Capillary Electrophoresis in Forensic Genetics

BIOLOGY/DNA/METHODS/ANALYTICAL TECHNIQUES

Capillary Electrophoresis in Forensic Genetics BR McCord, Florida International University, Miami, FL, USA E Buel, State of Vermont Forensic Laboratory, Waterbury, VT, USA ã 2013 Elsevier Ltd. All rights reserved.

Glossary Adenylation Tendency of the polymerase to add an additional base (often adenosine) to the terminal end of the amplified fragment. Capillary gel electrophoresis An analytical procedure in which DNA samples are separated by size based on their ability to migrate through an entangled polymer sieving matrix. CODIS A database of DNA profiles collected from crime scenes, convicted offenders, and missing persons. Electroosomotic flow Bulk flow created in a capillary due to the effects of wall charges in the presence of high electric fields. The effect can create reproducibility problems if the

Introduction Development of methods for amplification and detection of DNA fragments using polymerase chain reaction (PCR) has resulted in rapid and dramatic advances in forensic DNA typing. Using the PCR, it is possible to easily produce analytically significant amounts of a specified DNA product from trace quantities of DNA. In its forensic application, the PCR is used to demarcate and amplify known polymorphic sites on a distinct chromosome and produce discrete and easily characterized fragments of DNA. At present, the most widely utilized method for genetic analysis involves the simultaneous determination of a set of 13 or more short tandem repeats (STRs). Forensic STR loci consist of a repetitive motif of 4–5 bases, with the number of repeats varying from one individual to other. Introduction of PCR-based forensic assays has also resulted in a need for efficient and automated procedures for analysis of the reaction products. This requirement has been the driving force behind the development of capillary electrophoresis (CE) methods for DNA analysis. In CE, DNA separations are performed in thin, 50-mm, fused silica capillaries filled with a sieving buffer. These capillaries have excellent capabilities to dissipate heat, permitting much higher electric field strengths to be used than slab gel electrophoresis. As a result, separations in capillaries are rapid and efficient. Additionally, the capillaries can be easily refilled and manipulated for efficient and automated injections. Detection occurs via fluorescence through a window etched in the capillary. Both single-capillary and capillary-array instruments are available with array systems

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entangled polymer matrix improperly coats the capillary wall. Microfluidic electrophoresis The performance of electrophoresis in small channels etched into plastic and glass plates. PCR Polymerase chain reaction – an enzymatic technique used to amplify DNA fragments. STR Short tandem repeat – a sequence of 4–5 DNA bases repeated for multiple times at a particular genetic locus. The number of repeated sequences can vary between individuals. Stutter The tendency of STR amplifications to produce a smaller peak 1 repeat unit shorter than the main product due to slippage during amplification.

capable of running 16 or more samples simultaneously for increased throughput. The most common forensic markers used in forensic genetics are STRs. STRs are tandemly repeated nucleotide sequences of 2–6 base pairs in length. The number of repeated sequences varies between individuals and results in a high degree of length polymorphism. STRs are abundant throughout the human genome, occurring at an average rate of every 6–10 kb. Tetrameric and pentameric repeats are most commonly used in forensic analysis. These loci tend to produce less stutter than the di- or trimeric repeats and much work has been done to validate their use in forensic casework. A core set of 13 loci has been established by the Federal Bureau of Investigation for use in the Combined DNA Index System (CODIS) (Table 1). An additional set of STRs present on the Y chromosome has also been developed. These markers are particularly useful in resolving mixtures of male and female DNA, as these loci are not present in female cells. CE systems are ideal for separation of STRs because of the ready availability of information on size and intensity of each individual locus. When used with STR analysis, CE systems require specialized techniques. The high ionic strength of the PCR mixture inhibits CE injection methods requiring sample dilution with formamide or deionized water, and separations must be performed using highly viscous entangled polymer buffers for optimum resolution. Detection of STRs is carried out by measuring the fluorescence of dye-labeled primers that have been incorporated into each DNA strand during the amplification process. A single laser is used to detect as many as five different dye labels. Lastly,

