Multiplex Ligation-dependent Probe Amplification (MLPA) is a molecular diagnostic technique used in genetics. This method allows for the targeted examination of specific DNA sequences within a person’s genome. Its primary function is to accurately measure the number of copies of particular genes or segments of genes. MLPA helps identify genetic changes that can contribute to various inherited disorders.
What MLPA Detects
MLPA identifies “copy number variations” (CNVs) within the human genome. CNVs are instances where an individual has an atypical number of copies of a DNA segment, either too few (deletions) or too many (duplications). These variations can range from single exons to entire genes or larger chromosomal regions. Detecting these alterations is important because they often underlie the development of genetic conditions.
Many genetic disorders are linked to such deletions or duplications. For example, Duchenne muscular dystrophy, a muscle-wasting condition, frequently results from deletions or duplications within the DMD gene. Beckwith-Wiedemann syndrome, a disorder associated with overgrowth, can involve duplications in specific regions of chromosome 11. MLPA also aids in diagnosing hereditary cancer syndromes, such as those caused by changes in the BRCA1 and BRCA2 genes, where deletions or duplications can increase cancer risk.
The MLPA Process Explained
The MLPA technique involves a series of steps to quantify specific DNA sequences. The process begins with DNA preparation, where a patient’s DNA is extracted from a sample and then denatured by heating to separate its double strands into single strands. This denaturation is necessary to allow the MLPA probes to access the target DNA sequences.
A mixture of custom-designed MLPA probes is then added to the single-stranded DNA. Each probe consists of two half-probes, designed to bind specifically to adjacent target sequences on the patient’s DNA. These probes contain sequences complementary to the target DNA, universal primer binding sites, and a “stuffer” sequence that gives each probe a distinct length.
If the target DNA sequence is present and the two half-probes bind next to each other, DNA ligase joins them into a single, complete probe. This ligation only occurs with a perfect match between the probes and the target DNA. Unbound or non-ligated probes are not amplified.
The ligated probes are then copied millions of times using Polymerase Chain Reaction (PCR). A single pair of universal primers amplifies all ligated probes simultaneously, making the process efficient. The amplification products, known as amplicons, are fluorescently labeled for detection and measurement.
Finally, these amplified fragments are separated by size using capillary electrophoresis. Each fragment appears as a distinct peak. The amount or height of each peak is directly proportional to the number of copies of the original target DNA sequence present in the sample.
Advantages and Limitations of the MLPA Technique
MLPA offers several advantages in genetic diagnostics. Its multiplexing capability allows analysis of up to 60 different DNA sequences in a single reaction. This efficiency makes it a cost-effective approach for targeted genetic investigations compared to broader sequencing methods. The technique also provides accurate detection of single-exon deletions and duplications.
Despite its strengths, MLPA has limitations. It cannot detect small changes in the DNA code, such as single nucleotide variants (point mutations) or small insertions/deletions. These typically require DNA sequencing methods. MLPA also cannot identify balanced chromosomal rearrangements, where genetic material is rearranged but no net loss or gain occurs. As a targeted test, MLPA only detects changes in regions for which probes have been designed.
Interpreting MLPA Results
Interpreting MLPA results involves comparing the signal from a patient’s sample to that of reference samples with a known normal copy number. The outcome is typically presented as a ratio or dosage quotient for each analyzed gene segment. This ratio reflects the relative amount of the target DNA sequence in the patient’s sample compared to the reference.
A normal result usually shows a ratio close to 1.0 for a particular gene or exon, indicating the presence of two normal copies, consistent with most human genes. If a deletion is present, a heterozygous deletion, meaning one copy of the gene is missing, will yield a ratio around 0.5. A ratio of 0 indicates a homozygous deletion, where both copies of the gene are absent.
Conversely, a duplication, where an extra copy of a gene or segment exists, typically results in a ratio around 1.5. This indicates that three copies of the gene are present instead of the usual two. In some cases, more complex duplications might lead to higher ratios. Understanding these ratios is important, but a healthcare provider or genetic counselor should interpret all results within the context of the patient’s medical history and family background.