(412d) Rapid Separation of ?DNA Digests in Entangled Micelle Networks

The
ability to separate nucleic acids quickly and accurately via electrophoretic
means has been imperative in many medical and biological applications. In free
solution, DNA has a length invariant electrophoretic mobility. As such, DNA
electrophoresis is often carried out within a gel or gel-like sieving matrix. As
an alternative method, micelle-ELFSE (end-labeled free-solution
electrophoresis) achieves rapid, length-based separations in free solution by
attaching non-ionic micelles to the end of 5’-alkylated DNA. Separation
resolution is found to be highly sensitive to buffer thermal history and
composition. To increase the read length of micelle-ELFSE, surfactant
compositions and separation temperature were varied to systematically induce
micellar growth. This formulation effort led to a read length of 1150 bases using
a high concentration buffer (5x the overlap concentration) composed of 150 mM C12E5 and 3 M urea (6 wt%) at 33°C. Under these
conditions, unalkylated DNA is separable by a
sieving/reptation mechanism. Yet, end-alkylated DNA
is separated by an ELSFE mechanism as shown by its reversed elution order with
DNA length. As an example, the electropherogram of
Figure 1 has two dsDNA ladders mixed; one that is end-alkylated and another
that is not. The two ladders eluted independently: the unmodified DNA ladder
separates via sieving with short DNA eluting first, whereas an end-alkylated
ladder separates via ELFSE with long DNA eluting first.

Recent
efforts were successful in separating unalkylated and
alkylated kilobase (23kb) double stranded λDNA digests (Figure 2), demonstrating that, in
principal, micelle-ELFSE permits the use of very large micellar drag-tag for kilobase DNA separation despite the presence of
entanglement and sieving. One possible mechanistic explanation of the process
is that alkylated DNA’s alkyl group interact locally with nearby micelle
crosslinks, causing them to dissociate at a greater rate and negating the
sieving effects. In most cases, the mobility of alkylated DNA during
micelle-ELFSE can be accurately predicted by the standard ELFSE equation
assuming the drag-tag and DNA are hydrodynamically equivalent
(non-segregated) during the separation. We do observe deviations from the
non-segregated model for long kilobase DNA in high
concentrated buffers in which the mobility is better predicted by the
segregated model.  We discuss our efforts
to quantitatively model this behavior, and set upper limits for the lengths of
single- and double-stranded DNA that can be resolved in entangled micelle
networks. The ability to rapidly separate unalkylated
and alkylated kilobase λDNA
digests in free solution using standard benchtop CE instrument bypasses many
complications associated with the microarray fabrication process. As such, we compare
separation performance to other common kilobase DNA
separation methods such as entropic trap, nano-arrays,
microfluidic chip, etc.

We
envision many applications for kilobase DNA
separation via micelle-ELFSE. One such application involves using kilobase dsDNA saturated with intercalating fluorophore as
high-affinity probes for sub-fM at-line detection of
viral and bacterial contaminants in cell cultures. Micelle-ELFSE is an
excellent candidate for PCR-less detection of nucleic acid directly from the culture
media given that it’s fast, highly selective and compatible with high levels of
broad-spectrum lipophilic contaminants.

Figure 1. Simultaneous
separation of unalkylated dsDNA PCR prodcuts and alkylated dsDNA with a C16 alkane modification
to bind micelles in the running buffer (2 wt% C12E5).
Unalkylated ladder separates via sieving with shorter
DNA eluting first. Alkylated ladder separates via ELFSE with longer DNA eluting
first.

Figure 2. Simultaneous separation of unalkylated and alkylated HindIII
digest of λDNA. DNA lengths labeled with (*) has
a C18 alkane modification that binds to micelles in the running buffer.