miércoles, 7 de marzo de 2018

Multiplex Ligation-Dependent Probe Amplification (MLPA)

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By Catherine Shaffer, M.Sc.

Multiplex Ligation-dependent Probe Amplification (MLPA) is a method for detecting copy number changes using polymerase chain reaction (PCR).

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MLPA can detect copy number changes in up to 50 different DNA sequences in a single reaction, and it can distinguish sequences differing by only one nucleotide.

Applications of MLPA

The major application for MLPA is detection of copy number variations associated with disease. Complete or partial gene deletions and duplications account for up to 10% of hereditary diseases.

Heterozygous deletions and duplications are typically not detectable through sequencing or other methods used for point mutations. MLPA is also used to detect and characterize microdeletion syndromes, sub-telomeric deletions and duplications, and for analysis complicated by pseudo genes.

MLPA process

MLPA begins when DNA is denatured and incubated with MLPA probes. The probes consist of two oligonucleotides containing a primer sequence that hybridize to adjacent target sequences.

Next, a ligation reaction is carried out. In this step, the two probes hybridized to their target sequences are ligated together. The ligated probes are then amplified through PCR. The amplified products are separated from the sample using capillary electrophoresis, and the samples are analyzed for copy number.

Data analysis

Since it is the probes, not the target sequences amplified, data analysis is straightforward. Amplification products all have the same length, regardless of possible sequence variants in the probe target sequence. MLPA analysis begins with intra-sample normalization. Only relative peak areas are used, never absolute peak areas. This is accomplished through the use of reference probes.

After normalization, probe ratios in the experimental sample are compared to probe ratios in the reference sample. The resulting ratio is related to the sequence copy number. For example, a heterozygous deletion of a gene sequence would have a relative ratio of about 0.5, reflecting one copy per cell instead of two.

Probe ratios below 0.7 or above 1.3 are usually considered to show a heterozygous deletion or duplication. Reduced probe signal could also be caused by a change in the sequence detected by the probe. If the change is adjacent to the ligation site, that can prevent ligation of the two probes.

If there is a sequence change further away, up to 20 nucleotides from the ligation site, that could also produce a lower probe signal because the mismatch destabilizes binding of the probe. Copy number changes detected by a single probe always require confirmation by another method, usually sequencing, long-range PCR, or qPCR.

MLPA variations

Variations on MLPA have been developed, including reverse transcriptase MLPA (RT-MLPA) and methylation-specific MLPA (MS-MLPA). RT-MLPA is used to analyze mRNAs. As implied by its name, the procedure begins with reverse transcription of mRNA to complementary DNA (cDNA). The rest of the procedure is the same as standard MLPA.

MS-MLPA is used to identify methylation changes to DNA, which can be significant for detecting imprinting diseases and for tumor sample analysis. It differs from standard MLPA in that each MS-MLPA reaction generates two samples for capillary electrophoresis analysis. One is processed as a standard MLPA reaction and the other is incubated with methylation sensitive HhaI endonuclease while the probes are simultaneously ligated.

Unmethylated probes are digested and can’t be amplified by PCR, so will not generate a signal. In contrast, methylated probes are amplified, generating a signal. The copy number is determined by comparing the undigested reaction with the reference sample, and the methylation pattern is determined by comparing the undigested sample to its digested counterpart.