Fundamentals of Forensic DNA Typing Chapter 9 DNA Separation & Detection Fundamentals of Forensic DNA Typing Slides prepared by John M. Butler June 2009

Chapter 9 – DNA Separations and Fluorescence Detection Chapter Summary A multiplex PCR amplification of STR markers produces a complex mixture of DNA molecules that must be separated based on DNA size and fluorescent dye label to produce a coherent DNA profile. Original gel electrophoresis separation methods have been almost entirely replaced by capillary electrophoresis (CE) instruments over the past decade due to ease of use and automation. The most commonly used CE systems are the single capillary ABI Prism 310 Genetic Analyzer and the multi-capillary ABI 3100 or 3130xl. These CE instruments electrokinetically inject the negatively charged DNA molecules from a formamide-diluted sample of the PCR products mixed with an internal size standard. The size standard is labeled with a separate fluorescent dye to enable calibration of each analysis so that comparisons can be made between samples run at different times on the same instrument. A polymer solution inside the capillary permits resolution of DNA fragments differing by as little as a single basepair (bp) over a size range of approximately 100 to 400 bp. Fluorescent dyes are present on one strand of each PCR product due to incorporation of a PCR primer during multiplex PCR amplification. These dyes are excited by laser as they pass a detection point in the CE instrument. Since the four or five fluorescent dyes used in STR analysis have different chemical properties, they emit light at slightly different wavelengths enabling detection in different color channels. Because there is overlap with the emitted light from the different dyes, mathematical algorithms are used to perform a “matrix correction” or “spectral calibration” so that individual DNA peaks in an electropherogram appear to be labeled with a single color.

Gel Electrophoresis System - Voltage Gel Loading well + anode cathode Side view Top view Gel lanes DNA bands Buffer Gel stand John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 9.1 Figure 9.1 Schematic of a gel electrophoresis system. The horizontal gel is submerged in a tank full of electrophoresis buffer. DNA samples are loaded into wells across the top of the gel. These wells are created by a ‘comb’ placed in the gel while it is forming. When the voltage is applied across the two electrodes, the DNA molecules move towards the anode and separate by size. The number of lanes available on a gel is dependent on the number of teeth in the comb used to define the loading wells. At least one lane on each gel is taken up by a relative molecular mass size standard that is used to estimate the sizes of the sample bands in the other lanes.

Capillary Electrophoresis System Laser Inlet Buffer Capillary filled with polymer solution 5-20 kV - + Outlet Sample tray Detection window (cathode) (anode) Data Acquisition Sample tray moves automatically beneath the cathode end of the capillary to deliver each sample in succession John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 9.2 Figure 9.2 Schematic of capillary electrophoresis instruments used for DNA analysis. The capillary is a narrow glass tube approximately 50 cm long and 50 microns in diameter. It is filled with a viscous polymer solution that acts much like a gel in creating a sieving environment for DNA molecules. Samples are placed into a tray and injected onto the capillary by applying a voltage to each sample sequentially. A high voltage (e.g., 15,000 volts) is applied across the capillary after the injection in order to separate the DNA fragments in a matter of minutes. Fluorescent dye-labeled products are analyzed as they pass by the detection window and are excited by a laser beam. Computerized data acquisition enables rapid analysis and digital storage of separation results.

Sample Interpretation Mixture of dye-labeled PCR products from multiplex PCR reaction CCD Panel (with virtual filters) Argon ion LASER (488 nm) Color Separation Fluorescence ABI Prism spectrograph Size Processing with GeneScan/Genotyper software Sample Interpretation Sample Injection Sample Separation Sample Detection Sample Preparation Capillary (filled with polymer solution) John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 9.3 Figure 9.3 Schematic illustration of the separation and detection of STR alleles with an ABI Prism 310 Genetic Analyzer.

DNA Separation Modes Ogston Sieving Reptation (a) (b) Gel Gel Larger DNA molecules interact more frequently with the gel and are thus retarded in their migration through the gel Gel DNA Separation Modes (b) Ogston Sieving Reptation Small DNA molecules Long DNA molecules Gel John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 9.4 Figure 9.4 Illustration of DNA separation modes in gel electrophoresis. (a) Separation according to size occurs as DNA molecules pass through the gel, which acts as a molecular sieve. (b) Ogston sieving and reptation are the two primary mechanisms used to describe the movement of DNA fragments through a gel.

