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: 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
Ordering information

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
Ordering information

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

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
Step 1 : DNA extraction

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

Step 2 : PCR amplification

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
Ordering information

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
Ordering information
Step 3 : Capillary electrophoresis

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

Step 4 : Data analysis

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
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Brochure: DNA fragment analysis by capillary electrophoresis

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

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