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Multiplex ligation-dependent probe amplification
Multiplex ligation-dependent probe amplification (MLPA) is a variation of the multiplex polymerase chain reaction that permits amplification of multiple targets with only a single primer pair. It detects copy number changes at the molecular level, and software programs are used for analysis. Identification of deletions or duplications can indicate pathogenic mutations, thus MLPA is an important diagnostic tool used in clinical pathology laboratories worldwide.
Multiplex ligation-dependent probe amplification was invented by Jan Schouten, a Dutch scientist. The method was first described in 2002 in the scientific journal Nucleic Acid Research. The first applications included the detection of exon deletions in the human genes BRCA1, MSH2 and MLH1, which are linked to hereditary breast and colon cancer. Now MLPA is used to detect hundreds of hereditary disorders, as well as for tumour profiling.
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:
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.
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 two copies of the chromosome. If an extra copy is present in the test sample, the signals are expected to be 1.5 times the intensities of the respective probes from the reference. If only one copy is present the proportion is expected to be 0.5. If the sample has two copies, the relative probe strengths are expected to be equal.
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.
MLPA facilitates the amplification and detection of multiple targets with a single primer pair. In a standard multiplex PCR reaction, each fragment needs a unique amplifying primer pair. These primers being present in a large quantity result in various problems such as dimerization and false priming. With MLPA, amplification of probes can be achieved. Thus, many sequences (up to 40) can be amplified and quantified using just a single primer pair. MLPA reaction is fast, inexpensive and very simple to perform.
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Multiplex ligation-dependent probe amplification AI simulator
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Multiplex ligation-dependent probe amplification
Multiplex ligation-dependent probe amplification (MLPA) is a variation of the multiplex polymerase chain reaction that permits amplification of multiple targets with only a single primer pair. It detects copy number changes at the molecular level, and software programs are used for analysis. Identification of deletions or duplications can indicate pathogenic mutations, thus MLPA is an important diagnostic tool used in clinical pathology laboratories worldwide.
Multiplex ligation-dependent probe amplification was invented by Jan Schouten, a Dutch scientist. The method was first described in 2002 in the scientific journal Nucleic Acid Research. The first applications included the detection of exon deletions in the human genes BRCA1, MSH2 and MLH1, which are linked to hereditary breast and colon cancer. Now MLPA is used to detect hundreds of hereditary disorders, as well as for tumour profiling.
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:
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.
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 two copies of the chromosome. If an extra copy is present in the test sample, the signals are expected to be 1.5 times the intensities of the respective probes from the reference. If only one copy is present the proportion is expected to be 0.5. If the sample has two copies, the relative probe strengths are expected to be equal.
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.
MLPA facilitates the amplification and detection of multiple targets with a single primer pair. In a standard multiplex PCR reaction, each fragment needs a unique amplifying primer pair. These primers being present in a large quantity result in various problems such as dimerization and false priming. With MLPA, amplification of probes can be achieved. Thus, many sequences (up to 40) can be amplified and quantified using just a single primer pair. MLPA reaction is fast, inexpensive and very simple to perform.