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Fragment Analysis Workflow
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: How Does Fragment Analysis Work?
Workflow
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
5’ Labeled/unlabeled primer pairs
| Cat. No. | Size | HPLC yield | 5' Reporter dye | Quantity | Order |
|---|---|---|---|---|---|
| 450056 | 10,000 pmol | 5´ Labeled/Unlabeled Primer Pairs | 6-FAM, TET, VIC, HEX, NED, PET | 2 tubes | Order |
| 450059 | 80,000 pmol | 5´ Labeled/Unlabeled Primer Pairs | 6-FAM, TET, VIC, HEX, NED, PET | 2 tubes | Order |
| 450062 | 300,000 pmol | 5´ Labeled/Unlabeled Primer Pairs | 6-FAM, TET, VIC, HEX, NED, PET | 2 tubes | Order |
| 4304976 | 10,000 pmol | 5´ Labeled/Unlabeled Di-Repeats | 6-FAM, TET, VIC, HEX, NED, PET | 2 tubes | Order |
| 4304977 | 80,000 pmol | 5´Labeled/Unlabeled Di-Repeats | 6-FAM, TET, VIC, HEX, NED, PET | 2 tubes | Order |
| 4304978 | 300,000 pmol | 5´ Labeled/Unlabeled Di-Repeats | 6-FAM, TET, VIC, HEX, NED, PET | 2 tubes | Order |
| 4304979 | 10,000 pmol | 5´ Labeled/Unlabeled Di-Repeat + Tail | 6-FAM, TET, VIC, HEX, NED, PET | 2 tubes | Order |
| 4304981 | 80,000 pmol | 5´ Labeled/Unlabeled Di-Repeat + Tail | 6-FAM, TET, VIC, HEX, NED, PET | 2 tubes | Order |
| 4304982 | 300,000 pmol | 5´ Labeled/Unlabeled Di-Repeat + Tail | 6-FAM, TET, VIC, HEX, NED, PET | 2 tubes | Order |
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 fragments | Corresponding matrix standard | Recommended size standard | Possible applications |
| Dye set E5: dR110 (blue), dR6G (green), dTAMRA (yellow), dROX (red | DS-02 | GeneScan 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-30 | GeneScan 500 ROX Size Standard (red) | Custom fragment analysis |
| Dye set D: 6-FAM (blue), VIC (green), NED (yellow) | DS-31 | GeneScan 500 ROX Size Standard (red) | Custom fragment analysis |
| Dye set F: 5-FAM (blue), JOE (green), NED (yellow) | DS-32 | GeneScan 500 ROX Size Standard (red) | Microsatellite |
| amplified fragment length polymorphism (AFLP) | |||
| Dye set G5: 6-FAM (blue), VIC (green), NED (yellow), PET (red) | DS-33 | GeneScan 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-36 | GeneScan 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 | |||
|---|---|---|---|
 |  |  | |
| Throughput | Low | Med | High |
| 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.
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)
Video: How to set up a Sanger sequencing or fragment analysis run
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.
- 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
- 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
5’ Labeled/unlabeled primer pairs
| Cat. No. | Size | HPLC yield | 5' Reporter dye | Quantity | Order |
|---|---|---|---|---|---|
| 450056 | 10,000 pmol | 5´ Labeled/Unlabeled Primer Pairs | 6-FAM, TET, VIC, HEX, NED, PET | 2 tubes | Order |
| 450059 | 80,000 pmol | 5´ Labeled/Unlabeled Primer Pairs | 6-FAM, TET, VIC, HEX, NED, PET | 2 tubes | Order |
| 450062 | 300,000 pmol | 5´ Labeled/Unlabeled Primer Pairs | 6-FAM, TET, VIC, HEX, NED, PET | 2 tubes | Order |
| 4304976 | 10,000 pmol | 5´ Labeled/Unlabeled Di-Repeats | 6-FAM, TET, VIC, HEX, NED, PET | 2 tubes | Order |
| 4304977 | 80,000 pmol | 5´Labeled/Unlabeled Di-Repeats | 6-FAM, TET, VIC, HEX, NED, PET | 2 tubes | Order |
| 4304978 | 300,000 pmol | 5´ Labeled/Unlabeled Di-Repeats | 6-FAM, TET, VIC, HEX, NED, PET | 2 tubes | Order |
| 4304979 | 10,000 pmol | 5´ Labeled/Unlabeled Di-Repeat + Tail | 6-FAM, TET, VIC, HEX, NED, PET | 2 tubes | Order |
| 4304981 | 80,000 pmol | 5´ Labeled/Unlabeled Di-Repeat + Tail | 6-FAM, TET, VIC, HEX, NED, PET | 2 tubes | Order |
| 4304982 | 300,000 pmol | 5´ Labeled/Unlabeled Di-Repeat + Tail | 6-FAM, TET, VIC, HEX, NED, PET | 2 tubes | Order |
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 fragments | Corresponding matrix standard | Recommended size standard | Possible applications |
| Dye set E5: dR110 (blue), dR6G (green), dTAMRA (yellow), dROX (red | DS-02 | GeneScan 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-30 | GeneScan 500 ROX Size Standard (red) | Custom fragment analysis |
| Dye set D: 6-FAM (blue), VIC (green), NED (yellow) | DS-31 | GeneScan 500 ROX Size Standard (red) | Custom fragment analysis |
| Dye set F: 5-FAM (blue), JOE (green), NED (yellow) | DS-32 | GeneScan 500 ROX Size Standard (red) | Microsatellite |
| amplified fragment length polymorphism (AFLP) | |||
| Dye set G5: 6-FAM (blue), VIC (green), NED (yellow), PET (red) | DS-33 | GeneScan 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-36 | GeneScan 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 | |||
|---|---|---|---|
| | | |
| Throughput | Low | Med | High |
| 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.
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)
Video: How to set up a Sanger sequencing or fragment analysis run
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.
- 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
- 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
Brochure: DNA fragment analysis by capillary electrophoresis
Learn about our comprehensive solutions for each step of your fragment analysis workflow.
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