Sizing Dna Fragments

DNA fragments represented by peaks in capillary electropherograms or bands on a gel can be sized relative to an internal size standard that is mixed with the DNA samples. The internal size standard is typically labeled with a different colored dye so that it can be spectrally distinguished from the DNA fragments of unknown size. In the case of the ABI 310 and 4-dye chemistry AmpF/STR kits, the internal standard is usually the GS500-ROX (Figure 15.2). This size standard contains 16 DNA fragments, ranging in size from 35-500 base pairs (bp),

DNA Size

165.05 bp

147.32 bp

165.05 bp

147.32 bp

DNA fragment peaks are sized based on the sizing curve produced from the points on the internal size standard

DNA fragment peaks in sample

DNA fragment peaks are sized based on the sizing curve produced from the points on the internal size standard

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Data point

Figure 15.2 Peak sizing with DNA fragment analysis. An internal size standard, such as GS500-ROX (a), is analyzed along with the DNA sample and used to calibrate the peak data points to their DNA size (b). This standard is labeled with a different color fluorescent dye, in this case ROX (detected as red), so that it can be spectrally distinguished from the STR alleles which are labeled in other colors.

that have been labeled with the red fluorescent dye ROX. Alternatively, for 5-dye chemistries the DNA fragments are labeled with the orange dye LIZ to create the GS500-LIZ internal size standard.

For PowerPlex kit users, the internal lane standard ILS 600 is commonly employed (see Figure 5.5 bottom panel). This size standard contains 22 DNA fragments, ranging in size from 60-600 bp, that have been labeled with the red fluorescent dye CXR. The ILS 600 size marker is commonly used with the PowerPlex® 16 and PowerPlex® Y kits from Promega.

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Responses

  • kristian
    Can i use liz 500 powerplex?
    8 years ago

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