Describe how a point mutation in genomic DNA may be detected by MLPA in a single...

Describe how a point mutation in genomic DNA may be detected by MLPA in a single tube. Highlight the critical elements of probes and primers used to detect a “wild type” and single nucleotide variant. Ensure the role of enzyme(s) required for this assay are explained.

Expert Solution

Ans ) MLPA ,identifies the deletions/duplications and helps in detection of known point mutations, but not suitable for detection of unknown point mutation .

In the MLPA, the Probes function is to detect the reference (wild type) sequence or the SNP/point mutation sequence of interest. As we know, for detection of known point mutations, MLPA designed so that the ligation site is located directly at the site of the point mutation. Ligation will then only occur when the DNA sequence is a perfect complement to the designed probe, whether this is the reference or SNP/point mutation sequence. Only successfully ligated probes will be amplified, which gives the probes their specificity.

MLPA probes consists of two separate oligonucleotides and each of them containing one of the PCR primer sequences. It only happens when these two hemi probs are both hybridised to their adjacent targets that they can be ligated.And only the ligated probes will be amplified exponentially in a PCR reaction. Therefore, number of ligation probes depends upon the number of target sequences in the sample .

In case of point mutation, only the sequences that contain the SNP will be amplified.

Critical Elements of probe and primers:-

For detecting a point mutation using probes to detect SNP, one probe oligonucleotide will contain the sequence recognized by the forward primer, the other will contain the sequence recognized by the reverse primer.The two probe oligonucleotide will be complement to DNA sequence with point mutation.

Ligase enzyme catalyze the ligation of oligonucleotide probes after they hybridize with the DNA sequence.

gladiator answered 1 year ago

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