lect 27 Multiplex Ligation-Dependent Probe Amplification (MLPA) Lecture Notes

This set of vocabulary flashcards covers the terminology, components, methodology, and clinical applications of Multiplex Ligation-dependent Probe Amplification (MLPA) and its methylation-sensitive variant (MS-MLPA).

MLPA

Multiplex Ligation-dependent Probe Amplification; a technique used to detect the presence, absence, or duplication (deletions, insertions, and CNVs) of genomic regions like exons and large genes.

LPO (Left Probe Oligo)

An MLPA probe component whose $5'$ end contains a primer binding site not found in the genome and whose $3'$ end is specific for a genomic target.

RPO (Right Probe Oligo)

An MLPA probe component with a $5'$ end specific for a genomic target, a stuffer sequence, and a $3'$ end with a second non-genome targeting primer binding sequence.

Stuffer sequence

A non-genome targeting sequence included in the RPO to artificially define the size of the resultant amplicon, allowing multiple regions to be resolved by capillary electrophoresis.

Ligation

The step in which two probe oligonucleotides, hybridised to immediately adjacent sites on the genomic target, are joined by a ligase enzyme into a single template for PCR.

MLPA Step 1: Denaturation

The initial step of the MLPA method where the genomic DNA to be analysed is separated in the presence of oligo probes.

MLPA Step 2: Hybridisation

The process, typically occurring overnight, where the LPO and RPO bind to their specific adjacent targets on the denatured genomic DNA.

Capillary electrophoresis

The detection method used to resolve and identify MLPA products based on their molecular weight and fluorescent labels.

Intra-sample normalisation

The process of comparing the peak height of a target probe to the height of reference probe patterns within the same sample.

Inter-sample normalisation

A comparison between the intra-sample probe ratios of a test sample and a normal reference sample to detect copy number variations.

Heterozygous deletion ratio

The expected area ratio (reference to test) of $1:2$ when a single copy of an allele is lost.

MS-MLPA

Methylation-sensitive MLPA; a semi-quantitative method combining MLPA with methylation-sensitive restriction enzymes for methylation profiling.

Hha I

A methylation-sensitive endonuclease (4-base cutter) used in MS-MLPA that digests probes bound to unmethylated DNA, preventing their amplification.

Prader Willi/Angelman

Specific imprinting diseases that can be detected using the Methylation-sensitive MLPA (MS-MLPA) method.

MLH1

A mismatch repair gene whose transcriptional silencing via methylation is associated with cisplatin resistance, detectable by MS-MLPA.

Duchenne

An example of a large gene for which MLPA is well-suited to detect deletions or duplications across multiple exons.

MLPA input requirement

The technique requires a low amount of genomic DNA, specifically less than $100\text{ ng}$ ($50\text{ ng}$ is often sufficient).

Phe508del in CFTR

A specific mutation that can be detected using mutation-specific MLPA probes.