Sequences and Cell-free DNA Technologies

May 14, 2025 by Aldevron

Diving into the fidelity of cell-free DNA, Part Two

In my previous post, I discussed cell-free DNA sequence fidelity and one advanced method for error detection, an NGS approach that uses unique molecular identifiers (UMIs) to tag individual DNA strands with barcodes. In this post, I’ll follow up with a second advanced method, which relies on observing a phenotypic change of bacterial colonies to identify replication errors.

While I call this an advanced method, this is a much older technology than today’s sequencing approaches. This technique works on the same principle of blue/white colony screening employed for identifying colonies with a desired cloning product.

A LacZα fragment is included in a plasmid that is transformed back into E. coli cells. This fragment complements an incomplete β-galactosidase gene in the genome resulting in a functional β-galactosidase enzyme. When functional, this enzyme breaks down X-Gal into a blue substance and a corresponding blue colony. If the LacZα fragment does not complement the β-galactosidase gene, the colony will remain white.

Because the LacZα sequence is so well characterized, it is possible to calculate the error rate that takes into account only detectable errors within the LacZα sequence – those that change the phenotype of the colony. This method results in an observable difference that is hard to argue with. A downside is that this method can only be used with this specific sequence.

Once we calculate the error rate of cell-free DNA, the question then turns to how meaningful is this? An error rate of 1 x 10-6 represents one error every million bases. For a 10kb construct, one of every 100 molecules would have an error. Of these errors, many would be silent mutations or ones that do not cause a loss of function. When using the DNA for production of mRNA through IVT, one should also consider the error rate of the T7 RNA polymerase.

It is also worth considering sequence complexity and the variability of living systems. Certain sequences are more difficult to replicate in bacteria and often become truncated through the fermentation process. Things like encoded poly(A) tails or inverted terminal repeats (ITRs) commonly are truncated in E. coli.

Transposable elements can integrate from the bacterial genome into the plasmid. These can cause a much larger issue than the error discussed thus far as these will result in a variant in the consensus sequence. Cell-free DNA technologies are better able to handle these difficult segments.

When considering the error rate of cell-free DNA it is important to consider a number of factors. First, how was the fidelity measured and is it an accurate representation of the true error rate? Currently there is no established method across the industry to consistently measure error rates across different production techniques.

Second, what is the intended application of the DNA? For gene therapy approaches, higher fidelity may be more desired, while for vaccine programs it may not be as stringent.

Please reach out to us to discuss cell-free fidelity further and help to decide if it is a good fit for your program. You can also get more information by viewing my presentation from the 2025 DNA Process Development Symposium.

View the video presentation

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