Established Technology

Polyacrylamide gel electrophoresis (PAGE)

For each additional nucleotide unit added to a DNA molecule, the charge is increased proportional to the addition in mass. As a result, DNA fragments larger than about 10 bp possess essentially the same electrophoretic mobility. Since DNA possesses this constant mass-to-charge ratio, some form of separation matrix is needed to separate DNA fragments by their molecular size. In traditional slab gel electrophoresis, the requirement for a sieving matrix is met with polyacrylamide or agarose gels. The movement of larger DNA fragments is impeded relative to that of the smaller DNA fragments as the molecules migrate through the gel under the influence of an electric field.

A cross-linked polyacrylamide gel is prepared by polymerizing acrylamide and bisacrylamide. The physical properties and pore size of the gel are controlled by the proportion of polyacrylamide in the gel and its degree of cross-linking. High resolution, denaturing polyacrylamide gels are used for DNA sequencing and are capable of single base resolution. These sequencing gels have become popular for separating STR alleles {3}.

Polyacrylamide gels are no longer the only slab gel systems available for resolving STR alleles. White and Kusukawa (BioTechniques 1997, 22, 976-980) recently demonstrated that a small (10 cm long, 1 mm thick) agarose gel could have sufficient resolving power to type tetranucleotide repeats. Even dinucleotide repeats could be resolved with MetaPhor agarose and detected with SYBR Green staining.

Native vs. Denaturing separation systems

In native (non-denaturing) gel systems, DNA fragments separate in a double-stranded form. Native gels run faster than denaturing gels {2} and time is saved by not having to perform a denaturation step prior prior to loading the DNA samples. Unfortunately, single base resolution is not easily achieved under native conditions. Additionally, heteroduplex peaks may interfere with correctly calling alleles especially when performing multiplex PCR amplifications.

A denaturing slab gel system is typically produced by adding high levels of urea and/or formamide to the gel. With a denaturing system, the DNA fragments separate and may even travel through the gel matrix at slightly different velocities. When using silver stain detection, a double banding pattern (i.e., each individual strand) may be evident for each allele.

For more information, see Robertson, J.M. (1994) Evaluation of native and denaturing polyacrylamide gel electrophoresis for short tandem repeat analysis. Adv. Forensic Haemogenet. 5: 320-322.

Silver Staining

DNA fragments may be stained with silver nitrate following electrophoretic separation. Silver staining is less expensive than fluorescence detection methods because it does not require expensive intrumentation. However, both strands are detected, which gives a double banding pattern when high resolution denaturing gels are used. The double bands can make interpretation of mixtures more difficult with all of the extra bands.

For more information, see Bassam, et al. (1991) Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal. Biochem. 196: 80-83.

Fluorescent Labeling

Fluorescent labeling of DNA fragments may be performed in several ways. The most common method is to incorporate a fluorescent dye on the 5'-end of a PCR primer so that during PCR amplification either the forward or the reverse strand of DNA will be labeled. In a denaturing gel, the two DNA strands are separated during electrophoresis and only the fluorescently labeled strand is detected. This labeling strategy is most commonly used with automated fluorescent detection systems, such as the ABI 377. Alternatively, intercalating dyes, which insert between the stacked DNA bases, may be used as a post-amplification labeling procedure. SYBR Green, a fluorescent dye from Molecular Probes, may be used to fluorescently stain a gel following electrophoresis. Flatbed scanners may then detect and identify the DNA fragments.

For more information, see Mansfield, et al. (1993) Alternative labeling techniques for automated fluorescence based analysis of PCR products. BioTechniques 15: 274-279.

Flatbed Scanners

Scanners are available for post-electrophoresis detection of DNA fragments. The DNA strands may be fluorescently labeled with either intercalating dyes such as SYBR Green or end-labled with different color dyes, such as FAM, JOE, HEX, etc. Using a scanner for STR analysis has the potential of high throughput with digital data storage because multiple gels may be electrophoresed simultaneously off-line followed by a sequential feeding of the scanner to record the band positions. The Hitatchi FMBIO scanner is available with multiple wavelength detection.

TPOX allelic ladder with samples on either side

Figure courtesy of Margaret Kline (margaret.kline@nist.gov)

Promega multiplexes analyzed on the FMBIO scanner

Representative Publications:

Budowle, B., Koons, B.W., Keys, K.M., Smerick, J.B. (1996) Methods for typing the STR triplex CSF1PO, TPOX, and HUMTH01 that enable compatibility among DNA typing laboratories. Adv. Forensic Haemogenet. 6: 107-114.
 

