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

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Presentation on theme: "Fundamentals of Forensic DNA Typing Slides prepared by John M. Butler June 2009 Chapter 9 DNA Separation & Detection."— Presentation transcript:

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

2 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.

3 - 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 Gel Electrophoresis System

4 Laser Inlet Buffer Capillary filled with polymer solution 5-20 kV -+ Outlet Buffer 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 Capillary Electrophoresis System

5 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 Separation 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

6 (a) Larger DNA molecules interact more frequently with the gel and are thus retarded in their migration through the gel Gel (b) Ogston SievingReptation Small DNA molecules Long DNA molecules Gel John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 9.4 DNA Separation Modes

7 h ex h em 1 2 3 SoSo S’ 1 S1S1 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 Fluorescence and Excitation/Emission Spectra

8 Fluorescent dNTPs are incorporated into both strands of PCR product Ethidium bromide DNA labeled with intercalating dye Unlabeled DNA SYBR Green Intercalator inserts between base pairs on double-stranded DNA One strand of PCR product is labeled with fluorescent dye Fluorescent dye labeled primer (a) (b) (c) John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 9.6

9 FAM (blue) JOE (green) TAMRA (yellow) ROX (red) John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 9.7

10 520540560580 600 620640 WAVELENGTH (nm) 100 80 60 40 20 0 310 Filter Set F with color contributions 5-FAMJOENED ROX Laser excitation (488 nm, 514.5 nm) Laser excitation (488 nm, 514.5 nm) Normalized Fluorescent Intensity John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 9.8

11 Scan number Relative Fluorescence Units DNA size in base pairs Relative Fluorescence Units Region shown below (a) (b) John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 9.9

12 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

13 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

14 Capillaries Electrodes for Injection John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 9.12

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

16 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

17 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

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

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

20 ABI 3100 16-capillary array ABI 310 single capillary Capillary Electrophoresis Instrumentation

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

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

23 Profiler Plus  multiplex STR result DNA size (bp) COfiler  multiplex STR result FGA D21S11 D18S51 D8S1179 VWA D13S317 D5S818 Amel D3S1358 D7S820 TH01 Amel D16S539 D7S820 CSF1PO TPOX D3S1358

24 Autosampler Tray Pump Block capillary Detection window Syringe filled with POP-4 polymer Injection electrode Heated plate for temperature control Buffer (inlet) Buffer (outlet) Mechanical stepper motor Deionized water sample tubes

25 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

26 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?


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