Future Trends For Ce And Dna Analysis

6.1. Capillary Array Electrophoresis

Because CE is by nature a serial operation, a single capillary system cannot match the sample throughput available in a multi-lane slab gel experiment. Higher throughput, however, is available with capillary array electrophoresis (CAE), where multiple capillaries are run in parallel (69). Separation times are on the order of 15-50 min, but up to 96 samples can be analyzed simultaneously. Each DNA sample is analyzed in an individual capillary and migration times are adjusted using an internal lane standard. A recent demonstration of the throughput capabilities of CAE included the generation of over 8000 genotypes on a 48-capillary instrument in a matter of days (61). This type of CAE instrument will probably find application in laboratories which genotype a large number of samples.

6.2. Automation and CE

Automation of the entire process from extracted DNA to analyzed PCR product is an important issue for large laboratories. The potential of coupling CE analysis to the previous sample preparation steps of DNA extraction, PCR amplification, or restriction digestion has already been demonstrated with robotics (70,71) and microchip fluidics (46,71). The rapid speed of CE separations cannot be translated into sustainable high-throughput operations until the entire process, including sample preparation and data analysis, is fully automated.

6.3. Microchip CE Assays

The advent of photolithography has permitted micromachining of capillary channels in glass (73). Because of the small dimensions of the separation channels, separations may be performed even more rapidly than with conventional CE equipment. DNA restriction fragments have been separated in a matter of seconds using channels that are only a few centimeters in length (46,72,74,75,78,79). One of the major challenges in microchip CE analysis is sample preparation on a scale that is compatible with the small device. Integration of sample preparation steps with the separation portion is one solution to this dilemma (76,77). The recent integration of PCR and CE on a microchip (46) illustrates that such devices may soon be available. Figure 9 gives an example of the separation of a 4 locus multiplex on a microchip containing a 3-cm channel (78).

Detection Amplified Strs
Fig. 9. Microchip separation of a multiplex of 4 STRs: (A) allelic ladder (B) allelic ladder spiked with amplified sample. Conditions: 3-cm separation channel, 200 v/cm. Laser induced fluorescence detection was used with fluorescein labeled PCR products (78).

7. CONCLUSIONS

The advantages of CE, including single-sample analysis speed and automation, have resulted in its increasing visibility in molecular biology and forensic DNA laboratories. Multichannel fluorescence CE systems have become the instrument of choice for STR analysis in many DNA typing laboratories, primarily because of the appealing aspect of unattended operation, a fact that frees busy forensic scientists to work on other tasks. Recently developed commercial 96-capillary array instruments promise to improve dramatically sample throughput capabilities. CE is reliable and reproducible when performed carefully, and as the number and variety of applications increase, a greater role for CE in the future of DNA separations can be anticipated.

8. ACKNOWLEDGMENTS

Major funding for this work was provided by the National Institute of Justice under grants #93-IJ-0030 and 1999-IJ-CX-KO14. The authors would also like to thank Dr. Alice Isenberg for many helpful discussions.

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