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Table 1

13 STR loci approved for use with CODISa

STR locus

Chromosome

Repeat motif

Number of repeatsb

FGA VWA D3S1358 D21S11 D8S1179 D7S820 D13S317 D5S818 D16S539 CSF1P0 TPOX THO1 Amelogenind

4 12 3 21 8 7 13 5 16 5 2 11 X,Y

CTTTc TCTA TCTA TCTA TATC GATA TATC AGAT GATA AGAT AATG TCAT

18–30 11–22 11–20 25–36 8–19 6–15 8–15 7–16 5–15 6–15 6–13 3–11

a

Data obtained from STR base, published by NIST, http://ibm4.carb.nist.gov:8800/dna/ home.htm and from the Profiler and Profiler þ users manuals, Perkin-Elmer, Foster City, CA. b The range of repeats is approximate as new alleles are constantly being discovered. c FGA as well as other loci in this list have complex patterns of repeats. The most common is given. d Amelogenin is a sex-linked marker, which contains a six-base deletion in the X chromosome.

the serial nature of CE separations requires internal and external standardization to achieve highly precise measurements. CE separations are best described by dividing the process into three main steps: separation, injection, and detection.

Theory of CE Separation DNA fragments are difficult to separate under normal CE conditions due to their virtually constant charge-to-mass ratio. Therefore, analyses are performed using a replaceable sieving matrix, consisting of a water-soluble polymer dissolved in a suitable buffer. Such solutions are referred to as entangled polymer buffers and the DNA sieved based on its ability to fit within pores created within the polymer matrix. The fact that these polymer matrices are not rigid makes them different from rigid agarose or polyacrylamide gels traditionally used in DNA analysis. The advantage of using an entangled polymer buffer is that fresh polymer solution can be pumped into the capillary at the conclusion of each analysis, cleaning the capillary and limiting problems with carryover. Experiments carried out using a variety of entangled polymer buffers have shown that with careful optimization of molecular weight and concentration, high-resolution DNA separations can be produced. Several different mechanisms have been postulated to describe the separation of DNA in physical gels. These include transient entanglement coupling, Ogston sieving, and reptation. At low concentrations of polymer, separation takes place by means of a frictional interaction between the DNA and the polymer strands. This mechanism is known as transient entanglement coupling. At higher concentrations of polymer, individual polymer molecule strands begin to interact, producing a mesh. The polymer concentration at which this occurs is

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Electrophoretic flow

––

-

DNA– –

DNA

+ ––

DNA

Electroosmotic flow Figure 1 DNA is sieved through transient pores created in the polymer mesh. Smaller fragments are less impeded by the mesh and elute first. Movement of DNA strands occurs due to counteracting forces. The electric field results in migration of negatively charged DNA, whereas electroosmotic forces created by wall potentials produce a bulk flow in the opposite direction. Polymers like POP4 (4% polydimethylacrylamide) reduce electroosmosis by coating capillary walls producing a more reproducible separation.

known as the entanglement threshold. Above the entanglement threshold, DNA fragments separate by sieving through transient pores created in the polymer mesh (Figure 1). Fragments, which are larger than the average pore size, reptate or move in a snake-like manner through the mesh. The key to producing an acceptable separation is to specify a polymer concentration at which the size of these virtual pores approximates the radius of gyration of the DNA fragment (average size of a DNA fragment in solution). There are a number of key parameters involved in the development of a reliable separation of DNA using entangled polymers. In addition to concentration, the polymer length must be kept to a minimum to reduce viscosity and permit refilling of the capillary. Other important characteristics of entangled polymers include the relative stiffness and polydispersity of the polymer and its ability to coat the capillary walls. Uncoated silica capillaries have significant wall charge at the pH used to separate DNA. These charges can induce a bulk flow in the capillary walls when the electric fields are high. This effect is known as the electroosmotic flow (EOF) and can result in irreproducible changes in DNA migration from run to run. EOF is minimized by using polymers such as poly dimethyl acrylamide (POP), which coat capillary walls and neutralize wall charge effects. Furthermore, internal dye-labeled ladder standards are added to help compensate for any mobility shifts during the run. Another important issue in DNA separation is the flexibility of the molecule, which can be characterized by a parameter known as the persistence length. ssDNA is far more flexible (shorter persistence length) and produces superior separations when compared to dsDNA, which is quite stiff and interacts poorly with the polymer matrix. As a result, it is very important to denature the DNA, and maintain it in its single-stranded state throughout the separation. To do this, the DNA is denatured in formamide before injection, and separations are carried out at elevated temperatures and with high concentrations of denaturants, such as urea and pyrrolidinone, to maintain this denatured state. Generally speaking, dsDNA migrates faster and at lower resolution in standard CE systems. Its appearance can sometimes be observed in improperly denatured samples.

