Fragment Analysis

Take DNA analysis further

Fragment analysis comprises a series of techniques in which DNA fragments are fluorescently labeled, separated by capillary electrophoresis (CE), and sized by comparison to an internal standard. While DNA sequencing by CE is used to determine the specific base sequence of a particular fragment or gene segment, fragment analysis can provide sizing, relative quantitation, and genotyping information for fluorescently labeled DNA fragments produced by PCR using primers designed for a specific DNA target.

From straightforward sample preparation to peak analysis, we offer a wide range of products and services to simplify each step of the fragment analysis workflow.

Video Player is loading.
Current Time 0:00
Duration 0:00
Loaded: 0%
Stream Type LIVE
Remaining Time 0:00
 
1x
    • Chapters
    • descriptions off, selected
    • captions off, selected

      Video: How Does Fragment Analysis Work?

       

      Workflow

      COL33196-Frag-analysis-Workflow-(1)

      DNA extraction is a critical first step in the experimental workflow of DNA fragment analysis. The overall efficiency, quality, and size of the PCR product can be significantly affected by characteristics of the sample itself, and the method chosen for nucleic acid extraction and purification. 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.

      Learn more about our extraction kits:
      Tissue DNA 
      Blood DNA  
      Cellular DNA
      Plant DNA  
      Cell-free DNA

      Primers

      To perform fragment analysis on a CE system, primers must be designed that flank the region of interest. Typically, fluorescent dyes are attached to the primers, and the fragments are amplified by PCR before electrophoresis. We offer Applied Biosystems 5´ fluorescently labeled primers and primer pairs that have been validated for use in fragment analysis.

      Key features:

      • Custom 5´ fluorescently labeled primers
      • Choice of label includes 6-FAM, TET, VIC, HEX, NED, or PET dye
      • All oligos are desalted; larger scales of labeled primers have an option for HPLC purification
      • Choice of delivered scales

      5’ Labeled single primers

      Cat. No. SizeHPLC yield5′ Reporter dyeQuantityOrder
      45000710,000 pmolNA6-FAM, TET, VIC, HEX, NED, PET1 tubeOrder
      45000680,000 pmol40,000 pmol minimum6-FAM, TET, VIC, HEX, NED, PET1 tubeOrder
      450017300,000 pmol150,000 pmol minimum6-FAM, TET, VIC, HEX, NED, PET1 tubeOrder

      5’ Labeled/unlabeled primer pairs

      Cat. No.SizeHPLC yield5' Reporter dyeQuantityOrder
      45005610,000 pmol5´ Labeled/Unlabeled Primer Pairs6-FAM, TET, VIC, HEX, NED, PET2 tubesOrder
      45005980,000 pmol5´ Labeled/Unlabeled Primer Pairs6-FAM, TET, VIC, HEX, NED, PET2 tubesOrder
      450062300,000 pmol5´ Labeled/Unlabeled Primer Pairs6-FAM, TET, VIC, HEX, NED, PET2 tubesOrder
      430497610,000 pmol5´ Labeled/Unlabeled Di-Repeats6-FAM, TET, VIC, HEX, NED, PET2 tubesOrder
      430497780,000 pmol5´Labeled/Unlabeled Di-Repeats6-FAM, TET, VIC, HEX, NED, PET2 tubesOrder
      4304978300,000 pmol5´ Labeled/Unlabeled Di-Repeats6-FAM, TET, VIC, HEX, NED, PET2 tubesOrder
      430497910,000 pmol5´ Labeled/Unlabeled Di-Repeat + Tail6-FAM, TET, VIC, HEX, NED, PET2 tubesOrder
      430498180,000 pmol5´ Labeled/Unlabeled Di-Repeat + Tail6-FAM, TET, VIC, HEX, NED, PET2 tubesOrder
      4304982300,000 pmol5´ Labeled/Unlabeled Di-Repeat + Tail6-FAM, TET, VIC, HEX, NED, PET2 tubesOrder


      PCR master mix

      Invitrogen Platinum II Hot-Start PCR Master Mix helps you get to your fragment analysis run, faster. A unique combination of innovative buffer, high-performance Taq DNA polymerase, and Platinum hot-start technology enables exceptional PCR results, even in the toughest applications.