Automated Detection Systems

Various automated fluorescence detection systems have been used for separation, detection, and typing of STR alleles. These gel-based systems include the Applied Biosystems (ABI) 373A {2}, ABI 377 {134}, the LI-COR Model 4000 {90}, and Pharmacia Biotech's ALFexpress DNA sequencer {133}. In each of these systems, DNA fragments are detected via laser-induced fluorescence of fluorescent tags as the fragments pass a laser which scans across the gel during electrophoresis. An internal lane standard is run in a different color so that each sample may be compared on the same scale. While detection is automated in these systems, gels must still be poured and samples must be loaded manually. In the newer systems (e.g., ref. {134}), multiple samples may be detected in less than two hours. The ABI PRISM systems may detect multiple fluorophores which can permit multiplexing of STR loci that overlap in size.

Representative Publications:

Decorte, R. and Cassiman, J.-J. (1996) Evaluation of the ALF DNA sequencer for high-speed sizing of short tandem repeat alleles. Electrophoresis 17: 1542-1549.

Frazier, R.R.E., Millican, E.S., Watson, S.K., Oldroyd, N.J., Sparkes, R.L., Taylor, K.M., Panchal, S., Bark, L., Kimpton, C.P. and Gill, P.D. (1996) Validation of the Applied Biosystems Prism^TM 377 automated sequencer for forensic short tandem repeat analysis. Electrophoresis17: 1550-1552.

Roy, R., Steffens, D.L., Gartside, B., Jang, G.Y., Brumbaugh, J.A. (1996) Producing STR locus patterns from bloodstains and other forensic samples using an infrared fluorescent automated DNA sequencer. J. Forensic Sci. 41(3): 418-424.

Montagna, P., Pezza, A.L., Tavano, S., Spinella, A. (1994) Comparison of manual and automated detection for STRs analysis. Adv. Forensic Haemogenet. 5: 317-319.

Capillary Electrophoresis

Separation of STR alleles may be performed in a matter of minutes rather than hours with capillary electrophoresis (CE). The narrow capillary used in CE (e.g., 50 mm internal diameter) permits effective dissipation of heat generated from using high voltages, and these high voltages translate to more rapid DNA separations. Another important advantage includes full automation of the electrophoresis process with no need to pour the gel or manually pipet the samples onto the gel. McCord and coworkers {42}first demonstrated that alleles from D1S80 and SE33 could be separated efficiently with CE. More sensitive detection was demonstrated with laser-induced fluorescence of intercalating dyes bound to the DNA fragments {43}. Srinivasan et al. {52} used fluorescent intercalators to resolve D1S80 and apoB alleles. Williams and coworkers {46} examined conditions for separating HUMTH01 alleles with various CE instruments and buffer conditions. Butler et al. {44} was the first to demonstrate actual typing using internal standards to bracket the alleles of HUMTH01. An allelic ladder was first run with the internal standards to calibrate the DNA migration times followed by analysis of the samples with the same internal standards. This CE method was later used to compare 100 HUMTH01 samples {37} to results obtained by conventional PAGE silver staining. Complete correlation was observed between an established gel technique and CE {37}. Another approach has been demonstrated with dual wavelength detection, similar to that used with the ABI PRISM systems. Wang et al. {38} used energy-transfer primers to type HUMTH01 by coelectrophoresing the allelic ladder and samples labeled with different color dyes. The advantage of the ET primers includes better sensitivity, less spectral cross-talk, and better matching migration times between dye labels {38,40}. Recent work with the new ABI 310 Genetic Analyzer have been promising (McCord et al., Proceedings from the Seventh International Symposium on Human Identification (1996), pp. 116-122). Unfortunately the separation speeds available on other CE systems cannot be obtained due to the long capillary required by the 310. Yet, there are advantages with the 310 system, primarily in the automation and the available multiple color detection format.

Early on in the development of CE, one of the major concerns included sample preparation {46}. PCR-amplified samples had to be dialyzed to remove salts which interfered with the injection of DNA fragments onto the CE column. With the higher sensitivity of laser-induced fluorescence, sample preparation is no longer a major concern. Samples may be diluted in water or formamide and easily detected {37}. Another concern has been the overall sample throughput. While CE is rapid on a per sample basis, it is a sequential technique where only one sample is analyzed at a time. Therefore, throughput is on the same time scale as, or even slower than, conventional PAGE methods. However, new 96-capillary array instruments (e.g., MegaBACE 1000 and ABI Prism 3700) are being used in production sequencing laboratories to dramatically improve the throughput of sample processing.  It will probably only be a matter of time before these same instruments are routinely applied to STR genotyping.

Representative Early CE Publications:
McCord, B.R., Jung, J.M., Holleran, E.A. (1993) High resolution capillary electrophoresis of forensic DNA using a non-gel sieving buffer. J. Liq. Chromatogr. 16: 1963-1981.

Butler, J.M., McCord, B.R., Jung, J.M., Allen, R.O. (1994) Rapid analysis of the short tandem repeat HUMTH01 by capillary electrophoresis. BioTechniques 17: 1062-1070.

