Nucleic acids purification methods

Instead of specific amplification of one target to improve sensitivity, methods that amplify all genomic DNA or mRNAs are useful when the target is in short supply. For example, multiple-displacement amplification uses exonuclease-resistant random hexamers and a highly pro-cessive polymerase to amplify DNA nonspecificaily. Initial DNA denaturation is not necessary and the reaction proceeds isothermally. Similarly, messenger RNA can be generi-caUy amplified with a poly(T) primer modified with an RNA polymerase promoter. After reverse transcription, second-strand DNA synthesis, and transcription, antisense RNA is produced. Both whole genome and antisense RNA amplification are also useful as nucleic acid purification methods before amplification or detection. 

Classical gel electrophoresis has been used extensively for protein and nucleic acid purification and characterization [9, 10], but has not been used routinely for small molecule separations, other than for polypeptides. A comparison between TLC and electrophoresis reveals that while detection is usually accomplished off-line in both electrophoretic and TLC methods, the analyte remains localized in the TLC bed and the mobile phase is immediately removed subsequent to chromatographic development. In contrast, in gel electrophoresis, the gel matrix serves primarily as an anti-... 

It is often desirable to go through a postlysis separation/concentration step prior to chromatography. Concentration methods involve the use of ammonium acetate and polyethylene glycol precipitation to further remove host proteins and small nucleic acids. These methods also reduce the volume of the sample (or the process streams) prior to chromatographic purification. The separation may also involve centrifugation and filtration to remove cell debris. 

M. Muller, Considerations for the scale-up of plasmid DNA purification in Nucleic Acid Isolation Methods Eds. B. Bowlen, P. Dtirre), American Scientific Publishers, New York, 2003. 

Quake and coworkers [16] developed a PDMS microfluidic device (shown in Eig. 4c) for nucleic acid purification from a small number of bacterial or mammalian cells. This multilayer device contained fluidic channels and a system of membrane-actuated pneumatic valves and pumps, which enabled precise control of buffers, lysis agents and cell solution, and also allowed for parallel processing. Bacterial cells, dilution buffer and lysis buffer are first introduced into the chip and then transferred into the rotary mixer. Once mixed, the lysate is flushed over a DNA affinity column and drained. The DNA is recovered from the chip with an elution buffer for further analysis. We note that this is the only microfluidic chemical C3flome-try device to use a separation method other than solution-phase electrophoresis (i. e., solid phase extraction). 

This chapter lists some representative examples of biochemicals and their origins, a brief indication of key techniques used in their purification, and literature references where further details may be found. Simpler low molecular weight compounds, particularly those that may have been prepared by chemical syntheses, e.g. acetic acid, glycine, will be found in Chapter 4. Only a small number of enzymes and proteins are included because of space limitations. The purification of some of the ones that have been included has been described only briefly. The reader is referred to comprehensive texts such as the Methods Enzymol (Academic Press) series which currently runs to more than 344 volumes and The Enzymes (3rd Edn, Academic Press) which runs to 22 volumes for methods of preparation and purification of proteins and enzymes. Leading referenees on proteins will be found in Advances in Protein Chemistry (59 volumes. Academic Press) and on enzymes will be found in Advances in Enzymology (72 volumes, then became Advances in Enzymology and Related Area of Molecular Biology, J Wiley Sons). The Annual Review of Biochemistry (Annual Review Inc. Patio Alto California) also is an excellent source of key references to the up-to-date information on known and new natural compounds, from small molecules, e.g. enzyme cofactors to proteins and nucleic acids. 

In current practice the fluorescence assay is often followed by the use of hybridization techniques when more selectivity is required. We have for instance used the fluorescence techniques to obtain data on the nucleic acid content of malaria vaccine proteins produced in Escherichia coli. The rapid turnaround time of the fluorescence assay is particularly useful during the early stages of purification to determine the optimal process conditions. After the final process has been arrived at and a variety of methods used to assess the nucleic acid content (including the hybridization techniques), the fluorescence method can be developed for routine quality-control purposes. In certain cases, particularly at high protein concentrations, the dye may bind to the protein with... 

Nucleic acids may also be removed by treatment with nucleases, which catalyse the enzymatic degradation of these biomolecules. Indeed, nuclease treatment is quickly becoming the most popular method of nucleic acid removal during protein purification. This treatment is efficient, inexpensive and, unlike many of the chemical precipitants used, nuclease preparations themselves are innocuous and do not compromise the final protein product. 

Highly purified DNA may be obtained by repeating the chloroform-isoamyl alcohol extraction several times. The alcohol precipitation step may also be carried out many times. Various chromatographic methods including ion exchange have been applied to the purification of nucleic acids. 

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