A method for separating and/or preparing highly purified target substances from different biomaterials is difficult because natural biomaterials, such as tissue, cell, blood, bacteria, are complicated mixtures. However, isolation and purification of target substances from such biomaterials are often needed in diagnostics, biomedical research, and/or other applications. For example, in a natural state, deoxyribonucleic acids (DNAs) and ribonucleic acids (RNAs) are often mixed with other substances, such as proteins, lipids and carbohydrates; isolating and purifying these DNA and RNA molecules containing a target gene or a target gene transcript, respectively, is often necessary for investigating the gene.
With the fast advances in molecular biology and other related fields, there is a need for a new method for consecutive DNA and RNA isolation and purification from the same sample that is safe, effective, and suitable for automation and industrialization. It has been reported that certain silicon-containing materials can absorb target substances in the presence of binding agents or binding enhancers. The target substances can then be purified by being eluted from the silicon carrier after the impurities are eliminated. U.S. Pat. No. 6,218,531 discloses a method for isolating RNAs from lysed biomaterials with silicon binding carrier in the presence of chaotropic reagents.
The underlying mechanism for these nucleic acid isolation and purification methods is that silicon-containing materials can reversibly bind DNA, RNA and hybrid molecules of DNA and RNA in the presence of binding reagents. Some common chaotropic binding reagents include NaI, urea, guanidine hydrochloride, NaClO4, and KBr. Alcohol, such as 100% ethanol, is also a commonly used binding reagent for nucleic acid purification (see the background of European Pat. App. No. 0512676 A1 and U.S. Pat. No. 5,783,686).
Procedures for purification of nucleic acids using silicaceous matrices frequently involve washes of the support with bound nucleic acids in alcohol containing solutions to remove impurities. Preferably, a simple and robust process for nucleic acid purification is based on matrices where bound nucleic acids can be washed in non-alcohol containing solutions. In U.S. Pat. No. 6,355,792, a method for isolating and purifying nucleic acids comprising a solid carrier exposing hydroxyl groups where the nucleic acids are bound to the carrier material in a solution containing chaotropic agents in the acidic pH range, and eluted in the alkaline pH range is disclosed.
The binding of small RNA molecules, such as miRNA, onto silicaceous materials can be enhanced by addition of acetone or acetonitrile to the lysis buffer (U.S. Pat. App. No. 2009/0143570).
Procedures for sequential purification of DNA and RNA from the same sample have been devised. For example, WO2004/108925 discloses a method based on different affinities of RNA and DNA to a silicaceous matrix under different concentrations of ethanol in the binding buffer.
The recovery of RNA from complex mixtures with silica matrices may be adversely affected through competition for binding with DNA present in such mixtures. To alleviate these problems, it has been known to use a matrix for selective removal of DNA prior to binding RNA to enhance binding of the latter.
It should be noticed that, while using silicaceous materials as reversible absorbing materials for nucleic acids, the use of alcohols in wash buffers may result in alcohol contamination of the eluted end product. Such contamination may be detrimental to the performance of the end product in downstream processes.
Therefore, there is a need to provide a new, effective process for the recovery of different nucleic acid species from the same sample and where impurities can be removed by washing in aqueous buffer without alcohols, and the process is amenable to automation.
USPTO Backgrounds
USPTO Backgrounds