Williams, P.E., Marino, M.A., Del Rio, S.A., Turni, L.A., Devaney, J.M. (1994) Analysis of DNA restriction fragments and polymerase chain reaction products by capillary electrophoresis. J. Chromatogr. A 680: 525-540.
 

Work with ABI Prism 310 Genetic Analyzer

Since the introduction of the ABI Prism 310 Genetic Analyzer in 1995, a number of papers have been published demonstrating the effectiveness of this CE instrument to separate and genotype STR alleles.  Perhaps more importantly, the ABI 310 is being routinely used today in a number of forensic DNA laboratories around the world.  The ABI 310 offers four-color separation in addition to single-base resolution to approximately 350 nucleotides with a denaturing environment replaceable polymer solution.  As has been demonstrated by Buel and coworkers (1998) and Lazaruk et al. (1998) comparable separations have been obtained between gel systems and the ABI 310.  Samples are processed in a serial fashion in approximately 30 minutes each using the standard POP-4 polymer, 47-cm capillary, and GS STR POP4 (1mL) F Module.
 

Representative ABI 310 Publications:
Butler, J.M., Buel, E., Crivellente, F., McCord, B.R. (2004) Forensic DNA typing by capillary electrophoresis: using the ABI Prism 310 and 3100 Genetic Analyzers for STR analysis. Electrophoresis, in press.

Buel, E.; Schwartz, M.; LaFountain, M. J. (1998) Capillary electrophoresis STR analysis: Comparison to gel-based systems. J.Forensic Sci. 43: 164-170.

Isenberg, A. R.; Allen, R. O.; Keys, K. M.; Smerick, J. B.; Budowle, B.; McCord, B. R. (1998) Analysis of two multiplexed short tandem repeat systems using capillary electrophoresis with multiwavelength florescence detection. Electrophoresis 19: 94-100.

Lazaruk, K.; Walsh, P. S.; Oaks, F.; Gilbert, D.; Rosenblum, B. B.; Menchen, S.; Scheibler, D.; Wenz, H. M.; Holt, C.; Wallin, J. (1998) Genotyping of forensic short tandem repeat (STR) systems based on sizing precision in a capillary electrophoresis instrument. Electrophoresis  19: 86-93.

Wenz, H. M.; Robertson, J. M.; Menchen, S.; Oaks, F.; Demorest, D. M.; Scheibler, D.; Rosenblum, B. B.; Wike, C.; Gilbert, D. A.; Efcavitch, J. W. (1998) High-precision genotyping by denaturing capillary electrophoresis. Genome Res. 8: 69-80.
 

Emerging Technology

Capillary Array Electrophoresis

The real power of CE, in terms of sample throughput, is available with capillary array electrophoresis (CAE), where multiple capillaries are run in parallel. Separation times may be on the order of 20-40 minutes or longer, but with 48 or 96 samples analyzed simultaneously. Lanes do not have to be tracked like in a gel system because each DNA sample is in an individual capillary. CAE was first described in 1992 by Mathies and are now available in a commercial format through Molecular Dynamics (Sunnyvale, CA) and PE Applied Biosystems (Foster City, CA). A recent demonstration of the throughput capabilities of CAE included the generation of over 8,000 genotypes on a 48-capillary instrument in a matter of days {79}. This type of CAE instrument will probably find application in laboratories generating large databases of DNA profiles {77}.

Representative Publications:
Wang, Y., Ju, J., Carpenter, B., Atherton, J.M., Sensabaugh, G.F., Mathies, R.A. (1995) High-speed, high-throughput TH01 allelic sizing using energy transfer fluorescent primers and capillary array electrophoresis. Anal. Chem. 67: 1197-1203.

Mansfield, E.S., Vainer, M., Enad, S., Barker, D.L., Harris, D., Rappaport, E., Fortina, P. (1996) Sensitivity, reproducibility, and accuracy in short tandem repeat genotyping using capillary array electrophoresis. Genome Res. 6: 893-903.

Huang, X.C., Quesada, M.A., Mathies, R.A. (1992) DNA sequencing using capillary array electrophoresis. Anal. Chem. 64: 2149-2154

Microchip CE

The advent of photolithography has permitted micro-machining capillary electrophoresis channels in glass. 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 less than a few centimeters in length. PCR and CE have recently been integrated on a microchip (Woolley 1996). Such a device may someday prove useful in rapidly analyzing DNA fragments in a remote setting away from the traditional laboratory.

