LMPCRLigase Mediated Polymerase Chain Reaction
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[4] Nonstandard abbreviations: mtDNA, mitochondrial DNA; np, nucleotide position; MELAS, mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes; DM, diabetes mellitus; RFLP, restriction fragment length polymorphism; LMPCR, ligation-mediated PCR; PNA, peptide nucleic acid; [T.sub.m], melting temperature; and SNP, single-nucleotide polymorphism.
This site is also amplified by LMPCR in parallel as a control of the copy number of mtDNA.
We first performed the LMPCR amplification of the 3243 and 4427 sites, using the ApaI-digested total DNA of the 25D cells.
We then examined whether the existence of 1000-fold more DNA interferes with the LMPCR amplification of the 3243 site.
The relative relationship between the 3243 and 4427 signals was determined with samples taken at 36-40 PCR cycles (results not shown), indicating that the amplification of LMPCR was not saturated (that is, had not reached plateau), and therefore, the comparison between the 3243 and 4427 signals was reliable and valid.
The LMPCR method in this study detects the 3243 mutation signal weaker than the 0.01% control (Fig.
Although semiquantitative, the estimation of heteroplasmy by the LMPCR method in this study should be reliable for the following reasons: (a) this method overcomes the problem associated with heteroduplex formation (5) because we performed PCR after ApaI-digestion, and (b) the authentic ApaI restriction site is used as an internal standard, which corrects for the differences in digestion efficiency between samples.