As the demand for DNA typing results increases particularly with the national DNA databases being developed around the world (see Chapter 18), instrumentation capable of high volume sample processing will become more prevalent. Most laboratories are going the route of multi-capillary array electrophoresis systems (see Chapter 14), such as the ABI 3100 and ABI 3700, to meet high-throughput DNA testing needs. In this section, we will review another technology capable of high-throughput testing, which is currently not in use for STR typing.
Time-of-flight mass spectrometry is a technique capable of large-scale sample processing and has been demonstrated to work with STR analysis (Butler and Becker 2001). Mass spectrometry is a versatile analytical technique that involves the detection of ions and the measurement of their mass-to-charge ratio. Due to the fact that these ions are separated in a vacuum environment, the analysis times can be extremely rapid, on the order of seconds. Combined with robotic sample preparation, time-of-flight mass spectrometry offers the potential for processing thousands of DNA samples on a daily basis.
In order to get the DNA molecules into the gas phase for analysis in the mass spectrometer, a technique known as matrix-assisted laser desorption-ionization (MALDI) is used. When MALDI is coupled with time-of-flight mass spectrometry, this measurement technique is commonly referred to as MALDI-TOF-MS. Figure 17.3 shows a schematic of the MALDI-TOF-MS process.
The analysis of DNA using MALDI-TOF-MS proceeds as follows. A liquid DNA sample is combined with an excess of a matrix compound, such as 3-hydroxypicolinic acid (Butler et al. 1998). These samples are spotted onto a metal or silicon plate. As the sample air dries, the DNA and matrix co-crystallize. The sample plate is then introduced into the vacuum environment of the mass spectrometer for analysis. A rapid laser pulse initiates the ionization process.
Figure 17.3 Schematic of MALDI time-of-flight mass spectrometry. DNA samples are mixed with a matrix compound and dried as individual spots on a sample plate. The sample plate is then introduced to the vacuum environment of the mass spectrometer. A small portion of the sample is ionized by an ultraviolet (UV) laser pulse and the generated ions are accelerated through the ion optics. The ions separate by size as they pass down the flight tube. The mass-to-charge ratio (m/z) of each ion is detected as it impacts the detector.
Pulsed UV Laser
Field-free drift region (ions separate by size)
target Ion optics
Sample Plate (Target)
Sample Plate (Target)
The matrix molecules that surround the DNA protect it from fragmentation during the ionization process.
Each pulse of the laser initiates ionization of the sample and the subsequent separation of ions in the flight tube (Figure 17.3). The DNA ions travel to the detector in a matter of several hundred microseconds as they separate based on their mass. However, it takes several seconds to analyze each sample because multiple laser pulses are taken and averaged to form the final mass spectrum. Samples are analyzed sequentially by moving the sample plate underneath a fixed laser beam. Sample plates are now commercially available that can hold 384 (or more) samples at a time. Each sample plate can be analyzed in less than one hour depending on the number of laser shots collected for each sample and the pulse rate of the laser.
Time-of-flight mass spectrometry has the potential to bring DNA sample processing to a new level in terms of high-throughput analysis. However, there are several challenges for analysis of PCR products, such as STRs, using MALDI-TOF-MS. The most significant problem is that resolution and sensitivity in the mass spectrometer are diminished when either the DNA size or the salt content of the sample is too large.
However, STR markers have been successfully analyzed via MALDI-TOF mass spectrometry by redesigning the PCR primers to be closer to the repeat region, and thereby reducing the size of the amplified alleles (Ross and Belgrader 1997, Ross et al. 1998, Butler et al. 1998). The mass spectrum of an allelic ladder for the STR locus TH01, shown in Figure 17.4, demonstrates that STR alleles may be effectively detected with MALDI-TOF-MS. These alleles are 105 bp smaller than corresponding alleles amplified with AmpFlSTR® kit primers for the TH01 STR locus.
Another benefit to MALDI-TOF-MS besides sample analysis speed is accuracy. In fact, the high degree of accuracy for sizing STR alleles using this technique permits reliable typing without the use of an allelic ladder (Butler et al. 1998). Allelic ladders as well as internal sizing standards are necessary in elec-trophoretic separation systems to adjust for minor variations in peak migration times due to fluctuations in temperature and voltage (see Chapter 12).
With mass spectrometry, the actual mass of the DNA molecule is being measured, making it a more accurate technique than a relative size measurement as in electrophoresis. In fact, STR allele measurements taken almost a year apart on different instruments produced virtually identical masses (Butler and Becker 2001). Furthermore, a comparison study of MALDI-TOF-MS results with over 1000 STR alleles measured by conventional fluorescent methods using an ABI 310 demonstrated an excellent correlation between the two methods (Butler and Becker 2001).
Unfortunately, the expense of the MALDI-TOF-MS system, which is on the order of several hundred thousand dollars, and the previous wide-scale acceptance of fluorescent methodologies will likely keep mass spectrometry from becoming a major player in forensic DNA analysis of STR markers. However, it
Mass spectrum of a TH01 allelic ladder obtained with a time-of-flight mass spectrometer (Butler 1998). The ladder contains alleles 5, 6, 7, 8, 9, 9.3 and 10. It was generated by re-amplifying an AmpFlSTR® Green I allelic ladder mix using primers that bind close to the repeat region. The PCR product size of allele 10 is 83 bp with a measured mass of 20280Da. The separation time in the mass spectrometer for allele 10 is only 204 microseconds! The allele 9.3 and allele 10 peaks, which are a single nucleotide apart, differ by 1.5 microseconds on a separation time scale and can be fully resolved with this method.
is an effective means for analysis of single nucleotide polymorphisms (SNPs) and may have a role to play in forensic DNA analysis as SNPs become more widely accepted (see Chapter 8).
Laboratory automation is an important topic, especially since the demand for forensic DNA testing is increasing. Laboratories will take on more cases and have much larger amounts of samples to type because of DNA database laws. While the type of laboratory automation that is currently used by DNA typing laboratories varies widely from little to none, in the future automation will likely play an increasing role in primarily two areas: liquid handling and data analysis.
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