Fluorescence and Excitation/Emission Spectra hex hem 1 2 3 So S’1 S1 energy (a) Excitation Emission Wavelength (nm) 1 3 ex max em max Fluorescence (b) Stokes shift John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 9.5 Figure 9.5 Illustration of the fluorescence process and excitation/emission spectra. (a) In the first step of the fluorescence process, a photon (hνex) from a laser source excites the fluorophore (dye molecule) from its ground energy state (S0) to an excited transition state (S’1). The fluorophore then undergoes conformational changes and interacts with its environment resulting in the relaxed singlet excitation state (S1). During the final step of the process, a photon (hνem) is emitted at a lower energy. Because energy and wavelength are inversely related to one another, the emission photon has a longer wavelength than the excitation photon. (b) Excitation and emission spectra differ in wavelength by an amount known as the ‘Stokes shift.’

(a) Ethidium bromide DNA labeled with intercalating dye Unlabeled DNA SYBR Green Intercalator inserts between base pairs on double-stranded DNA (b) Fluorescent dNTPs are incorporated into both strands of PCR product John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 9.6 (c) Figure 9.6 Methods for fluorescently labeling DNA fragments. Double-stranded DNA molecules may be labeled with fluorescent intercalating dyes (a). The fluorescence of these dyes is enhanced upon insertion between the DNA bases. Alternatively a fluorescent dye may be attached to a nucleotide triphosphate and incorporated into the extended strands of a PCR product (b). The most common method of detecting STR alleles is the use of fluorescent dye labeled primers (c). These primers are incorporated into the PCR product to fluorescently label one of the strands. One strand of PCR product is labeled with fluorescent dye Fluorescent dye labeled primer

FAM JOE TAMRA ROX (blue) (green) (yellow) (red) John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 9.7 Figure 9.7 ABI dyes used in four-color STR detection. The circle portion of the dye highlights the succinimidyl ester, which is used for dye attachment to the fluorescent oligo. In the AmpFlSTR kits, TAMRA has been replaced by NED (also a yellow dye). PowerPlex 1.1 and 2.1 STR kits use fluorescein and TMR, which are very similar to FAM and TAMRA, respectively. The red dye ROX is used to label an internal sizing standard and is the same as CXR used in PowerPlex kits.

with color contributions Normalized Fluorescent Intensity 520 540 560 580 600 620 640 WAVELENGTH (nm) 100 80 60 40 20 310 Filter Set F with color contributions 5-FAM JOE NED ROX Laser excitation (488 nm, 514.5 nm) Normalized Fluorescent Intensity John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 9.8 Figure 9.8 Fluorescent emission spectra of ABI dyes used with AmpFlSTR kits. The virtual filters for ABI 310 Filter Set F is represented by the four boxes centered on each of the four dye spectra. Each dye filter contains color contributions from adjacent overlapping dyes that must be removed by a matrix deconvolution. The dyes are excited by an argon ion laser, which emits light at 488 and 514.5nm.

(a) (b) Scan number Region shown below Relative Fluorescence Units John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 9.9 DNA size in base pairs Relative Fluorescence Units (b) Figure 9.9 STR Data from ABI Prism 310 Genetic Analyzer. This sample was amplified with the AmpFlSTR SGM Plus kit. Raw data prior to color separation (a) compared with GeneScan 3.1 color separated allele peaks (b). The red-labeled peaks are from the internal sizing standard GS500-ROX.

Outlet buffer reservoir Inlet buffer reservoir Capillary Heat plate Detection window electrode Autosampler Gel block Syringe (with polymer) Outlet buffer reservoir Inlet buffer reservoir Sample tray Samples John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 9.10 FIGURE 9.10 Photograph of ABI Prism 310 Genetic Analyzer with a single capillary.

