Rapid DNA sequencing by horizontal ultrathin gel electrophoresis.

A horizontal polyacrylamide gel electrophoresis apparatus has been developed that decreases the time required to separate the DNA fragments produced in enzymatic sequencing reactions. The configuration of this apparatus and the use of circulating coolant directly under the glass plates result in heat exchange that is approximately nine times more efficient than passive thermal transfer methods commonly used. Bubble-free gels as thin as 25 microns can be routinely cast on this device. The application to these ultrathin gels of electric fields up to 250 volts/cm permits the rapid separation of multiple DNA sequencing reactions in parallel. When used in conjunction with 32P-based autoradiography, the DNA bands appear substantially sharper than those obtained in conventional electrophoresis. This increased sharpness permits shorter autoradiographic exposure times and longer sequence reads.     

Capillary gel electrophoresis for rapid, high resolution DNA sequencing.

Capillary gel electrophoresis has been demonstrated for the separation and detection of DNA sequencing samples. Enzymatic dideoxy nucleotide chain termination was employed, using fluorescently tagged oligonucleotide primers and laser based on-column detection (limit of detection is 6,000 molecules per peak). Capillary gel separations were shown to be three times faster, with better resolution (2.4 x), and higher separation efficiency (5.4 x) than a conventional automated slab gel DNA sequencing instrument. Agreement of measured values for velocity, resolution and separation efficiency with theory, predicts further improvements will result from increased electric field strengths (higher voltages and shorter capillaries). Advantages of capillary gel electrophoresis for automatic DNA sequencing instruments and for genomic sequencing are discussed.     

Two-label peak-height encoded DNA sequencing by capillary gel electrophoresis: three examples.

We report a modification to the peak-height encoded DNA sequencing technique of Tabor and Richardson. As in the original protocol, the sequencing reaction uses modified T7 polymerase with manganese rather than magnesium to produce very uniform incorporation of each dideoxynucleoside. To improve sequencing accuracy, two fluorescently labeled primers are employed in separate sequencing reactions. As an example, one sequencing reaction uses a FAM-labeled primer with dideoxyadenosine triphosphate and dideoxycytosine triphosphate; the concentrations of ddATP and ddCTP are adjusted to produce a 2:1 variation in the relative intensity of fragments. The second sequencing reaction uses a TAMRA labeled primer with dideoxythymidine triphosphate and dideoxyguanidine triphosphate; the concentrations of ddTTP and ddGTP are adjusted to produce a 2:1 variation in relative intensity of fragments. The pooled reaction products are separated by capillary gel electrophoresis and identified by one of three different detector systems. Use of a 2:1 peak height ratio typically produces a sequencing accuracy of 97.5% for the first 350 bases; a 3:1 peak height ratio improves accuracy to 99.5% for the first 400 bases. For these experiments, capillary electrophoresis is performed at an electric field of 200 V/cm; two to three hours are required to separate sequencing fragments up to 400 nucleotides in length.     

Misincorporation during DNA synthesis, analyzed by gel electrophoresis.

A method has been developed for simultaneous comparison of the propensity of a DNA polymerase to misincorporate at different points on a natural template-primer. In this method elongation of a [5'-32P] primer, annealed to a bacteriophage template strand, is carried out in the presence of only three dNTPs (highly purified by HPLC). Under these conditions the rate of primer elongation (monitored by gel electrophoresis/autoradiography) is limited by the rate of misincorporation at template positions complementary to the missing dNTP. Variations in the rate of elongation (revealed by autoradiographic banding patterns) reflect variations in the propensity for misincorporation at different positions along the template. The effect on primer elongation produced by addition of a chemically modified dNTP to 'minus' reactions reveals the mispairing potential of the modified nucleotide during DNA synthesis. By use of this electrophoretic assay of misincorporation we have demonstrated that the fidelity of E. coli DNA polymerase I varies greatly at different positions along a natural template, and that BrdUTP and IodUTP can be incorporated in place of dCTP during chain elongation catalyzed by this enzyme.     

Transient orientation of linear DNA molecules during pulsed-field gel electrophoresis.