      Key features:

      • Universal primer annealing at 60°C enables co-cycling of all assays, reducing tedious optimization steps
      • An engineered Taq polymerase enables 4x faster DNA synthesis, inhibitor resistance, and robust amplification
      • Platinum hot-start technology allows for room temperature reaction setup
      • A 2X master mix format helps reduce pipetting errors with fewer pipetting steps
      Catalog # Name Size Price (USD) Qty
      14000013 Platinum™ II Hot-Start PCR Master Mix (2X), 200 reactions Each
      389.65

      Online Exclusive

      412.00
      Save 22.35 (5%)
      14000012 Platinum™ II Hot-Start PCR Master Mix (2X), 50 reactions Each
      106.65

      Online Exclusive

      111.00
      Save 4.35 (4%)
      14000014 Platinum™ II Hot-Start PCR Master Mix (2X), 1000 reactions Each
      1,544.65

      Online Exclusive

      1,650.00
      Save 105.35 (6%)

      Matrix and size standards

      To prepare for capillary electrophoresis, a spectral calibration with the corresponding matrix standard to the group of dye you select for labeling your DNA fragments must be performed on your genetic analyzer to accurately detect the dyes with which your primers are labeled. Each unknown sample is mixed with the size standard and Hi-Di Formamide before proceeding with electrophoresis. Size standards allow sizing of sample peaks and correct for injection variations.

      Refer to the following chart for the recommended Applied Biosystems matrix standard and GeneScan size standard for your workflow.

      Selecting the right matrix and size standards

      Dye set used to label DNA fragmentsCorresponding matrix standardRecommended size standardPossible applications
      Dye set E5:
      dR110 (blue), dR6G (green), dTAMRA (yellow), dROX (red
      DS-02GeneScan 120 LIZ Size
      Standard (orange)
      SNaPshot multiplexing
      GeneScan 600 LIZ Size
      Standard v2.0 (orange)
      Microsatellite, LOH, chimerism, inter-simple sequence repeat (ISSR), restriction fragment length polymorphism (RFLP), terminal-RFLP (T-RFLP), methylation-sensitive mobility shift assay (MSMSA)
      Dye set D:
      6-FAM (blue), HEX (green), NED (yellow)
      DS-30GeneScan 500 ROX Size Standard (red)Custom fragment analysis
      Dye set D: 6-FAM (blue), VIC (green), NED (yellow)DS-31GeneScan 500 ROX Size Standard (red)Custom fragment analysis  
      Dye set F:
      5-FAM (blue), JOE (green), NED (yellow)
      DS-32GeneScan 500 ROX Size Standard (red)Microsatellite
      amplified
      fragment length polymorphism (AFLP)
      Dye set G5:
      6-FAM (blue), VIC (green),
      NED (yellow), PET (red)
      DS-33GeneScan 600 LIZ Size
      Standard v2.0 (orange)
      Microsatellite
      Custom fragment analysis
      Dye set J6:
      6-FAM (blue), VIC (green), NED (yellow), SID (violet), TAZ (red)
      DS-36GeneScan 600 LIZ Size
      Standard v2.0 (orange)
      Custom fragment analysis

      Ordering information


      Genetic analyzers and on-instrument reagents

      A range of platforms to meet your throughput’s need
       
      ThroughputLowMedHigh
       Learn more ›Learn more ›Learn more ›


      On-instrument consumables

      Universal all-in-one cartridge

      The Applied Biosystems SeqStudio cartridge has an integrated system design that includes a capillary array, POP-1 universal polymer, buffer, and pump. The POP-1 universal polymer allows for flexibility to perform simultaneous Sanger sequencing and fragment analysis with one cartridge on the same plate in a single run. The cartridge is removable and can be stored on the instrument up to six months. Just load your samples, insert the cartridge, and start the run.

      SeqStudio cartridge

      Polymer

      Using the right Applied Biosystems polymer can help reduce your re-run rate and enables high-quality reproducible data. We offer different polymer chemistries for different needs:

      POP-4:  Human identification applications, other fragment analysis applications, short DNA fragments (<500 bp)
      POP-6:  Mid-sized DNA fragments (500–700 bp)
      POP-7:  Long DNA fragments (>700 bp)

      Order polymer ›


      Video: How to set up a Sanger sequencing or fragment analysis run

      Video Player is loading.
      Current Time 0:00
      Duration 0:00
      Loaded: 0%
      Stream Type LIVE
      Remaining Time 0:00
       
      1x
        • Chapters
        • descriptions off, selected
        • captions off, selected

          We offer a wide portfolio of Applied Biosystems software solutions for viewing and analyzing your fragment analysis results. Access, analyze, and share data anytime, anywhere, with Connect, our cloud-based platform.