Outlet buffer reservoir Inlet buffer reservoir Mechanical pump (with polymer) Capillary array Oven Detection window electrodes Autosampler Lower gel block Polymer bottle Outlet buffer reservoir Inlet buffer reservoir Sample tray Fan John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 9.11 Figure 9.11 Photograph of ABI 3130xl Genetic Analyzer with a 16-capillary array.

Capillaries Electrodes for Injection John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 9.12 Figure 9.12 Photo of a 3100 capillary array illustrating the 16 glass capillaries (top) and the electrodes surrounding the capillaries (bottom) that enable electrokinetic injection of DNA samples from 2 columns of an 8x12 96well microtiter plate. Electrodes for Injection

The polymerase chain reaction (PCR) is used to amplify STR regions and label the amplicons with fluorescent dyes using locus-specific primers Scanned Gel Image 8 repeats 10 repeats Locus 1 8 repeats 9 repeats Locus 2 Capillary Electropherogram

Transfer of DNA Samples Following PCR, a small portion of the sample is transferred for analysis This aliquot of the sample is mixed with a molecular size marker (termed an internal size standard) that permits calibration of sizing measurements

Sample Plates Spun Down via a Centrifuge Sample plates are spun to remove bubbles that would interfere with the injection (loading) process onto the capillary electrophoresis instrument

ABI 3130xl DNA Analysis Instrument Import sample names Determine run conditions (voltages and times to be used based on laboratory protocols)

Data Collection on ABI 3130xl Instrument Data analysis is performed on an Applied Biosystems (ABI) 3130xl capillary electrophoresis instrument DNA Profile

Capillary Electrophoresis Instrumentation ABI 310 single capillary ABI 3100 16-capillary array Not too much detail here 4 – 5 dye chemistry used

A DNA Profile is Produced by Separating DNA Molecules by Size and Dye Color LASER Excitation (488 nm) The labeled fragments are separated (based on size) and detected on a gel or capillary electrophoresis instrument ~2 hours or less Fragment size ranges from 100 - 350 base pairs Peaks represent labeled DNA fragments separated by electrophoresis This ‘profile of peaks’ is unique for an individual – a DNA type

ABI 310 Data Before and After Matrix is Applied Source: AFDIL training slides

Profiler Plus multiplex STR result DNA size (bp) COfiler multiplex STR result FGA D21S11 D18S51 D8S1179 VWA D13S317 D5S818 Amel D3S1358 D7S820 TH01 D16S539 CSF1PO TPOX AmpFlSTR Profiler Plus and COfiler STR Data Collected on an ABI 310 Capillary Electrophoresis System. The STR loci that are surrounded by a box are common to both multiplex mixes and are therefore useful as a quality assurance measure to demonstrate sample concordance.

Mechanical stepper motor Autosampler Tray Pump Block capillary Detection window Syringe filled with POP-4 polymer Injection electrode Heated plate for temperature control Buffer (inlet) (outlet) Mechanical stepper motor Deionized water sample tubes Schematic of ABI Prism 310 Genetic Analyzer. The capillary stretches between the pump block and the injection electrode. The mechanical stepper motor pushes polymer solution in the syringe into the pump block where it enters and then fills the capillary. Samples placed in the autosampler tray are sequentially injected onto the capillary. Electrophoretic separation occurs after each end of the capillary is placed in the inlet and outlet buffer and a voltage is applied across the capillary. A laser (not shown) is used to detect fluorescently labeled DNA fragments as they pass by the capillary detection window.

ELECTROPHORESIS and DETECTION steps are simultaneous Replace capillary Refill syringe with polymer solution Fill buffer vials Prepare samples (denature, cool, and mix with size standard) Prepare sample sheet and injection list Automated Sample Injection, Electrophoresis and Data Collection Genotype STR alleles Size DNA Fragments Perform Data Analysis GeneScan Software Genotyper Software Manually inspect the data Performed only once per batch of ~96 samples Allelic ladder every tenth injection ELECTROPHORESIS and DETECTION steps are simultaneous Sample processing steps using ABI 310 Genetic Analyzer.

Chapter 9 – Points for Discussion What is electro-osmotic flow and how does it impact DNA separations in a capillary? What component of a PCR reaction is labeled with a fluorescent dye to enable detection of amplified STR alleles?