The transient orientation of lambda DNA and lambda-DNA oligomers has been measured during pulsed field gel electrophoresis. The DNA becomes substantially aligned parallel to the electric field E. In response to a single rectangular pulse, orientation shows an overshoot with a peak at 1 second, then a small undershoot, and finally a plateau. When the field is turned off, the orientation dissipates in two distinct exponential phases. Field inversion leads to periods of orientation with intervening periods of reduced orientation as the chains reverse direction. Field inversion pulses applied to linear oligomers of lambda-DNA show that orientation responses slow down but increase in amplitude as molecular weight increases, for a given field. Because DNA stretching and alignment parallel to E are expected to correlate with DNA velocity, the velocity in response to a pulsed field is also expected to exhibit an overshoot.     

DNA sequencing with direct blotting electrophoresis.

A method for transferring the DNA molecules of sequencing reaction mixtures onto an immobilizing matrix during electrophoresis has been developed. A blotting membrane moves with constant speed across the end of a very short, denaturing gel and collects the molecules according to size. A constant distance between bands for molecules differing in length by one nucleotide is obtained over a large range (approximately 600 nucleotides with a 5% gel), simplifying the determination of DNA sequences considerably. Reliable sequences of 500 nucleotides can be read and sequence features up to greater than 1000 nucleotides are revealed in a single experiment. The sequencing of a potential Z-DNA-forming fragment from Escherichia coli DNA is given as an example and possible further developments are discussed.     

Minimal electrophoresis time for DNA sequencing.

This study presents a mathematical approach that allows one to determine the shortest electrophoresis time and migration path length required for DNA sequencing. The calculation was applied to the capillary electrophoresis of a DNA sequencing separation and showed that acceptable resolution could be obtained using a shorter path length than anticipated.     

Automated DNA sequencing: ultrasensitive detection of fluorescent bands during electrophoresis.

A simple system has been designed enabling ultrasensitive on-line detection of fluorescently labelled macromolecules, e.g. nucleic acids, proteins and peptides during electrophoretic separations in gels. An important application is the automated DNA sequence determination without radioactivity. Drying of gels, film exposure and handling are not necessary. A sulphydryl containing M13 sequencing primer has been synthesised and end-labelled in a reaction with fluorescein iodoacetamide. This is then used in the dideoxy reactions. In particular no moving parts or complicated software are required for data collection and analysis. Compared to our first automated device detection sensitivity has been improved by a factor of thirty to about 3 X 10(-18) mol per band. The resolution has increased to about 400 bases in 5 hours, with the possibility to read up to about 500 bases when they are properly labelled. Gels shorter than 20 cm may be used for resolution of about 300 bases. The single gel system may be upgraded for simultaneous running and reading of six or ten sequencing samples.     

Pulsed-field gel electrophoresis of circular DNA.

Mobility of supercoiled (form I) and nicked circular (form II) plasmid DNAs was determined on two major forms of pulsed-field electrophoresis, CHEF and OFAGE. Plasmids with molecular lengths ranging from 2.30 to 17.8 kilobase pairs (kb) were used with Saccharomyces cerevisiae chromosomes as standards. Agarose gel concentrations were varied from 0.3 to 2.0 percent, with higher percentage gels resolving forms I and II of smaller plasmids. The pulsing range of 3.7 to 240 seconds resulted in quite variable Saccharomyces chromosomal mobilities on both 0.5 and 1.0 percent gels, while both form I and II of all plasmid DNAs showed relatively constant mobilities with some increase at the shortest pulse times. Using a 30 second pulse time and gel concentrations of at least 1.0 percent, the usual order of migration of plasmid forms for a 17.8 kb plasmid could be changed. We interpret this result as an increase in the relative mobility of form II in our pulsed-field gel conditions.     

The acceleration of linear DNA during pulsed-field gel electrophoresis

The velocity and orientation of T4 and lambda DNA have been measured for the first 20 s during pulsed-field gel electrophoresis in order to clarify the DNA motions that occur. For a square pulse with field strength E = 10 V/cm, the velocity of lambda DNA increases gradually to 10.5 microns/s in 1.0 s, declines to 8.6 microns/s, and then rises to a plateau value of 9.3 microns/s after 4 s. T4 DNA behaves similarly, but more slowly. Parallel measurements of fluorescence-detected linear dichroism show that the DNA becomes substantially aligned with its chain axis parallel to the electrophoretic field E after the pulse is applied. The alignment also shows an overshoot, an undershoot, and a plateau comparable to those seen for velocity. When the field strength increases, both the velocity and the alignment reach their peaks more quickly. For all field strengths and both molecular weights, the velocity peak occurs when the molecular center of mass has moved 0.3 to 0.5 L, where L is the chain contour length. A qualitative model is provided.     

