J. T. Petty, Z. Haung, R. Habbersett,(1) J. H. Jett,(1) and R. A. Keller
Life Sciences Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545
We have demonstrated that our flow cytometry based ultrasensitive fluorescence detection can be used to size DNA fragments ranging from 564 base pairs to 167 kilobase pairs. Size analysis is accomplished by staining the fragments with intercalating dimeric nucleic acid stains, POPO3 or TOTO, which bind stoichiometrically to DNA. When the stained fragments pass through the exciting laser beam, the amount of fluorescence emitted is proportional to the number of base pairs in the fragment.
Small size range samples consist of restriction digests of Lambda phage DNA along with the complete Lambda genome. Large fragment samples consist of a Lambda digest, full length Lambda, and the T4 and T5 phage genomes to provide fragments ranging in size from 17 to 167 kilobases. Samples are analyzed at a concentration (10[-13] M) at which the probability of more than one fragment being present in the probe volume at any one time is very small. Data are collected by recording the time history of detected photons. The integrated intensities from the individual fragments are assembled in a histogram. Histograms are fitted to a sum of Gaussians plus an exponential background function and the centroids of the peaks are plotted versus the known fragment sizes. Resulting calibration curves are linear over the size ranges analyzed. This result is to be compared with the pulsed field gel electrophoresis which results in a highly nonlinear calibration curve.
The size range of DNA fragments that have been analyzed by these techniques is from 0.564 kilobases to 167 kilobases, covering over 2 orders of magnitude. Data collection is accomplished in less than three minutes. Since each fragment is counted, an absolute measure of the number of fragments in each size class is obtained from the measurement. The amount of material required to produce a statistically well defined distribution is small. Data are collected for approximately 10,000 fragments which translates to a mass of approximately 2x10[-l3] grams for the small fragment size range.
We are currently constructing a simple, inexpensive apparatus to perform these measurements. Excitation will be accomplished with a 30 mw diode pumped Nd:YAG laser that emits at 532 nm. Photon detection will be with a solid state avalanche photodiode which provides a logic signal to a multichannel scaler board that resides in a PC. The whole apparatus will be contained in a cubic foot plus the personal computer.
Applications of fragment sizing to the analysis of the size and stability of vector inserts will be discussed.
This work supported by Los Alamos National Laboratory LDRD funds and the DOE/OHER Human Genome Program.
(1) Chemical Science and Technology Division