What should a no DNA control look like in MLPA?
No DNA control sample (ligation-independent background) MLPA control reactions that completely lack sample DNA, or in which the Ligase-65 enzyme is omitted, should theoretically not yield any amplification products larger than 100 nt. In practice, however, no DNA controls are often not completely blank in MLPA as the PCR easily finds something to amplify. Compared to conventional PCR, MLPA is more prone to the generation of aspecific long amplification products in the absence of sample. This is because the unlabeled reverse PCR primer can bind to its/the reverse complementary sequence contained/present in the M13-derived oligonucleotide (the RPO), which is thus always linearly amplified even without a ligation event., As a result, a complementary copy of nearly all M13-derived probe oligonucleotides is made in each MLPA PCR cycle, resulting in an estimated 40 x 150,000,000 molecules per cycle – even when no sample DNA was present or no ligation occurred. Furthermore, if after this firs
