Overview of DNA extraction and purification technology

Extraction and purification of plasmid DNA

Plasmids are a class of double-stranded, closed-loop DNA ranging in size from 1 kb to over 200 kb. In general, the plasmid can be stably and stably in an extrachromosomal free state and has an autonomous replication function; but under certain conditions, it can also reversibly integrate into the host chromosome, replicate with the replication of the chromosome, and be transmitted through cell division. Descendants.

Plasmids have become the most commonly used vector for nucleic acid cloning and sequencing. The yield and purity of plasmid DNA extraction is directly related to the success or failure of downstream experimental procedures. There are many methods for extracting plasmid DNA. The most widely used method is the alkaline lysis-neutralization method. The basic principle is: when the bacteria are lysed in NaOH and SDS solution, the protein and DNA are denatured; when the neutralizing solution is added, The plasmid DNA molecule can be rapidly renatured and dissolved, and remains in the supernatant during centrifugation; the protein and chromosomal DNA can not be renatured and flocculent, and can be precipitated during centrifugation.

The purification method of plasmid DNA usually utilizes two properties of relatively small plasmid DNA and covalent ring closure, such as cesium chloride-ethidium bromide gradient equilibrium centrifugation, ion exchange chromatography, gel filtration chromatography, polyethylene dioxide A method such as fractional precipitation of alcohol. But these methods are relatively expensive or time consuming. For small-volume plasmid DNA, residual protein and RNA can be removed by simple steps such as phenol, chloroform extraction, RNase digestion and ethanol precipitation. The purified plasmid DNA can be used for enzyme digestion, transformation, probe labeling, sequencing, etc. Claim. However, this method takes a long time and requires contact with toxic solvents such as phenol and chloroform, and the extracted plasmid DNA is sometimes not completely digested by the restriction endonuclease. Therefore, in the pursuit of efficiency, this method has been gradually obtained with high quality. It is replaced by a quick and convenient centrifugal adsorption column purification method.

Purification of linear DNA fragments

The enzymes, salts, residual primers, dNTPs and other DNA fragments contained in the PCR product or the enzymatically reacted DNA fragment may affect the effect of the subsequent reaction, and thus need to be subjected to purification treatment. For the single band observed by electrophoresis, ethanol precipitation recovery or simple column recovery method can be used; for DNA fragments with non-specific bands or other bands, it is necessary to carry out gel recovery and purification. At present, the commercially available DNA fragment recovery kit mostly uses a silica gel matrix material to specifically adsorb DNA, and elutes with high salt/ethanol to remove impurities. Due to the different silica matrix materials used in various kits, the recovery efficiency of DNA fragments is also very different.

Extraction and purification of genomic DNA

Genomic DNA is relatively stable, so its extraction method is relatively simple. Usually, the cells are treated with a surfactant to release genomic DNA. After digestion with protease and RNase, a certain amount of genomic DNA can be obtained by organic solvent extraction, ethanol precipitation or column purification. Currently, commercial genomic DNA extraction kits are divided into two types: solution type and centrifugal adsorption column type. The solution type product is economical and has high yield, and can obtain high molecular weight DNA suitable for establishing a library, and can recover DNA in a rare sample as much as possible; but the operation process is cumbersome and requires high experimental skill of the operator. The centrifugal adsorption column type product is convenient and rapid to use, and the extracted DNA has high purity, but the price is high, the yield is low, and the obtained fragment is about 20 kb, which is not suitable for establishing a DNA library or as a template for a long fragment PCR product.