Key Points:
Fragment analysis (FA) is a molecular biological technique. Nowadays the fragment analysis is widely used in genotyping, DNA fingerprinting and mutation detection in medical, environmental and agricultural research fields.
The FA technology implementation takes four steps, and traditional FA technology is different from the advanced FA technology, which is more efficient, systematic and economic.
The related application of fragment analysis contain clinical aspects, Paternity test and other aspects such as prenatal diagnosis, individual identification, etc.

The Definition of the Fragment Analysis
Fragment analysis (FA) is a molecular biological technique. The fragments are composed of unequal number of nucleotides, which is produced by the Polymerase Chain Reaction(PCR) process, using DNA or cDNA as the templates. During the process of fragments analysis, we mark the fragments to analyze by the way of the different size of fragments and fluorescence labeling. Nowadays the fragment analysis is widely used in genotyping, DNA fingerprinting and mutation detection in medical, environmental and agricultural research fields.

The Principle of FA Technology
Firstly, we extracted Nucleic acids (DNA, RNA) from biological samples, such as tissue, blood and sputum. Secondly, prepare the corresponding templates and primers, and multiple PCR was carried out. Then DNA fragment was analyzed by the way of gel electrophoresis, PAGE electrophoresis, HPLC, DHPLC, capillary electrophoresis and other methods. Finally, processed the obtained data.
Advanced fragment analysis (AFA) is a leading technology in fragment analysis. The principle is to design primers for different targets. Each target product has at least 1bp difference. We marked the end part of the third parties synthesized primers which have different sizes with different colors to carry out multiple polymerase chain reaction (PCR), which can result in the nucleic acid fragments of all the targets with fluorescent labeling. After pretreatment, capillary electrophoresis was performed, and the products with different fragment sizes were identified and distinguished by fluorescence detector. Using software analysis data to determine the following results:
1) Size: the analysis software creates a standard curve for each sample using the standard size of each sample. The relative size of the fragments was obtained by comparing the fluorescence labeled fragments with the standard curve.
2) Genotype: analysis software assigns alleles based on user-defined genetic locus.
Characteristics of FA Technology
Based on gel electrophoresis, traditional gene fragment analysis is based on the separation and analysis of nucleic acid products by using fragment size and charge. Although the application is common and low-cost, it has the following shortcomings: tedious operation, low resolution, the inability to accurately measure the base number of gene fragments; EB pollution, seriously endangering the health of human.
The core of advanced FA detection technology is the separation of multiple PCR technology and capillary electrophoresis system. Multiple primers are included in the same PCR reaction system, and the primers specifically combine the corresponding target genes to carry out multiple PCR. The capillary electrophoresis system can be used to separate and identify the PCR products of different fragment length. At the same time, the quantitative analysis of different products can be carried out according to the size of the peak area. Advanced FA includes the following advanced technologies:
Multiplexing Allele Specific Assay (MASA): it can be used to analyze as many as 15 SNP loci in a single reaction and can be used for pharmacogenomics analysis.
High Sensitivity Pathogen Specific(HSPS): detect and identify multiple low copy numbers of pathogens as the same time in a response system, and are used for multiple detection of infectious pathogens.
Traditional PCR only uses a pair of primers, and produces a nucleic acid fragment by PCR amplification, which is mainly used for identification of single pathogenic factors.
Multiple PCR characteristics: (1) high efficiency, simultaneous detection of a variety of pathogenic microbes in the same PCR reaction tube, or typing of multiple types of target genes, especially the detection of a variety of pathogens by one drop of blood. (2) systematic, multiple PCR is suitable for the detection of groups of pathogens, such as hepatitis virus, intestinal pathogenic bacteria, and sex Disease, anaerobes without spore, combat infected bacteria and bacteriological agents at the same time. (3) economic simplicity, multiple pathogens are detected simultaneously in the same reaction tube. It will save time, save reagents, money and provide more accurate diagnostic information for clinical.

Characteristics of capillary electrophoresis: the volume is small (the volume of a 100 cm x 75 m tube is only 4.4 mu L); the side / section area ratio is large, so the heat dissipation is fast, the high electric field (100-1000 V/cm) can be borne, and the free solution and gel can be used as the supporting medium; the electroosmotic percolation can be produced in the solution medium.Thus, capillary electrophoresis has the following advantages:
(1) high efficiency: the number of tower plates is between 10-10 pieces of /m. When CGE is used, the number of capillaries can reach 10 pieces of /m, which is far higher than that of high performance liquid chromatography.
(2) fast: it usually takes ten minutes to complete the separation, and it takes three minutes faster.
(3) trace: the sample size required for sampling is nL;
(4) the instrument is simple and easy to automate.
(5) the operation is convenient;
(6) a wide range of applications.
Compared with direct sequencing: a certain gene site is easier to obtain, but it is difficult to obtain a complete sequence of genes, and no direct sequence comparison can be made if it is not available. If the marker is very close to the gene locus, it is possible to infer the existence of the mutant gene by marker analysis. Marker analysis is much faster than direct gene sequencing.

Author's Bio: 

This article is written by scientists from Creative Peptides, a professional peptide supplier with twenty years’ history. Creative Peptides also offers peptide synthesis service and various biologic and chemical techniques such as custom conjugate , bioconjugation , Epitope Mapping , Stable Isotope Label , etc.