Fragment analysis is used to describe genetic marker analysis experiments which rely on detection of changes in the length of a specific DNA sequence to indicate the presence or absence of a genetic marker. In genetic marker analysis, the sequence of the gene is not directly analyzed, but the presence of a particular allele or mutant version of the allele of the gene is inferred from the presence or absence of a linked DNA sequence which can serve as a marker for the allele. Genetic markers are usually polymorphic genetic sequences contained in or near an allele of interest, such as microsatellite or RFLPs, which allow the chromosomal alleles to be distinguished.
We offer DNA Fragment Analysis services where fluorescently labeled fragments are detected using our Applied Biosystems Genetic Analyzer and then interpreted using the GeneMapper® v4.0 analysis software
For analysis of fragments larger than 500bp, please send your enquiries to us.
- We are using 500-ROX (for Dye Set D) and 500-LIZ (for Dye Set G) DNA Size Standard. They consist 16 DNA fragments, sized as 35, 50, 75, 100, 139, 150, 160, 200, 250, 300, 340, 350, 400, 450, 490 and 500 bp. Each band is single-stranded and fluorescently labeled with ROX and the size fragments are evenly distributed for very accurate size calling.
- High throughput and medium throughput of Genetic Analyzers are both available for use in our Fragment Analysis services:
- ABI3730XL (50cm 96-capillary, 1 hour complete 95 loadings, POP7)
- ABI3100 (50cm 16-capillary, 1 hour complete 15 loadings, POP7)
We support Dye Sets D and G5:-
Dye Set G5
|LIZ (Size Standard)
Labeled primers may be ordered with 1st BASE Custom Oligos. Do note that labeled primers need to be at least TOP Purified.
- Customers may take up our Spectral Optimization Service to optimize the loading of samples in order to obtain optimal signal strength for data analysis. We will include an optimizing step by loading the sample based on separate loci or number of labeled primer(s) at several dilution factors with our size marker to determine the optimal amount of sample for subsequent loading.
Spectral optimization can be carried out for each loci/ labeled primer before actual run on Genetic Analyzer, which is inclusive of pooling of different tubes or plates into a single loading (especially for new users in multiplexing).
For customers who have already performed the multiplex reaction into a single well or a single tube, the level of the spectral optimization will be limited because we are unable to separate the labeled primers to determine the optimum peaks individually.
If spectral optimization is not required, the customer has to indicate the Dilution Factor to be used in the Order Form. The loading volume in our Genetic Analyzer is 10uL (which consist of your PCR sample, our DNA size marker and deionized formamide) from each individual well. If you need us to run 1 uL of your undiluted sample, 1/10 means the dilution factor required is 10. If the sample needs to be diluted at 50 times before loading, 1uL of the diluted sample to be injected into our machine will means the dilution factor of 500.
Applications of Fragment Analysis:
- Microsatellite Analysis: Simple Sequence Repeats (SSRs), Short Tandem Repeats (STRs)
- AFLP Analysis (amplified fragment length polymorphisms)
- PCR amplification of genomic DNA using validated conditions
- SNP analysis (single nucleotide polymorphisms)
- Any analysis of fragment size and allele calling
- Electrophoresis of fluorescent PCR amplicons using ABI3100 or ABI3730xl