          Available on Connect

          • Peak Scanner Module—offers peak identification and fragment sizing
          • Microsatellite Analysis Module—enables analysis of a mixture of DNA fragments separated by size. This analysis provides a profile of the separation, precisely calculates the sizes of the fragments, and determines the microsatellite alleles present in the sample

           Downloadable

          • Gene Mapper Software—flexible genotyping software package that provides DNA sizing and quality allele calls. Specializes in multi-application functionality, including AFLP, LOH, and microsatellite SNP genotyping analysis

          DNA extraction is a critical first step in the experimental workflow of DNA fragment analysis. The overall efficiency, quality, and size of the PCR product can be significantly affected by characteristics of the sample itself, and the method chosen for nucleic acid extraction and purification. 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.

          Learn more about our extraction kits:
          Tissue DNA 
          Blood DNA  
          Cellular DNA
          Plant DNA  
          Cell-free DNA

          Primers

          To perform fragment analysis on a CE system, primers must be designed that flank the region of interest. Typically, fluorescent dyes are attached to the primers, and the fragments are amplified by PCR before electrophoresis. We offer Applied Biosystems 5´ fluorescently labeled primers and primer pairs that have been validated for use in fragment analysis.

          Key features:

          • Custom 5´ fluorescently labeled primers
          • Choice of label includes 6-FAM, TET, VIC, HEX, NED, or PET dye
          • All oligos are desalted; larger scales of labeled primers have an option for HPLC purification
          • Choice of delivered scales

          5’ Labeled single primers

          Cat. No. SizeHPLC yield5′ Reporter dyeQuantityOrder
          45000710,000 pmolNA6-FAM, TET, VIC, HEX, NED, PET1 tubeOrder
          45000680,000 pmol40,000 pmol minimum6-FAM, TET, VIC, HEX, NED, PET1 tubeOrder
          450017300,000 pmol150,000 pmol minimum6-FAM, TET, VIC, HEX, NED, PET1 tubeOrder

          5’ Labeled/unlabeled primer pairs

          Cat. No.SizeHPLC yield5' Reporter dyeQuantityOrder
          45005610,000 pmol5´ Labeled/Unlabeled Primer Pairs6-FAM, TET, VIC, HEX, NED, PET2 tubesOrder
          45005980,000 pmol5´ Labeled/Unlabeled Primer Pairs6-FAM, TET, VIC, HEX, NED, PET2 tubesOrder
          450062300,000 pmol5´ Labeled/Unlabeled Primer Pairs6-FAM, TET, VIC, HEX, NED, PET2 tubesOrder
          430497610,000 pmol5´ Labeled/Unlabeled Di-Repeats6-FAM, TET, VIC, HEX, NED, PET2 tubesOrder
          430497780,000 pmol5´Labeled/Unlabeled Di-Repeats6-FAM, TET, VIC, HEX, NED, PET2 tubesOrder
          4304978300,000 pmol5´ Labeled/Unlabeled Di-Repeats6-FAM, TET, VIC, HEX, NED, PET2 tubesOrder
          430497910,000 pmol5´ Labeled/Unlabeled Di-Repeat + Tail6-FAM, TET, VIC, HEX, NED, PET2 tubesOrder
          430498180,000 pmol5´ Labeled/Unlabeled Di-Repeat + Tail6-FAM, TET, VIC, HEX, NED, PET2 tubesOrder
          4304982300,000 pmol5´ Labeled/Unlabeled Di-Repeat + Tail6-FAM, TET, VIC, HEX, NED, PET2 tubesOrder


          PCR master mix

          Invitrogen Platinum II Hot-Start PCR Master Mix helps you get to your fragment analysis run, faster. A unique combination of innovative buffer, high-performance Taq DNA polymerase, and Platinum hot-start technology enables exceptional PCR results, even in the toughest applications.

          Key features:

          • Universal primer annealing at 60°C enables co-cycling of all assays, reducing tedious optimization steps
          • An engineered Taq polymerase enables 4x faster DNA synthesis, inhibitor resistance, and robust amplification
          • Platinum hot-start technology allows for room temperature reaction setup
          • A 2X master mix format helps reduce pipetting errors with fewer pipetting steps

          Matrix and size standards

          To prepare for capillary electrophoresis, a spectral calibration with the corresponding matrix standard to the group of dye you select for labeling your DNA fragments must be performed on your genetic analyzer to accurately detect the dyes with which your primers are labeled. Each unknown sample is mixed with the size standard and Hi-Di Formamide before proceeding with electrophoresis. Size standards allow sizing of sample peaks and correct for injection variations.

