detection. An aberration in the pattern of Methylation in the imprinting region or the change in the Methylation pattern in the CpG island which is present in close proximity to the promoter region of the TSG of tumors. (Piotr Kozlowski.et.al.2008)
Segmental Duplication of genes is a case where the gene is duplicated somewhere else in the genome and are highly homologous. A MLPA assay for this segmental duplication is a great challenge. These gene sequences occur more than once in a haploid genome with a length over 1 kb. Typical examples are BRCA1 and PKD1 where MLPA was successful. (Piotr Kozlowski.et.al.2008)
mRNA Profiling can be done by another MLPA derivative namely Real Time MLPA (RT-MLPA). A RT-MLPA uses RT-MLPA probe mix which contains reverse transcriptase enzyme since direct detection of mRNA is not possible due to inability of ligase-65 to ligate DNA( Since DNA oligonucleotide and RNA are annealed) .(Piotr Kozlowski.et.al.2008)
The mouse transgenes that have been employed frequently include cre recombinase, enhanced GFP and Te2/onc. A MLPA assay has been designed for genotyping these transgenes with exactly the same strategy and competitor oligonucleotides were used. The assay was able to discriminate the genotypes in a reliable fashion that uses competitor oligonucleotides in transgenic mouse lines that were dissimilar. (Piotr Kozlowski.et.al.2008)
Fig 2: The figure illustrate the MLPA steps for each of the different applications.
Taken from Piotr Kozlowski.et.al. New applications and developments in the use of multiplex ligation-dependent probe amplification.
There are a few areas that did not have great influence by the MLPA assay. Expression profiling along with the detection of small mutations are a few areas that were not greatly exploited. Though paper shouten.et.al explained the use of MLPA for sensitive detection of small mutation it still is relatively un-exploited. This might be due to the fact that presence of other simple methods for small mutation detection. (Piotr Kozlowski.et.al.2008)
Another application is the use array hybridization combined with the MLPA technique for detection of the probe. It increases the possibility for high multiplexing along with the use of several multiple probes that would have been used for different individual MLPA assay thus minimizing the time from several to a single assay. (Piotr Kozlowski.et.al.2008)
Advantages and Limitations:
MLPA boasts itself for providing the customers with many advantages like simple to use, low cost, high sensitivity, high throughput, high reproducibility with relatively low sample volume and simplified quality control. (www.mlpa.com)
The limitations are mainly the relative intensity of the obtained signals that differ as a result of characteristic feature of the input DNA that depend on time of extraction, method of extraction, extent of degradation etc. Another important limitation is the failure to detect the inversion or translocation defects. (Piotr Kozlowski.et.al.2008)
MLPA vs Other techniques:
MLPA vs Multiplex PCR: Duchene muscular dystrophy (DMD) is a X-linked disorder that consists of a mutant DMD gene. A milder form of DMD is Becker muscular dystrophy (BMD). A thorough investigation on the DMD/BMD was done on 150 male patients diagnosed with either of the diseases. The multiplex PCR was able to detect deletions in 103 cases out of 150 with a percentage of 68.7 which comprised of various percentage of single and multiple deletions. Though mPCR was able to detect deletion for 68.7% a precise or exact diagnosis was obtained for only 40% of the patients. MLPA assay was able to precisely diagnose 75% of the total patients and thus increasing the percentage of detection from 40 to 75. mPCR could not pick up duplications and deletion outside the hot-spot region.(sakthi, 2010)
MLPA vs FISH Analysis:
Approximately 5% of the patients affected by unexplained mental retardation carried micro deletions that were not observed by conventional cytogenetic analysis. 12 patients carrying this microdeletion confirmed by FISH was assayed using MLPA. There was a perfect concordance with the result from FISH and that obtained from MLPA. Deletions and duplications were the areas where FISH failed along with quality related problem of metaphase spread.( Eun Hae Cho 2008)
Another comparison was done between the two methods with chronic lymphoid leukemia (CLL). There was a concordance of 93.6% with a 52 chromosomal alterations detected by each. Further MLPA detected a copy number gain on 18q21.1 and 19 and also new micro deletion at 19q13.43 and 19p13.2 loci which FISH failed.( Abdool.et.al 2010)
MLPA vs Denaturing High Performance Liquid Chromatography (DHPLC): A comparative study between DHPLC and MLPA was done for mutation screening of EXT1 and EXT2. Multiple osteochondromas (MO) is an autosomal dominant disorder that is often related to mutation in the EXT1 and EXT2 genes. The mutation screening was done on a set of 63 patients and the DHPLC was able to give a positive result for mutation in 43 patients. When MLPA assay was carried out a additional 5 mutations was found leading upto 48 patients. The DHPLC did not pick up 4deletion and one translocation. (Ivy Jennes, 2008).
Conclusion: The review can be summarized by quoting that the Multiplex Ligation-dependent Probe Amplification stands out as a strong competitor to other methods not only in detection of abnormalities but even in other categories like ease of use, low cost, good reproducibility, high sensitivity and high throughput. With the bringing in automation, increased multiplexing and precision in measuring even low copy number variation with increased accuracy it can replace other methods for detection methods with ease. (Piotr Kozlowski.et.al.2008)