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Injection and Sample Preparation In order to inject a DNA sample, each individual capillary is dipped into a sample vial containing a mixture of PCR products and formamide. A positive voltage is then applied to the capillary to move the negatively charged DNA into the capillary orifice. This type of injection is known as an electrokinetic injection and typically voltages of 3–15 kV are applied over a period of 3–10 s depending on type of the instrument and number of capillaries used. The quantity of DNA onto the capillary by this technique can be described by the following formula:   QDNA ¼ pr 2 ½DNA Et mep þ meor where E is the field strength, t is the time, r is the capillary radius, mep is the electrophoretic flow, and meof is the EOF. From this equation, it is easy to see that longer injections at higher field strengths will inject larger quantities of DNA. However, it should be noted that other ions present in the sample matrix will compete with DNA for injection. Thus, the quantity of DNA injected is also a function of the ionic strength of the solution, as well as the mobility of other negative ions that might be injected instead of DNA. In addition, longer injections can induce band broadening with an overall loss of resolution. In general, electrokinetic injections produce narrow injection zones but are highly sensitive to the sample matrix. The injection of PCR products can produce such problems because of the high ionic strength of the sample matrix (>50 mM Cl). To overcome this problem, PCR samples are typically diluted in deionized formamide. This process serves two purposes: it reduces ionic strength and dentures the DNA, permitting efficient and selective injections. This dilution step increases the quantity of DNA injected through a process known as stacking. Stacking, also called field amplified injection, occurs when the ionic strength of the sample zone is lower than that of the buffer. Since the current through the system is constant, the lack of charge carriers in the sample zone produces a strong electric field that ends abruptly at the interface between the low-ionic strength sample zone and the higher-ionic strength buffer in the capillary (Figure 2).

DNA molecules stack and focus at this interface. Stacking allows a large sample zone to be loaded onto the capillary with a minimum of band broadening increasing both the sensitivity and the efficiency of the injection.

Sample Injection Interestingly, it is possible to further enhance DNA signal intensity by dialyzing the PCR product with spin filtration- or float membrane-based methods. These techniques are known as post-PCR purification methods and are utilized to accentuate the stacking process and can greatly improve sensitivity. Laboratories performing such processes must take care to carefully validate these procedures as they can produce variable results when used with low levels of DNA template. In such cases, replicate analysis and careful attention to contamination controls are necessary.

Detection and Data Analysis Fluorescence detection of DNA by CE methods is achieved by derivatizing the DNA using dyes to produce fluorescent adducts. The dye molecules are covalently bound to the DNA fragments during the PCR process. To do this, a dye molecule is added to the 50 end of one member of each primer pair. Upon completion of the PCR, the targeted DNA molecules are labeled with a fluorophore. By using a variety of different dyes in a single multiplexed reaction, individual loci can be isolated, amplified, and labeled with specific dyes. The dyes used in these reactions absorb at similar wavelengths, but emit at different wavelengths. Thus, a single laser can be used to excite four or more dyes. A multichannel analyzer is then used to identify the specific PCR product by means of the wavelength of emission of the bound dye and the length of the fragment. To minimize interference with other dye-labeled products, the internal sizing standard is labeled with a different fluorescence dye than that of the product. Modern CE systems with five dye capability permit detection of labeled STRs in four fluorescent channels while the fifth channel is reserved for the internal standard. For such systems, specific algorithms have been developed to deconvolute the fluorescence signals and avoid interferences from overlapping dyes.