          Refer to the following chart for the recommended Applied Biosystems matrix standard and GeneScan size standard for your workflow.

          Selecting the right matrix and size standards

          Dye set used to label DNA fragmentsCorresponding matrix standardRecommended size standardPossible applications
          Dye set E5:
          dR110 (blue), dR6G (green), dTAMRA (yellow), dROX (red
          DS-02GeneScan 120 LIZ Size
          Standard (orange)
          SNaPshot multiplexing
          GeneScan 600 LIZ Size
          Standard v2.0 (orange)
          Microsatellite, LOH, chimerism, inter-simple sequence repeat (ISSR), restriction fragment length polymorphism (RFLP), terminal-RFLP (T-RFLP), methylation-sensitive mobility shift assay (MSMSA)
          Dye set D:
          6-FAM (blue), HEX (green), NED (yellow)
          DS-30GeneScan 500 ROX Size Standard (red)Custom fragment analysis
          Dye set D: 6-FAM (blue), VIC (green), NED (yellow)DS-31GeneScan 500 ROX Size Standard (red)Custom fragment analysis  
          Dye set F:
          5-FAM (blue), JOE (green), NED (yellow)
          DS-32GeneScan 500 ROX Size Standard (red)Microsatellite
          amplified
          fragment length polymorphism (AFLP)
          Dye set G5:
          6-FAM (blue), VIC (green),
          NED (yellow), PET (red)
          DS-33GeneScan 600 LIZ Size
          Standard v2.0 (orange)
          Microsatellite
          Custom fragment analysis
          Dye set J6:
          6-FAM (blue), VIC (green), NED (yellow), SID (violet), TAZ (red)
          DS-36GeneScan 600 LIZ Size
          Standard v2.0 (orange)
          Custom fragment analysis


          Genetic analyzers and on-instrument reagents

          A range of platforms to meet your throughput’s need
           
          ThroughputLowMedHigh
           Learn more ›Learn more ›Learn more ›


          On-instrument consumables

          Universal all-in-one cartridge

          The Applied Biosystems SeqStudio cartridge has an integrated system design that includes a capillary array, POP-1 universal polymer, buffer, and pump. The POP-1 universal polymer allows for flexibility to perform simultaneous Sanger sequencing and fragment analysis with one cartridge on the same plate in a single run. The cartridge is removable and can be stored on the instrument up to six months. Just load your samples, insert the cartridge, and start the run.

          SeqStudio cartridge

          Polymer

          Using the right Applied Biosystems polymer can help reduce your re-run rate and enables high-quality reproducible data. We offer different polymer chemistries for different needs:

          POP-4:  Human identification applications, other fragment analysis applications, short DNA fragments (<500 bp)
          POP-6:  Mid-sized DNA fragments (500–700 bp)
          POP-7:  Long DNA fragments (>700 bp)

          Order polymer ›


          Video: How to set up a Sanger sequencing or fragment analysis run

          Video Player is loading.
          Current Time 0:00
          Duration 0:00
          Loaded: 0%
          Stream Type LIVE
          Remaining Time 0:00
           
          1x
            • Chapters
            • descriptions off, selected
            • captions off, selected

              We offer a wide portfolio of Applied Biosystems software solutions for viewing and analyzing your fragment analysis results. Access, analyze, and share data anytime, anywhere, with Connect, our cloud-based platform.

              Available on Connect

              • Peak Scanner Module—offers peak identification and fragment sizing
              • Microsatellite Analysis Module—enables analysis of a mixture of DNA fragments separated by size. This analysis provides a profile of the separation, precisely calculates the sizes of the fragments, and determines the microsatellite alleles present in the sample

               Downloadable

              • Gene Mapper Software—flexible genotyping software package that provides DNA sizing and quality allele calls. Specializes in multi-application functionality, including AFLP, LOH, and microsatellite SNP genotyping analysis

              frag_analysis_brochure

              Brochure: DNA fragment analysis by capillary electrophoresis

              Learn about our comprehensive solutions for each step of your fragment analysis workflow.

              Download brochure

              books-transparent-sm

              Build your knowledge

              Get bite-sized answers to your everyday Sanger sequencing and fragment analysis questions

              Learn about the history of sequencing and how to pick the right platform for your research needs