High speed DNA sequencing by capillary electrophoresis.

A major challenge of the Human Genome Initiative is the development of a rapid, accurate, and efficient DNA sequencing technology. A major limitation of current technology is the relatively long time required to perform the gel electrophoretic separations of DNA fragments produced in the sequencing reactions. We demonstrate here that it is possible to increase the speed of sequence analysis by over an order of magnitude by performing the electrophoresis and detection in ultra thin capillary gels. An instrument which utilizes these high speed separations to simultaneously analyze many samples will constitute a second generation automated DNA sequencer suitable for large-scale sequence analysis.     

Fast gel electrophoresis to analyze DNA-protein interactions

A rapid method for electrophoresis of DNA-protein complexes is described. Popular "gel-shift" assays are performed using Pharmacia PhastSystem with its convenience of pre-cast gels and buffer strips and microprocessor-controlled high-resolution separation. Using this system, the products of a DNA binding reaction (DNA-protein complexes) can be separated from "free" DNA in less than one hour. DNA fragments as well as oligonucleotides have been used as targets with partially purified extracts containing sequence-specific DNA binding proteins. We present here a comparison of results of gel-shift assays obtained by conventional techniques and by our rapid "PhastShift" method which reduces the time, effort and technical expertise necessary to obtain reproducible results.     

On the movement and alignment of DNA during 120 degrees pulsed-field gel electrophoresis.

The displacement per pulse of lambda, T4, and G DNA during pulsed-field agarose gel electrophoresis has been measured for a fine mesh of pulse durations T between 0.02 and 120 s. The slopes of these curves show that the DNA moves by two distinct processes, designated 1 and 2, depending upon the pulse duration T. Process 1 operates at short T and causes dx/dT to decrease gradually with increasing T. This process is independent of molecular weight M. Process 2 is effective at longer T and causes dx/dT to rise sharply in sigmoidal fashion at a value of T which increases as M1.2, finally reaching a plateau of 1.4 microns/s for E = 4 V/cm. The shape of the dx/dT curve and its dependence on M lead directly to 4 zones of separation in plots of mobility vs M for different T. The alignment of the 3 DNAs during PFGE was measured by fluorescence-detected linear dichroism for E between 4 and 10 V/cm. These results are used in developing a molecular understanding of the mobility data.     

Transverse agarose pore gradient gel electrophoresis of DNA.

Transverse agarose pore gradient gels were prepared on GelBond in the concentration range of nominally 0.2-1.5% SeaKem GTG agarose, using density stabilization by glycerol and incorporation of a dye to define the gel concentration at each point on the pore gradient gel. The distribution of the dye was evaluated by photography, video-acquisition and digitization of the gradient mixture and by densitometry of the gel. The gel was applied to the electrophoresis of a 1-kb standard ladder of DNA fragments, using standard submarine apparatus. The method extends to agarose gel electrophoresis the benefits of semi-automated analysis of 'Ferguson curves' described in application to polyacrylamide gel by Wheeler et al. (J. Biochem. Biophys. Methods 24, 171-180).     

Orientation and stretching of DNA chains during pulsed field gel electrophoresis

The quantitative analysis of the decay of birefringence accompanying gel electrophoresis leads to the characterization of two phenomena respectively attributed to alignment of the tube and to overstretching of the chain in its tube. The major contribution to the molecular orientation is given by the overstretching which relaxes according to a stretched exponential law. The other process, slow, is characterized by a reptation time and a mean orientation factor in good agreement with the biased reptation model without overstretching.     

Scrambling of bands in gel electrophoresis of DNA.

Under certain conditions of agarose gel electrophoresis, larger DNA molecules migrate faster than smaller ones. This anomalous mobility of DNA, which can lead to serious errors in the measurement of DNA fragment lengths, is related to near-zero velocity conformations which can trap DNA chains during electrophoresis. Intermittent electric fields can be used to alter the chain conformations so as to restore the monotonic mobility-size relationship which is necessary for a correct interpretation of the gel. These data are in agreement with the results of a computer simulation based on a theoretical model of electrophoresis.