Figure 3: Heteroduplex analysis of E. coli ITS.
From: Microbial Typing by Machine Learned DNA Melt Signatures
(a) Clustal Omega Multiple Sequence Alignment of E. coli ATCC 25922 (GenBank Accession number CP009072) ITS short (361 bp) and ITS long (453 bp) sequences. (b) ITS homoduplex-heteroduplex profiles obtained after 20 (1), 25 (2), 30 (3), 35 (4) and 40 (5) number of PCR cycles. Slow migrating bands (at 800 and 1000 bp) were visible starting from cycle number 25, suggesting the heteroduplex nature of the bands. Expected homoduplex bands were at 540 bp (361 bp ITS short + 179 bp of 3′ of 16S and 38 bp of 5′ 23S), and 632 bp (453 bp + 179 bp). Lane M contains 100-bp DNA marker. (c) Agarose gel electrophoresis showing E. coli ITS PCR products treated (2) and untreated (1) with mung bean nuclease, an enzyme that recognizes and cleaves single stranded DNA, even when it is located in double-stranded DNA products. The loss of the higher molecular weight bands confirms the heteroduplex nature of the bands, leaving the true homoduplexes. Lane M contains 100-bp DNA marker. (d) ITS HRM analysis on 20 colonies resulted in 2 distinct melt curve groups (ITS short and ITS long). Combinations of the amplicons from each group did not recreate the original E. coli melt curve.
