Chapter 10 DNA Detection Methods: Fluorescent Dyes and Silver Staining ©2002 Academic Press

Methods for Fluorescently Labeling DNA Intercalating Dyes (post-PCR) Dye-labeled nucleotide insertion during PCR Dye-labeled primer insertion during PCR 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 Fluorescent dNTPs are incorporated into both strands of PCR product ©2002 Academic Press

Fluorescent Dyes Used in 4-Color Detection FAM (Blue) JOE (Green) TAMRA (Yellow) ROX (Red) ©2002 Academic Press

WAVELENGTH (nm) FAMJOENEDROX Laser excitation (488, nm) Laser excitation (488, nm) Normalized Fluorescent Intensity Fluorescent Emission Spectra for ABI Dyes ABI 310 Filter Set F with color contributions between dyes ©2002 AP, Elsevier

These values are used by the GeneScan  Analysis Software to separate the various dye colors from one another. The letters B, G, Y, and R represent the dye colors Blue, Green, Yellow, and Red, respectively. Matrix File Table from an ABI 310 ©2002 Academic Press

Sample Detection CCD Panel Color Separation Ar+ LASER (488 nm) Fluorescence ABI Prism spectrograph Capillary or Gel Lane Size Separation Labeled DNA fragments (PCR products) Detection region Principles of Sample Separation and Detection ©2002 Academic Press

(prior to separation of fluorescent dye colors) Raw Data from the ABI Prism 310 ©2002 Academic Press

DNA size in base pairs Relative Fluorescence Units (after separation of fluorescent dye colors) Processed Data from the ABI Prism 310 Internal sizing standard peaks are highlighted and labeled with a red dye (ROX) ©2002 Academic Press

FGA D21S11 D18S51 D8S1179 VWA D13S317 D5S818 Amel D3S1358 D7S820 DNA size (bp) Profiler Plus  TH01 Amel D16S539 D7S820 CSF1PO TPOX D3S1358 DNA size (bp) COfiler  Fluorescence Profiler Plus and COfiler STR Kits ©2002 Academic Press

FMBIO II Detection of STR Alleles DNA samples are loaded onto a polyacrylamide gel STR alleles separate during electrophoresis through the gel Sample Separation Sample Detection (Post- Electrophoresis) 505 nm scan to detect fluorescein-labels 585 nm scan to detect TMR-labels ©2002 Academic Press

FMBIO II 3-Color Detection Wavelength (nm) fluorescein TMR CXR Laser excitation (532 nm) Relative Fluorescent Intensity Filters Used to Detect PowerPoint 1.1 and 2.1 Dyes ©2002 Academic Press

PowerPlex 1.1 and 2.1 Data Gel Image Taken with FMBIO II Instrument ©2002 Academic Press