Fig 6. Cloning an earlier ending of 16S rRNA with 11 mismatches and a poly-A tail.

A: Using our newA and mtF2006 primers in PCR to amplify the newA-primed RT product from HEK293 cells, we obtained a cDNA that contains the 2006-2274^th-bp region of the mtDNA in AY195786.2, followed by a 16-nt sequence (shaded) that has four mismatches (in lowercase letters) to all mtDNA references we can find, and then a poly-A tail (underlined). Our newA primer contains 17 As (shaded), which constitute part of the poly-A tail, and a 23-nt sequence that is our newC primer (boldfaced). The 2006-2274^th-bp region contains 11 single-nt mismatches (shaded). B: Alignment of our sequence with BC000845.1, which is a normal mt sequence and is 24-nt longer (shaded) than our sequence before a poly-A tail. Note that within the 2006-2274^th-bp region, the 11 mismatches (shaded) occur only at A and C, compared with the BC000845.1 or with all mtDNA references we can find. C: Image of mtDNA isolated from HEK293 cells using the simple method we developed. The mtDNA that migrated much slower than the 12-kb band in the linear DNA markers (M) in a 1% agarose gel was excised from the gel and purified for PCR, cloning and sequencing.