Multiplex Ligation-dependent Probe Amplification

Multiplex ligation-dependent probe amplification (MLPA) is a variation of the polymerase chain reaction that permits multiple targets to be amplified with only a single primer pair. Each probe consists of a pair of primers that straddle the target site of interest and are subsequently ligated into a complete target sequence. The advantage of splitting the target probe is that only the ligated sequences, not the original sample DNA, are amplified. If the target probe sequences were not split in this way, the primers at both ends would cause the probes themselves to be amplified regardless, and the product would have no relation to the sample DNA. One of the MLPA primer pair is tagged with a DNA sequence of predefined length, known as a stuffer sequence, together with the specific target sequence. During electrophoresis, the stuffer sequence ensures that a given amplicon occurs at a known position along the gel. Different stuffer sequence lengths are therefore used to separate the various target amplicons. This avoids the resolution limitations of multiplex PCR. The probes are also tagged with a fluorescent label so that the intensity of the measured signal is related to the quantity of the target amplicon.

Various techniques including DGGE (Denaturing Gradient Gel Electrophoresis), DHPLC (Denaturing High Performance Liquid Chromatography), and SSCA (Single Strand Confirmation 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.

Relative ploidy
An important use of MLPA is to determine relative ploidy. For example, probes may be designed to target various regions of chromosome 21 of a human cell. The signal strengths of the probes are compared with those obtained from a reference DNA sample known to have 2 copies of the chromosome. If an extra copy is present in the test sample, the signals are expected to be 3/2 times the intensities of the respective probes from the reference. If only one copy is present the proportion is expected to be 1/2. If the sample has two copies the relative probe strengths are expected to be equal.

Dosage quotient analysis
Dosage quotient analysis is the usual method of interpreting MLPA data. If a and b are the signals from two amplicons in the patient sample, and A and B are the corresponding amplicons in the experimental control, then the dosage quotient DQ = (a/b) / (A/B). Although dosage quotients may be calculated for any pair of amplicons, it is usually the case that one of the pair is an internal reference probe.

Applications of MLPA
MLPA has a variety of applications including detection of mutations and single nucleotide polymorphisms, analysis of DNA methylation , relative mRNA quantification , chromosomal characterisation of cell lines and tissue samples , detection of duplications and deletions in human cancer predisposition genes such as BRCA1, BRCA2, hMLH1 and hMSH2 and aneuploidy determination. MLPA has potential application in prenatal diagnosis both invasive and noninvasive.