Transition between Two Regimes Describing Internal Fluctuation of DNA in a Nanochannel

Abstract

We measure the thermal fluctuation of the internal segments of a piece of DNA confined in a nanochannel about 50100 nm wide. This local thermodynamic property is key to accurate measurement of distances in genomic analysis. For DNA in 100 nm channels, we observe a critical length scale 10 m for the mean extension of internal segments, below which the de Gennes' theory describes the fluctuations with no fitting parameters, and above which the fluctuation data falls into Odijk's deflection theory regime. By analyzing the probability distributions of the extensions of the internal segments, we infer that folded structures of length 150250 nm, separated by 10 m exist in the confined DNA during the transition between the two regimes. For 50 nm channels we find that the fluctuation is significantly reduced since the Odijk regime appears earlier. This is critical for genomic analysis. We further propose a more detailed theory based on small fluctuations and incorporating the effects of confinement to explicitly calculate the statistical properties of the internal fluctuations. Our theory is applicable to polymers with heterogeneous mechanical properties confined in non-uniform channels. We show that existing theories for the end-to-end extension/fluctuation of polymers can be used to study the internal fluctuations only when the contour length of the polymer is many times larger than its persistence length. Finally, our results suggest that introducing nicks in the DNA will not change its fluctuation behavior when the nick density is below 1 nick per kbp DNA.

Distribution of distances between DNA barcode labels in nanochannels close to the persistence length

“…6 and similar data in the supplementary material. 31 The most likely discrepancy between our measurements and prior work 19 is the advancement in the barcoding technology; we were able to confirm the molecular weight scaling for the variance only by studying a very wide range of molecular weights with the large data set afforded by our high throughput method.…”

“…A previous experimental study 19 of barcode label distances in somewhat larger channels also indicated that the variance about the mean extension is larger than the prediction for the Odijk regime in Eq. (12).…”

“…However, the theory surrounding DNA confinement for channel sizes D ≈ l p is contentious. [16][17][18][19][20][21][22] It would be greatly beneficial to have a fundamental understanding of the thermodynamics of DNA confinement in circa 50 nm channel sizes to guide the development of the emerging genome barcoding technology.…”

Resolution limit for DNA barcodes in the Odijk regime

“…Several competing technologies have arisen to accomplish this task: using a receding contact line and subsequent binding to the surface (molecular combing), [8][9][10] extensional flow (direct linear analysis), [11][12][13] and nanochannel confinement. [5][6][7]14,15 The genomic distance between neighboring barcodes is then determined by first locating the centers of the sequence-specific labels and then integrating the total backbone fluorescence intensity between these two locations. Nanochannels provide a particularly attractive approach to make such measurements since a confined chain fluctuates about its equilibrium extension; making multiple, statistically independent measurements of the distance reduces the sampling error.…”

“…If we consider a sequence-specific probe that emits light with wavelength k ¼ 573 nm (Ref. 14) and a completely extended DNA molecule with a rise of 0.34 nm per base pair (bp), then two probes would have to be separated by at least 843 bp if we take k/2 as an estimate of the resolution limit for diffraction limited optics. However, as we can see from simulation data in Fig.…”