Sample injection

Sample Preparation Capillary Buffer

+

5 kV

+ + +

Sample zone DNADNA- DNAFigure 2 DNA is injected using an applied voltage. Because electric field is inversely proportional to ionic strength, DNA and other negatively charged ions move rapidly to the interface between the low conductivity sample zone and buffer. This process is known as stacking.

Purified template DNA can be amplified to yield products from a single STR locus or multiple primers can be added to simultaneously amplify multiple STR loci. PCR cocktails are commercially available for multiplex PCRs that include primers for as many as 15 different loci. The products of these reactions are labeled and simultaneously amplified with as many as four different fluorescent dyes. The PCR products are then prepared for CE injection by mixing approximately 1 ml of PCR product with 24 ml of deionized formamide. In addition, a red or orange dye-labeled internal standard is used to permit allele sizing. Care must be taken when using formamide as an injection solvent because the products of its decomposition are ionic and can inhibit the injection process. For this reason, the

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conductivity of the formamide should be tested and control samples should be run before routine analysis. To yield denatured fragments ready for electrophoresis, the prepared sample solution is typically heated at 95  C, and then snap-cooled in an ice bath. Because formamide is such a strong denaturant and can denature properly prepared DNA samples without heating, this heating and cooling step is skipped in many laboratories. Deionized water can be substituted for formamide but in this case, snap cooling is absolutely necessary as long-term DNA stability is compromised in deionized water. Because the quantity of DNA injected is dependent on the ionic strength of the sample solution, it is also possible to greatly enhance DNA injection using what are known as postPCR cleanup procedures. Such procedures should be used with caution as low conductance solutions can be prone to carryover effects and may also result in detection of stochastic amplification. Proper validation of such procedures is important, and consistency can be maintained through good quantification controls and regulation of sample ionic strength.

R¼

397

2ðt2  t1 Þ w1 þ w2

where t is the migration time of the peak and w is the peak width. Since the peak at base line is difficult to determine, the shape of the CE peak can be assumed to be Gaussian with a width of 4s and the above equation can be converted to: R¼

1:18ðt2  t1 Þ wh1 þ wh2

where wh is the peak width at half height. System resolution can also be quickly evaluated using a mixture containing STR alleles that vary by one base. The STR system THO 1 has a variant (allele 9.3) that is one base less than expected. This allele can be mixed in equal proportions with the nonvariant allele 10. After this mixture is run on the capillary, the evaluation of the relative height of the peaks versus the valley point between them yields a ratio that can be monitored to obtain the relative resolution of the system.

Analytical Separation

Genotyping

Electrokinetic injection is routinely employed to apply samples onto the capillary column. Injection time may be varied within certain ranges to affect the amount of sample applied to the column without adversely affecting resolution. Varying the injection time from 1 to 10 s has been shown to increase sample input while maintaining the resolution of the system. A particular advantage of CE is the ability to quickly reanalyze more dilute samples by simply increasing the injection time. As a result, many laboratories will validate two different injection times, one shorter and the other longer. The separation media employed for analysis includes polydimethyl acrylamide, urea, pyrrolidine, and EDTA in a TAPS buffer at pH 8.0. The polymer provides the separation, the TAPS buffer maintains the ionic strength and pH, the urea and pyrrolidine keep the DNA denatured, and the EDTA sequesters metals which can affect DNA resolution. Most forensic laboratories have opted to purchase prepared polymer solutions for reasons of quality control and simplicity of use. Varying the polymer concentration, through the purchase of the appropriate separation media or by preparation, allows the user to fine-tune resolution. The STRs under current use contain alleles that generally differ by 2, 4, or 5 base repeat units, however, variants which contain deletions of a single base can occur in these systems. As a result, it is important to design separation systems that can also resolve variant alleles. In situations where increased resolution of alleles is necessary, column length or polymer concentration can be increased, however, both of these procedures can increase migration times, a concern for laboratories with large numbers of samples. Monitoring the resolution of a system allows the analyst to recognize degradation in column performance or inappropriate sample preparation. As the column ages through continued use or inadequate maintenance, sample resolution may deteriorate. Samples prepared in formamide that has not been sufficiently deionized will also show poor resolution. Resolution between two peaks can be calculated using the standard equation:

