What is mlpa

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. 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. Thus, many sequences upto 40 can be amplified and quantified using just a single primer pair. Sigma Genosys. It is performed in many laboratories worldwide, and can be applied to detect copy number changes like deletions or duplications of a gene, identify the methylation status of DNA, detect single nucleotide polymorphisms SNPs and point mutations, and quantify mRNA.

Therefore, it is used in many research and diagnostic fields, such as cytogenetics, cancer research, and human genetics, among others. We can also observe a typical electropherogram obtained through MLPA analysis showing a deletion of exon 46 red arrow.

Hybridization involves hybridizing the DNA sample to specific probes. Because it is a multiplex technique, you can analyze each sample by up to 60 probes simultaneously, thus targeting different sites!

These probes have a primer sequence that binds to the PCR-primer in the amplification process. All different probes will have the same primer binding sequence. Additionally, the probes also have a hybridization sequence complementary to the target site that will allow the probe to bind to the DNA. Both probes will hybridize on adjacent sites on the DNA strand.

One of the probes from the pair contains a stuffer sequence, which is different in length for each target site. The length of the stuffer sequence changes between different probes, allowing multiplexing. So, you can expect each amplification product to have a unique length!

The ligation step will bind the two probes together. In this step, a specific enzyme called DNA ligase is used. It binds the probes that are already hybridized on adjacent sites of the DNA strand at the target site. Previous genetic tests were negative or only identified a single variant in a gene or condition that is associated with autosomal recessive inheritance. MLPA can be used to diagnose specific disorders associated to: Deletions or duplications of specific regions, genes or exons Imprinting alterations.

Uniparental disomy UPD Small rearrangements small intragenic deletions. Request Information. An MLPA probe set is designed so that the length of each of its amplification products is unique. The length increases in a stepwise-fashion, with the total size range lying between nucleotides.

This size range provides optimal fragment separation and low background on sequence type gels. The only equipment needed to perform MLPA is a capillary electrophoresis device, and a thermocycler.

MLPA reactions can be performed as a one tube reaction, as non-ligated oligonucleotides do not need to be removed.