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Multiplex Ligation-Dependent Probe Amplification - Coggle Diagram
Multiplex Ligation-Dependent Probe Amplification
Describe Multiplex Ligation-dependent Probe Amplification (MLPA)
Discuss Advantages & Disadvantages of MLPA
Pros
Detection down to nucleotide level
Cheaper than CGH
High throughput
100ng genomic DNA required
Cons
Sensitive to contaminants in genomic DNA preps
Doesn't detect alterations not represented in probe set
Not suited to genome wide screening
In cell mixtures, estimates of copy number averaged for whole sample
Applications of MLPA
Detection of exon deletions/duplications in e.g. BRCA1, MSH2, MLH1 genes
Detection of trisomies e.g. Down syndrome
Characterisation of chromosomal aberrations in cell lines & tumour samples
SNP & mutation detection
DNA methylation analysis
Methylation sensitive MLPA
Can detect presence, absence, duplication of a genomic region
Suited to detection of larger alterations due to multiplexing
Process
2 oligomer probes
binding adjacent to one another
Ligated
to form a single DNA molecule that becomes template for detection by PCR
Two sequences immediately adjacent - ligate can occur & detect by PCR
Two sequences not adjacent - no ligation, no amplification
Reagents
Genomic DNA (template) to be assessed
Multipartite primers
Left Probe Oligo (LPO)
Right Probe Oligo (RPO)
Ligase
Primers for terminal sequences in multipartite primers (one fluorescently labelled) to allow fragments to be resolved
Taq, dNTPs
Steps
Denaturation
Hybridisation
Ligation
Amplification (by PCR)
Detection (capillary electrophoresis)