Multiple STR loci are determined during a single analysis by adjusting the fragment length of each PCR product and by labeling sets of different primers with dyes that fluoresce at differing wavelengths. Primers are carefully designed to produce DNA with allele sizes that do not overlap. This permits the analysis of 3–4 STR loci, which are labeled with the same color. Figure 3 illustrates this procedure by showing the result of the amplification of a male DNA sample using the Profiler Plus STR kit (Life Technologies™). Each lane in the electropherogram consists of 3 STR loci labeled with a different dye. In addition, a sex marker, amelogenin, is shown in the green lane. Numbers under the individual peaks indicate the allele number and its relative intensity. STR loci are identified by their size and their dye label. The task of separating and identifying the alleles is performed using CE instruments equipped with detector arrays capable of analyzing all dyes simultaneously. The peaks resulting from this analysis can be genotyped through the use of software supplied by the instrument manufacturer. Typically, electrophoresis is conducted by combining amplified products with an internal size standard that is labeled with a fluorescent dye that is different from those used to tag the STR loci. During each analysis a sizing ladder (not shown in Figure 3) is added to provide an internal reference to standardize the electrophoretic run and permit the calculation of the base sizes of the detected peaks. The calculated sizes can be compared to those sizes obtained from a previously run allelic ladder, a control sample containing a mixture of all possible alleles. Once allele sizes are determined, a table is prepared of all detected alleles at each locus in a sample. If the sample is from a single source, the frequency of the genotype in a given population can then be calculated by multiplying together the component frequencies calculated for each locus. The resultant frequencies can be quite small. For example, the power of discrimination for the Profiler Plus STR kit is approximately 1 in 1011.

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Figure 3 An analysis of a male sample amplified using the Profiler plus multiplex amplification kit. The results have been split into three panels to aid in analysis. Each gray panel indicates a different STR locus and is identified above the panel. The dark gray zones within the lighter gray indicate potential locations of alleles for each locus. A sex typing locus (Amelogenin) produces a 6-base deletion in the X chromosome.

Mixture Analysis Mixtures (samples that contain DNA from more than one individual) must be anticipated in the analysis of forensic specimens. These specimens may be a composite of body fluids from different individuals and will produce complex DNA profiles. To complicate the analysis of mixtures, STR patterns from one individual may contain imbalanced peaks and PCR artifacts known as stutter. Stutter peaks are amplified products resulting from the ‘slippage’ of DNA polymerase during amplification where the enzyme and growing DNA chain are out of alignment with the target DNA. The resulting fragment is usually four bases less than the true allelic peak although weaker signals consisting of sets of peaks four bases apart may also be seen. Some loci have yielded stutter peaks of >10% of the height of the true peak. In the interpretation of mixtures, the possibility of stutter peaks must be taken into account and interpretations should be adjusted based on the amount of stutter observed at a particular locus. Typically, a mixture is suspected when peak heights rise above typical stutter values for a particular locus. Another problem in the interpretation of mixtures is that peaks obtained from a heterozygous locus may vary by as much as 30%. Deviations of this size, although uncommon, must be considered in the evaluation of mixtures. Differences in the expected peak ratio in a sample that presents a heterozygous pattern can indicate a mixed sample whose alleles have coelectrophoresed. Fortunately, when multiple

