MLPA quantifies the presence of particular sequences in a sample of DNA, using a specially designed probe pair for each target sequence of interest. The process consists of multiple steps:
# The sample DNA is denatured, resulting in single-stranded sample DNA.
# Pairs of probes are hybridized to the sample DNA, with each probe pair designed to query for the presence of a particular DNA sequence.
# Ligase is applied to the hybridized DNA, combining probe pairs that are hybridized immediately next to each other into a single strand of DNA that can be amplified by PCR.
# PCR amplifies all probe pairs that have been successfully ligated, using fluorescently labeled PCR primers.
# The PCR products are quantified, typically by (capillary) electrophoresis.
Each probe pair consists of two oligonucleotides, with sequence that recognizes adjacent sites of the target DNA, a PCR priming site, and optionally a "stuffer" to give the PCR product a unique length when compared to other probe pairs in the MLPA assay. Each complete probe pair must have a unique length, so that its resulting amplicons can be uniquely identified during quantification, avoiding the resolution limitations of multiplex PCR. Because the forward primer used for probe amplification is fluorescently labeled, each amplicon generates a fluorescent peak which can be detected by a capillary sequencer. Comparing the peak pattern obtained on a given sample with that obtained on various reference samples, the relative quantity of each amplicon can be determined. This ratio is a measure for the ratio in which the target sequence is present in the sample DNA.
Various techniques including DGGE (Denaturing Gradient Gel Electrophoresis), DHPLC (Denaturing High Performance Liquid Chromatography), and SSCA (Single Strand Conformation Analysis) effectively identify SNPs and small insertions and deletions. MLPA, however, is one of the only accurate, time-efficient techniques to detect genomic deletions and insertions (one or more entire exons), which are frequent causes of cancers such as hereditary non-polyposis colorectal cancer (HNPCC), breast, and ovarian cancer.  MLPA can successfully and easily determine the relative copy number of all exons within a gene simultaneously with high sensitivity.