References >> MLPA®MLPA® Probes
MLPA® (Multiplex Ligation-dependent Probe Amplification) is a simple, high throughput and easy to perform method developed by MRC Holland that allows detection of DNA copy number changes of up to 40 sequences in a single reaction. This relatively simple technique is based on the semi-quantitative polymerase chain reaction principle and can be applied for detecting copy number changes and methylation quantification of the genomic DNA or for mRNA profiling.
MLPA® Probes are of Two Types:
MLPA® probes consist of two oligonucleotides, each containing a PCR primer sequence and a sequence complementary to the target, known as the hybridization sequence.
The two probes hybridize immediately adjacent to each other. When the probes correctly hybridize to the target sequence they are ligated by a thermo stable ligase enzyme. The PCR primers exponentially amplify the ligated probes. One of the primers is labeled with a fluorescent dye to visualize the amplification product of the probe. MLPA® based detection assays can be run in a single tube as non-ligated probes do not need to be removed.
Sequence type electrophoresis is used to separate the resulting PCR products. Each MLPA® probe length is designed such that it can be easily identified when the amplification product of the PCR is run through a gel. The difference in size is achieved with the help of the stuffer sequence.
Length Criteria: Length of the hybridization sequence should be greater than 21 bases and the two hybridization sequence should be adjacent to each other.
Amplicon Length: The designed probes should have a unique amplicon length.
Tm Criteria: Tm of the probes should be 72 +/- 5 °C.
Avoid Homology: The designed probes should be unique. Homologous regions should be avoided by performing a BLAST search.
Secondary Structures: The designed probes should be tested for hairpins, self dimer, runs. Ideally the probe should have as few secondary structures as possible.
Unique Ligation Site: To avoid mispriming, the ligation site and the surrounding nucleotides should be non-homologous to all the selected sequences.
Nucleotide Following the PCR Primer: First nucleotide following the PCR primer sequence should, preferably, not be adenine as it results in low peak size for the probe amplification product.
MLPA® is a variation of the polymerase chain reaction. It facilitates the amplification and detection of multiple targets with a single primer pair. In a standard multiplex PCR reaction, each fragment needs a unique amplifying primer pair. These primers being present in a large quantity result in various problems such as dimerization and false priming. With MLPA®, one does not amplify the target DNA, but only the probes. Thus, many sequences (upto 40) can be amplified and quantified using just a single primer pair. Compared to other techniques, MLPA® reaction is fast, cheap and very simple to perform.