allelic systems are evaluated, other loci may show three or four peaks, establishing the specimen as a mixture, and can be used to determine if the altered ratio could be due to an overlapping allele. Figure 4 illustrates the analysis of a sample of mixed DNA. The balance observed between loci is an additional consideration in the assessment of mixtures. Although commercially available STR amplification kits attempt to achieve a balance across all loci, the efficiency of amplification of larger loci can decrease, particularly in situations where degraded DNA is present. Under such circumstances, shorter PCR products will predominate as few of the longer fragments of template have survived. In some situations, minor peaks can disappear later in the electropherogram because of poor amplification or degradation. Nonallelic peaks, occasionally, also cause problems in the interpretation of a mixed sample. These peaks may be the result of unbound dye, electrical interferences, or other sample artifacts. There are also PCR artifacts such as adenylation, which produce peaks one base less than the true allelic peak. In most STR systems, the amplification process promotes the nontemplate addition of a nucleotide (usually an A) to the end of the PCR product. This yields an amplicon that has been increased in size by one base. Under some conditions, such as excessive amount of template DNA or Taq inhibitors, the complete conversion of all the products to the n þ 1 state may not occur. Often

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Figure 4 The analysis of a 1:1 mixture of male and female DNA using the Profiler plus STR kit. Peak ratios at each locus vary depending on the number of shared alleles.

this condition can be rectified by increasing the extension time for the amplification. Finally, mutations or rare genetic events may give rise to unusual profiles that can be mistaken as a mixture. In addition, mutations at primer sites may lead to genotyping variations at particular loci, due to differences in the location of primer annealing sites used by various manufacturers of STR amplification kits.

Analysis of Mitochondrial DNA There are circumstances in forensic analysis in which there is insufficient nuclear DNA to perform PCR. These cases involve samples such as shed hairs or those that are highly degraded. In these circumstances, there may still be enough mitochondrial DNA (mtDNA) to permit PCR amplification. The DNA present in mitochondria is approximately 16 000 bases long and contains a section known as the control region that contains a number of polymorphic sites which are usually point

mutations. In this procedure, certain hypervariable segments of the control region are PCR amplified and sequenced. These sequences are then compared to a known standard in order to identify polymorphic sites. CE is used in this process both to determine if the amplified product is present in sufficient quantity and in the subsequent sequencing. The quantification step is carried out before the sequencing step. A small portion of the amplified product is analyzed in its native state using a short 27-cm capillary at 15 000 V. or a short channel etched in a microfluidic CE chip. In the analysis, an intercalating dye is added to the amplified sample to provide a fluorescent product. Total analysis time is below 4 min. The peak intensity of the amplified DNA is compared to an internal standard to determine the quantity of amplified material. Figure 5 illustrates this separation. The electropherogram is checked to determine if any contaminants, such as primers or extraneous amplified product, is present in the sample. These materials can interfere with the sequencing reactions and

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2000

Multipole PRC products caused by G-stretch stutter

Fluorescence

1500

1000

1000 High-molecularweight marker

Low-molecularweight marker

1500

500

0 30

35

40

45

50

55

60

65

70 75 Time (s)

80

85

90

95

100 105 110 115

Figure 5 The analysis and quantitation of mitochondrial DNA in under 100 s using an Agilent 2100 Bioanalyzer. The quantity of DNA produced is determined with reference to the internal standard. The presence of additional peaks around the main peak indicates the presence of multiple PCR products caused by G stretch stutter and indicates reamplification of this particular sample may be necessary to produce an accurate sequence. Copyright by Agilent Technologies – Reproduced with Permission.

Figure 6 Microfluidic DNA analyzer: This system, currently under development by James Landers at the University of Virginia, combines DNA extraction, PCR amplification, STR separation, and fluorescent detection in a single integrated device.

reduce the quality of the result. The results of this analysis are used to adjust the amount of template used for the sequencing reaction. The products are then analyzed on CE-based sequencers using the same separation principles detailed above.

