Fragment analysis separates and analyzes amplified PCR products according to fragments using a primer marked by a fluorescent label.
Linkage mapping
Pathogen sub‐typing
Loss of Heterozygosity (LOH)
Animal breeding
Genetic diversity
Inter‐simple sequence repeat (ISSR)
Human, animal, and plant typing
Microsatellite instability
Multilocus Variant Analysis (MLVA).
DNA Fragments will be labeled with fluorescent dyes during amplification using polymerase chain reaction (PCR) on a thermal cycler. Multiple different colored fluorescent dyes can be detected in one sample (Multiplex). 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.
The fragments will be separated by size using capillary electrophoresis. The data is analyzed using a software to determine size and genotype.
Size: The analysis software uses the size standard 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).
Different ways to use Fragment 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: Paternity test - Forensics - Disease Linkage - Animal Typing - Microsatellite Instability.
A Single Nucleotide Polymorphism (SNP) marker consists of a single base pair that varies in the known DNA sequence, creating up to four alleles or variations of the marker.
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 genomic DNA samples 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. Applications include: Loss of heterozygosity (LOH) in tumor samples - Copy number variation (CNV) - Aneuploidy assays - Large chromosomal deletion detection.