A New Method Of Nucleic Acid Purification Using GoldMag^? Particles As A Carrier

Nucleic acid purification is the most technically demanding and labor-intensive procedures performed in molecular biology research and associated diagnostic methods.The principle of nucleic acid purification is to obtain high purity and ensure the integrity of nucleic acid.There are some nucleic acid purification methods such as phenol-chloroform extraction,high concentrations of salt,as well as ion exchange chromatography and adsorption chromatography.However,these methods have disadvantages including safety concenrns(need hazardous reagent),tedious processes (multi-step centrifugation)and can not always guarantee the isolation of nucleic acid with high throughput and large-scale automation.Super-paramagnetic particles have already been used in purification of nucleic acid,in so much the method has advantages such as simple,rapid,and can be accomplished without centrifugation, and can be used to isolate nucleic acids with high-throughput and automation.The nucleic acid purification kits using magnetic particles have been commercialized in USA,Germany,etc.So far,associated research in this field is still in its preliminary stages within the domestic environment.Using GoldMag(?)particles as a carrier,a novel method of total nucleic acid (including genomic DNA and total RNA)purification from rat liver was established: GTC(Guanidin Thiocyanate)lysis buffer was chosen to treat the liver,homogenized, and then GoldMag(?)particles were added;After incubation for 5 min the complex particles of GoldMag(?)particles and nucleic acids were washed and then the nucleic acids was eluted from the particle surface.The total nucleic acid has characteristic peak in the wavelength of 260 nm.The bands for genomic DNA,28S rRNA,18S rRNA were clearly evident by agarose electrophoresis detection and the ratio of 28S rRNA and 18S rRNA was about 2:1.The method also can be used in total nucleic acid purification from rat heart,spleen,lung and kidney as well as other biological materials such as cultured cells,bacteria,plant and whole blood.In order to evaluate the method,the recovery and the coefficients of variation (CVs)within-and between-purification were tested,the results indicated that the coefficients of variation was from 5.06%to 16.29%and from 4.22%to 20.5% respectively.The recovery ratio was about 80%.The total nucleic acid from rat liver was used as template for amplification ofβ-actin gene via reverse transcription-polymerase chain reaction(RT-PCR)and the agarose electrophoresis result showed that the target DNA molecule with 240 bp can be synthesized, indicating that no enzyme inhibitor exist in the whole purification process.Without eluting step,the total RNA was obtained by treatment the complex of GoldMag(?)particle and nucleic acids with DNase,40μg total RNA was isolated from 10 mg rat liver and the ration of OD₂₆₀/OD₂₈₀was about 2.0,and the purified total RNA can be used in RT-PCR.In the same manner,after treated with RNase,the genomic DNA was obtained.The yield of purified DNA from 10 mg rat liver was about 5μg,and the ration of OD₂₆₀/OD₂₈₀was about 1.78.The oligo(dT)coupled with functional magnetic particles was used to isolate mRNA fron total nucleic acids purified by the above method.In conclusion,a novel total nucleic acid purification method from animal tissue using GoldMag(?)as carrier was established.Without eluting the total nucleic acid,by treating the complex of magnetic particles and nucleic acid with DNase,and after further purification processes,the total RNA was obtained,we can also obtain DNA in the same manner.Using the total nucleic acid as material,mRNA was isolated using functional magnetic particles which was coupled with oligo(dT)₂₀.The purified DNA and RNA demonstrated high purity,so the method is promising in terms of nucleic acid purification.