Future Applications Presently, a number of researchers are developing smaller, more compact systems based on microchip technology. By using photolithography, multiple channels can be etched into silicon wafers and the entire capillary array can be placed on a glass or plastic chip. Tightly focused sample injections on a microchip permit fast electrophoretic separations using relatively short channels. A further advantage of this technique is that sample preparation and detection apparatus can be built into the chip design. Thus, the entire process from DNA extraction to PCR to

separation to detection can be integrated into a single device. Figure 6 shows an example of an integrated microfluidic device in which laboratory procedures such as extraction, amplification, separation, and detection are all combined.

Conclusions CE is a technique which provides the DNA analyst with much flexibility. CE systems utilize replaceable physical gels, which are pumped into the capillary at the beginning of each analysis. DNA quantitation, genotyping, and sequencing are all possible using this technique. Sample injection, separation, and analysis are easily automated. Multichannel fluorescence detection permits multiplex PCRs to be simultaneously analyzed, greatly conserving precious forensic samples. Capillary array systems

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further increase throughput. The resulting data can be analyzed to detect the presence of mixtures, processed, and stored in a database known as CODIS. Present systems under development will utilize microfluidic chips in which integration of the entire process of DNA analysis from extraction to analysis is possible.

See also: Biology/DNA: DNA Databases; DNA Extraction and Quantification; Introduction to Nonhuman DNA Typing; Low-Template DNA Testing; MiniSTRs; Mitochondrial DNA; Mixture Interpretation (Interpretation of Mixed DNA Profiles with STRs Only); Short Tandem Repeats; Single-Nucleotide Polymorphisms; Biology/DNA/Botany: Cannabis DNA Typing Methods; Biology/DNA/Wildlife: DNA and Endangered Species; Methods: Capillary Electrophoresis: Basic Principles; Capillary Electrophoresis in Forensic Biology; Capillary Electrophoresis in Forensic Chemistry; Field-Deployable Devices.

Further Reading Buel E, Schwartz MB, and LaFountain MJ (1998) Capillary electrophoresis STR analysis: Comparison to gel-based systems. Journal of Forensic Sciences 43: 164–170.

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Butler J, Buel E, Crivelente F, and McCord B (2004) Forensic DNA typing by capillary electrophoresis. Electrophoresis 25(10–11): 1397–1412. Easley CJ, Karlinsey JM, Bienvenue JM, et al. (2006) Totally-integrated genetic analysis in an electrophoretic microchip with sample in-answer out capability. Proceedings of the National Academy of Sciences 103(51): 19272–19277. Heller C (ed.) (1997) Analysis of Nucleic Acids by Capillary Electrophoresis. Wiesbaden: Friede Vieweg. Jensen M (2004) Use of the Agilent 2100 Bioanalyzer and the DNA 500 Labchip in the Analysis of PCR Amplified Mitochondrial DNA, Application Note. http://www.chem. agilent.com/Library/applications/5989-0985EN.pdf. Lazaruk K, Walsh PS, Oaks F, et al. (1998) Genotyping of forensic short tandem repeat (STR) systems based on sizing precision in a capillary electrophoresis instrument. Electrophoresis 19: 86–93. Moreno L and McCord B (2008) Separation of DNA for forensic applications. In: Landers J (ed.) Handbook of Capillary Electrophoresis, pp. 761–784. Boca Raton, FL: CRC Press. Rodriguez I, Lesaicherre M, Tie Y, et al. (2003) Practical integration of polymerase chain reaction amplification and electrophoretic analysis in microfluidic devices for genetic analysis. Electrophoresis 24: 172–178. Wallin JM, Buoncristiani MR, Lazaruk K, Fildes N, Holt CL, and Walsh PS (1998) TWGDAM validation of the AmpFlSTR BluePCR amplification kit for forensic casework analysis. Journal of Forensic Science 43: 854–870. Woolley AT, Hadley D, Landre P, deMello AJ, Mathies RA, and Northrup MA (1996) Functional integration of PCR amplification and capillary electrophoresis in a microfabricated DNA analysis device. Analytical Chemistry 68: 4081–4086.