Fragment analysis generates a size estimate for DNA fragments relative to a size standard of DNA fragments with known lengths. The size standard is combined with the sample of interest and co-injected on the capillary electrophoresis system.
The facility uses the 3730 and 3130xl platforms to deliver this service. Please contact the Centre Manager for details on sample requirements for fragment analysis.
What is Fragment Analysis
- Labeling fragments with fluorescent dyes. Multiple different colored fluorescent dyes can be detected in one sample. One of the dye colors is used for a labeled size standard present in each sample. The size standard is used to extrapolate the base-pair sizes of the sample product peaks.
- Amplifying the labeled fragments using polymerase chain reaction (PCR) on a thermal cycler.
- Separating the fragments by size using capillary electrophoresis.
- Analyzing the data using software to determine:
- Size : The analysis software uses the size standard in each sample to create a standard curve for each sample. It then determines the relative size of each dye‐ labeled fragment in the sample by comparing fragments with the standard curve for that specific sample.
- Genotype : The analysis software assigns allele calls based on user‐ defined makers (loci).
Fragment Analysis Applications
Microsatellite (STR) analysis.
Microsatellite markers (loci), also known as short tandem repeats (STRs), are polymorphic DNA loci consisting of a repeated nucleotide sequence. In a typical microsatellite analysis, microsatellite loci are amplified by PCR using fluorescently labeled forward primers and unlabeled reverse primers. The PCR amplicons are separated by size using electrophoresis. Applications include:
- Linkage mapping
- Animal breeding
- Human, animal, and plant typing
- Pathogen sub‐typing
- Genetic diversity
- Microsatellite instability
- Loss of Heterozygosity (LOH)
- Inter‐simple sequence repeat (ISSR)
- Multilocus Variant Analysis (MLVA)
A Single Nucleotide Polymorphism (SNP) marker consists of a single base pair that varies in the known DNA sequence, thereby creating up to four alleles or variations of the marker. Applications include:
- SNaPshot® Multiplex Kit
Several AFLP®‐based technologies use restriction enzyme length polymorphism and polymerase chain reaction (PCR) to generate a fingerprint for a given sample, allowing differentiation between samples of genomic DNA based on the fingerprint. Applications include:
- Microbial genome typing
- Animal or plant genome typing
- Creation of genetic maps of new species
- Genetic diversity and molecular phylogeny studies
- Establishment of linkage groups among crosses
Relative fluorescence applications compare peak height or area between two samples. Common techniques include:
- Qualitative Fluorescence (QF) PCR
- Quantitative Multiplex PCR of Short Fluorescent Fragments (QMPSF)
- Multiplex Ligation‐dependent Probe Amplification (MLPA)
- LOH in tumor samples
- Copy Number Variation (CNV)
- Aneuploidy detection
Fragment Analysis Service Request Form