DNA mapping using microfluidic stretching and single-molecule detection of fluorescent site-specific tags

Abstract

We have developed a rapid molecular mapping technology--Direct Linear Analysis (DLA)--on the basis of the analysis of individual DNA molecules bound with sequence-specific fluorescent tags. The apparatus includes a microfluidic device for stretching DNA molecules in elongational flow that is coupled to a multicolor detection system capable of single-fluorophore sensitivity. Double-stranded DNA molecules were tagged at sequence-specific motif sites with fluorescent bisPNA (Peptide Nucleic Acid) tags. The DNA molecules were then stretched in the microfluidic device and driven in a flow stream past confocal fluorescence detectors. DLA provided the spatial locations of multiple specific sequence motifs along individual DNA molecules, and thousands of individual molecules could be analyzed per minute. We validated this technology using the 48.5 kb lambda phage genome with different 8-base and 7-base sequence motif tags. The distance between the sequence motifs was determined with an accuracy of +/-0.8 kb, and these tags could be localized on the DNA with an accuracy of +/-2 kb. Thus, DLA is a rapid mapping technology, suitable for analysis of long DNA molecules.

Figure 1

Schematics of the Direct Linear Analysis (DLA) technology. (A) A cross-section of the microfluidic DNA stretching microchip (top). A typical design, one of a few, is presented. See text for the exact parameters of the chip used in this study. (B) Optical scheme (side view). The excitation and detection are arranged within a confocal fluorescence microscope. The excitation laser beams are directed into the microscope objective with a dichroic mirror that reflects the light with 532 nm (beam ExI) and 633 nm (beams ExII and ExIII) wavelengths, but is transparent to the fluorescence emission excited by these beams. The emission is further split by another dichroic mirror and bandpass filters. Fluorescence excited by the green laser is delivered by optical fiber to the photon-counting avalanche photodiode (APD) for signal detection in data channel 1. Fluorescence excited by red beams ExII and ExIII is directed to the APDs of data channels 2 and 3, respectively. (C) Typical raw data traces from data channels 1–3 for a single tagged DNA molecule. The red and blue traces arise from fluorescence of the intercalating dye when the DNA backbone travels through the excitation spots ExII and ExIII, respectively. The green spikes are detected when the DNA-bound PNA tags pass through the excitation spot ExI and emit bursts of photons.

Figure 2

Properties of detected molecules of λ phage DNA (48.5 kb) in a representative experiment. (A) The distribution of DNA lengths (sizes of DNA projections X onto the movement direction) for detected DNA molecules. (B) The distribution of burst sizes B (total number of photons per detected molecule) of the stained DNA backbone (data channel 2). The peak corresponding to full-size λ DNA is interpolated with a best fit Gaussian curve. Scatter plots of burst size B vs. DNA length X (C) and of average intensity of DNA backbone I vs. X (D). C and D include points representing single observed DNA molecules stained with the TOTO-3 intercalating dye. Because the points are concentrated in small regions, we have chosen to present their color-coded density rather than the points themselves. The solid red lines in C and D correspond to the center of the Gaussian distribution B_λ interpolating the full-size DNA molecules shown in B. A total of 2373 individual molecular traces are represented in this data file. The data points included in the red box in D were used to calculate one of the DNA maps presented in Figure 3D. This selection contains ∼400 molecules. The corresponding interval, including molecular traces with projections between 16.25 and 17.25 μm, is outlined in red in A.

Figure 3

Mapping of bisPNA tag H-2TMR on λ phage DNA. (A) Theoretical λ DNA (48.5 kb) map. Motif H has sequence (amino terminus)TCCTTCTC(carboxyl terminus) (Table 1), corresponding to the target (5′)GAGAAGGA(3′). Positions of motif target sites are shown with green dashes. These motifs are separated by 17.2 kb. (B) Actual fluorescence data traces from a single motif tagged λ DNA molecule. Motif tag fluorescence (green trace) and intercalated DNA fluorescence (red trace) are detected in data channels 1 and 2, respectively (see also Fig. 1C). The vertical gray line shows the position of the center of the molecule (CM). (C) A map obtained by summing of ∼500 single molecule traces. Gaussian fits to the tag position data are represented by the red line. (D) Oriented maps measured in five different experiments with different sample preparations to illustrate reproducibility. Only Gaussian fits are presented. The maps are aligned in the middle of the interpolated central peak. The maps are normalized to the higher of the two peaks. The average measured geometrical distance between the motif targets is presented.

Figure 4

Theoretical and measured maps of λ phage DNA obtained with H-2TMR, H-1TMR, and bis7 bisPNA tags (Table 1). Motif H has sequence (amino terminus)TCCTTCTC(carboxyl terminus) and motif bis7 has sequence (N)TCCTTCT(C); their corresponding targets are (5′)GAGAAGGA(3′) and (5′)AGAAGGA(3′). (A) Theoretical motif map obtained using either H or bis7. (B–E) Experimental maps obtained with H-2TMR, H-1TMR, and bis7 tags. The data sets used to obtain the maps B, D, and E included 2562, 7794, and 1909 total individual molecular traces, respectively. Map C was obtained using a subset of only 57 molecular traces from the same data set used on map B. These 57 molecules belonged to a very narrow range of burst and linear sizes, representing a small spot within the red box on the graph Figure 2D. All experimental maps are aligned using their centers of molecules (CM, at 0 microns). The center of the theoretical map is aligned with the experimental CM. Theoretical map was scaled using 0.336 μm/kb ratio (see text for details).

Figure 5

Theoretical and measured maps of λ phage DNA obtained with S-1TMR, S-2TMR, S-1Alx, and S-2Alx bisPNA tags (Table 1). These tags all have the same sequence motif (amino terminus)TTTCTCTT(carboxyl terminus), which recognizes the target (5′)AAGAGAAA(3′). (A) Theoretical map obtained for this motif. (B–E) Experimental maps obtained with S-1TMR, S-2TMR, S-1Alx, and S-2Alx bisPNA tags, respectively. The data sets used to obtain the maps B, C, D, and E included 2279, 2275, 145, and 943 total individual molecular traces, respectively. See Figure 4 caption for details.