Fragment analysis is a kind of Molecular biology technology, we can analysis a large number of fragments through their size, or get some fragments infected by the fluorescence while others not. In that way, we can distinguish them clearly. The fragment analysis is so effective that it’s widely used in genotypic typing, DNA fingerprinting and mutation detection in medical, environmental and agricultural research fields.

In more detail, to analysis the fragments exactly, we extract the nucleic acids (DNA, RNA) from biological samples, such as tissue, blood and sputum. Then we need to conduct the Polymerase Chain Reaction (PCR) several times to get a sufficient number of Nucleic acids. After that, we have a few different ways to analysis the DNA fragments, such as gel electrophoresis, PAGE electrophoresis, HPLC, DHPLC, capillary electrophoresis, etc. I’ll give a real knockdown to the PCR and gel electrophoresis in the following paragraphs.

Polymerase chain reaction (PCR)

PCR, short for Polymerase Chain Reaction, is a molecular biological technology for amplifying and amplifying specific DNA fragments, which can make many copies of a specific DNA region in vitro.

The general process includes three steps: first, we need to heat the DNA chains strongly to separate it. This provides a single chain template for the next step. Next, we cool it down to enables primers to bind to complementary sequences on single strand template DNA. Last, raise the reaction temperature properly, the Taq polymerase will be extended to primer to synthesize the new DNA chain. For details, please see the following picture:

In the Polymerase Chain Reaction, there’re three points should be noticed. First, the Taq polymerase is necessary. Because the Taq polymerase has a characteristic heat stability that other polymerases don’t. Second, PCR primers are in need as well. The region of DNA that will be copied or amplified is decided by the primers. So, the primers play a great role in the PCR. Last but not least, during the PCR experiment, the temperature in extremely important. When the temperature is too high, the nucleic acids may get inactivation, which will lead to a fail.

Gel electrophoresis

Gel electrophoresis can separate DNA fragments according to their size, which is usually used for analytical use, but it can also be used as a preparation technique to detect some of the purified molecules before the detection of some methods, such as mass spectrometry (MS), polymerase chain reaction (PCR), cloning technology, DNA sequencing, or immunoblotting.

The principle of gel electrophoresis is hardly simple. When a molecule is placed in an electric field, they move to the appropriate electrode at a certain speed, the rate of migration of the electrophoretic molecule under the action of the electric field, called the mobility of the electrophoresis. It is directly proportional to the intensity of the electric field and the number of net charges carried by the electrophoretic molecule itself. That is to say, the greater the electric field intensity and the more the net charge carried by the electrophoresis molecule, the faster the migration rate is. Therefore, when the nucleic acid molecules are placed in the electric field, they migrate to the direction of the positive electrode. At certain electric field intensity, the migration rate of DNA molecules, that is, the mobility of electrophoretic, depends on the size and configuration of the nucleic acid molecule itself, and the small molecular weight of the DNA molecule is faster than the large one. This way, we can get them separated.

The schematic diagram of the device is as follows:
As we can see, there are a few wells indentations in the device. When it’s working, DNA samples were loaded into the wells, and then it’ll be pulled through the gel by the electric current. When the gel is dyed with DNA dye, the DNA fragment can be regarded as a band. Each band represents a group of DNA fragments of the same size.

Microsatellite genotyping

Microsatellite, also known as short tandem repeat (STR) or simple repeat (SSR), is a simple repeat that is widely distributed in the genome of eukaryotes. Microsatellite loci are usually detected by PCR amplification and electrophoresis, and the alleles are isolated according to the size of the fragments; the amplified allelic microsatellites can be detected by various methods. The traditional method uses polyacrylamide gel electrophoresis plus radiation. The method of developing or silver dyeing is time consuming and inefficient. Based on the 5-year experience of micro-reading genes, the ABI genetic analyzer was used to detect the fluorescently labeled DNA fragments, and the molecular weight internal standard was used to calculate the length of the DNA fragments, making the STR typing more efficient and more accurate.

Summary

In general,the steps of Fragment Analysis include the PCR, which can make many copies of a specific DNA region, and the gel electrophoresis, which can separate DNA fragments according to their size and prepare for deeper research.

Author's Bio: 

CD Genomics was established in 2004, we are aiming at providing the research community with high quality Next Generation Sequencing, high throughput microarray services. Due to the demand for our services has being increased; CD Genomics has already updated its technology platform to mainstream NGS and microarray instruments. At present, our senior bioinformaticians have ever viewed more than ten thousands of trace files and accumulated abundant experience with our Illumina HiSeq2000/2500, Illumia Miseq, Ion Torrent PGM, PacBio RS and ABI 3730/3730XL analyzers. We continue to work hard to offer you the same dependable services to pharmaceutical and biotech companies, as well as academia and government agencies for the purpose of satisfying all your sequencing or array needs.