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Fundamentals of Forensic DNA Typing

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Title: Slide 1 Author: John M. Butler Last modified by: John M. Butler Created Date: 6/29/2009 10:57:18 PM Document presentation format: On-screen Show – PowerPoint PPT presentation

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Title: Fundamentals of Forensic DNA Typing


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

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
Gel Electrophoresis System
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, Figure 9.1
4
Capillary Electrophoresis System
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, Figure 9.2
5
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, Figure 9.3
6
DNA Separation Modes
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, Figure 9.4
7
Fluorescence and Excitation/Emission Spectra
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, Figure 9.5
8
(a)
(b)
Fluorescent dNTPs are incorporated into both
strands of PCR product
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, Figure 9.6
(c)
9
JOE (green)
FAM (blue)
TAMRA (yellow)
ROX (red)
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, Figure 9.7
10
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, Figure 9.8
11
(a)
Scan number
Region shown below
Relative Fluorescence Units
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, Figure 9.9
(b)
12
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, Figure 9.10
13
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, Figure 9.11
14
Capillaries
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, Figure 9.12
Electrodes for Injection
15
The polymerase chain reaction (PCR) is used to
amplify STR regions and label the amplicons with
fluorescent dyes using locus-specific primers
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

20
Capillary Electrophoresis Instrumentation
ABI 3100 16-capillary array
ABI 310 single capillary
21
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
22
ABI 310 Data Before and After Matrix is Applied
Source AFDIL training slides
23
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24
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25
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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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