More recently, Dieter Schmalzing and coworkers at the MIT Whitehead Institute have demonstrated rapid STR separations using a single color (Schmalzing 1997) and a dual-wavelength laser-induced fluorescence detection system (Schmalzing 1999).  Tetranucleotide repeat alleles from the four-locus multiplex of CSF1PO, TPOX, TH01, and VWA were separated in as little as 45 seconds using a 2.6-cm long separation channel. Single base resolution needed to resolve TH01 alleles 9.3 and 10 was accomplished by decreasing the separation speed to ~10 minutes (Schmalzing 1999).  Samples are genotyped by mixing the PCR products with standard allelic ladders in lower concentrations.

Capillary array electrophoresis microchips have also been developed to increase sample throughput as well.  A 96-channel device with the separation channels constructed in a radial format has been used to separate restriction digests in less than 120 seconds for all samples (Shi 1999).  Genotyping information can therefore be generated at a rate of less than two seconds per sample!

Representative Early Microchip CE Publications:
Woolley, A.T. and Mathies, R.A. (1994) Ultra-high-speed DNA fragment separations using microfabricated capillary array electrophoresis chips. Proc. Natl. Acad. Sci. USA 91: 11348-11352.

Jacobson, S.C. and Ramsey, J.M. (1996) Integrated microdevice for DNA restriction fragment analysis. Anal. Chem. 68: 720-723.

Woolley, A.T., Hadley, D., Landre, P., deMello, A.J., Mathies, R.A., Northrup, M.A. (1996) Functional integration of PCR amplification and capillary electrophoresis in a microfabricated DNA analysis device. Anal. Chem. 68: 4081-4086.

More Recent Microchip CE Publications:
Schmalzing, D.; Koutny, L.; Adourian, A.; Belgrader, P.; Matsudaira, P.; Ehrlich, D. (1997) DNA typing in thirty seconds with a microfabricated device. Proc.Natl.Acad.Sci.USA 94, 10273-10278.

Schmalzing, D., Koutny, L., Chisholm, D., Adourian, A., Matsudaira, P., Ehrlich, D. (1999) Two-color multiplexed analysis of eight short tandem repeat loci with an electrophoretic microdevice.  Anal. Biochem. 270: 148-152.

Shi, Y., Simpson, P.C., Scherer, J.R., Wexler, D., Skibola, C., Smith, M.T., Mathies, R.A. (1999) Radial capillary array electrophoresis microplate and scanner for high-performance nucleic acid analysis. Anal. Chem. 71: 5354-5361.
 

MALDI-TOF Mass Spectrometry

Mass spectrometry offers unprecented speed for DNA fragment analysis. The mass of a DNA fragment may be obtained in a fraction of a second. Many improvements have been made in recent years to overcome the difficulties of fragmentation and poor ionization of large DNA fragments. Great strides have been made in improving the reproducibility of the technique. Nevertheless, MALDI-TOF detection of STR alleles in the size range of 150 bp have been reported (Taranenko 1998). Scientists from GeneTrace Systems (Alameda, CA) have demonstrated that STR systems, including HUMTH01, FES/FPS, F13A1, and CSF1PO, may be efficiently detected by mass spectrometry using proprietary chemistry (Becker 1997) and by redesigning the PCR primers to be closer to the repeat region (Ross 1997, Butler 1998). As part of a research effort funded by the National Institute of Justice, GeneTrace demonstrated that reliable genotyping of STRs could be performed in less than 5 seconds per sample with a sample throughput of several thousand samples per instrument per day (Butler 1999).

Representative Publications:
Monforte, J.A. and Becker, C.H. (1997) High-throughput DNA analysis by time-of-flight mass spectrometry. Nature Medicine 3: 360-362.

Becker, C.H., Li, J., Shaler, T.A., Hunter, J.M., Lin, H., Monforte, J.A. (1997) Genetic analysis of short tandem repeat loci by time-of-flight mass spectrometry. Proceedings from the Seventh International Symposium on Human Identification (Promega 1996), pp. 158-162.

Ross, P.L., Belgrader, P. (1997) Analysis of short tandem repeat polymorphisms in human DNA by matrix-assisted laser desorption/ionization mass spectrometry.  Anal Chem 69: 3966-3972.

Taranenko, N.I., Golovlev, V.V., Allman, S.L., Taranenko, N.V., Chen, C.H., Hong, J., and Chang, L.Y. (1998) Matrix-assisted laser desorption/ionization for short tandem repeat loci. Rapid Commun.Mass Spectrom. 12: 413-418.

Butler, J.M., Li, J., Shaler, T.A., Monforte, J.A., Becker, C.H. (1998) Reliable genotyping of short tandem repeat loci without an allelic ladder using time-of-flight mass spectrometry.  Int. J. Legal Med. 112: 45-49.

Butler, J.M. and Becker, C.H. (1999) Improved analysis of DNA short tandem repeats with time-of-flight mass spectrometry. Final Report on NIJ Grant 97-LB-VX-0003. National Institute of Justice, August 1999.
 

A Comparison of DNA Separation Technologies
 

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