Relative fluorescent quantitation (or quantitative fluorescence PCR (QF-PCR)) is a technique used in a variety of fragment analysis applications that requires accurate peak height comparisons across multiple samples. Applications that utilize this technique include screening for loss of heterozygosity (LOH) using microsatellites or SNPs, aneuploidy assays, and detecting large chromosomal deletions.
Figure 1. Detecting Loss of Heterozygosity (LOH). Electropherogram of a microsatellite marker in DNA from a healthy (top panel) and tumor sample (bottom panel) run on a Life Technologies 3130 Series Genetic Analyzer. The reduced peak height in the tumor sample indicates potential LOH in the sample.
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Step-by-Step Guide to Relative Fluorescent Quantification
 DNA extraction is a critical first step in the experimental workflow of DNA Sequencing and Fragment analysis. The overall quality, accuracy and length of the DNA sequence read can be significantly affected by characteristics of the sample itself, and the method chosen for nucleic acid extraction. Ideal methods will vary depending on the source or tissue type, how it was obtained from its source, and how the sample was handled or stored prior to extraction. Recommended Products: DNA Isolation |
 Amplify microsatellite loci of interest by PCR using fluorescent labeled primer Recommended Products: Design Primer and Perform PCR |
 Add aliquot of PCR product to size standard and Hi-Di Formamide mix; heat denature and proceed with electrophoresis. Recommended Products: Prepare Sample for AnalysisSizing InformationTo learn more about our GeneScan Size standards used in sizing experiments please see our table below.
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 During capillary electrophoresis, the products of the PCR are injected electrokinetically into capillaries filled with polymer. High voltage is applied so that the fluorescent DNA fragments are separated by size and are detected by a laser/camera system. Which Electrophoresis Instrument (Genetic Analyzer) Is Right for You? |
Which Data Analysis Software Is Right for You? |
DNA extraction is a critical first step in the experimental workflow of DNA Sequencing and Fragment analysis. The overall quality, accuracy and length of the DNA sequence read can be significantly affected by characteristics of the sample itself, and the method chosen for nucleic acid extraction. Ideal methods will vary depending on the source or tissue type, how it was obtained from its source, and how the sample was handled or stored prior to extraction. Recommended Products: DNA Isolation |
Amplify microsatellite loci of interest by PCR using fluorescent labeled primer Recommended Products: Design Primer and Perform PCR |
Add aliquot of PCR product to size standard and Hi-Di Formamide mix; heat denature and proceed with electrophoresis. Recommended Products: Prepare Sample for AnalysisSizing InformationTo learn more about our GeneScan Size standards used in sizing experiments please see our table below.
|
During capillary electrophoresis, the products of the PCR are injected electrokinetically into capillaries filled with polymer. High voltage is applied so that the fluorescent DNA fragments are separated by size and are detected by a laser/camera system. Which Electrophoresis Instrument (Genetic Analyzer) Is Right for You? |
Which Data Analysis Software Is Right for You? |
Step-by-Step Guide to Relative Fluorescent Quantification
DNA extraction is a critical first step in the experimental workflow of DNA Sequencing and Fragment analysis. The overall quality, accuracy and length of the DNA sequence read can be significantly affected by characteristics of the sample itself, and the method chosen for nucleic acid extraction. Ideal methods will vary depending on the source or tissue type, how it was obtained from its source, and how the sample was handled or stored prior to extraction. Recommended Products: DNA Isolation |
Amplify microsatellite loci of interest by PCR using fluorescent labeled primer Recommended Products: Design Primer and Perform PCR |
Add aliquot of PCR product to size standard and Hi-Di Formamide mix; heat denature and proceed with electrophoresis. Recommended Products: Prepare Sample for AnalysisSizing InformationTo learn more about our GeneScan Size standards used in sizing experiments please see our table below.
|
During capillary electrophoresis, the products of the PCR are injected electrokinetically into capillaries filled with polymer. High voltage is applied so that the fluorescent DNA fragments are separated by size and are detected by a laser/camera system. Which Electrophoresis Instrument (Genetic Analyzer) Is Right for You? |
Which Data Analysis Software Is Right for You? |
DNA extraction is a critical first step in the experimental workflow of DNA Sequencing and Fragment analysis. The overall quality, accuracy and length of the DNA sequence read can be significantly affected by characteristics of the sample itself, and the method chosen for nucleic acid extraction. Ideal methods will vary depending on the source or tissue type, how it was obtained from its source, and how the sample was handled or stored prior to extraction. Recommended Products: DNA Isolation |
Amplify microsatellite loci of interest by PCR using fluorescent labeled primer Recommended Products: Design Primer and Perform PCR |
Add aliquot of PCR product to size standard and Hi-Di Formamide mix; heat denature and proceed with electrophoresis. Recommended Products: Prepare Sample for AnalysisSizing InformationTo learn more about our GeneScan Size standards used in sizing experiments please see our table below.
|
During capillary electrophoresis, the products of the PCR are injected electrokinetically into capillaries filled with polymer. High voltage is applied so that the fluorescent DNA fragments are separated by size and are detected by a laser/camera system. Which Electrophoresis Instrument (Genetic Analyzer) Is Right for You? |
Which Data Analysis Software Is Right for You? |
Highlighted products for Fluorescent Quantification:
Visistat reference component
For Research Use Only. Not for use in diagnostic procedures.