Methods and reagents: Carriers for precipitating nucleic acids Methods and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methds-reagnts, available on the Internet. This month's column provides some tips for the precipitation of DNA and RNA samples. For details on how to partake in the newsgroup, see the accompanying box. A recurring question on methds-reagnts concerns the best method of precipitating DNA or RNA when preparing them for enzymatic reactions. The most common technique for precipitation of DNA is with the addition of 0.1 vols of 3 M sodium acetate (pH 5.5) and either 2-2.5 vols of ethanol or 0.8-1.0 vols of isopropanol. The mixture is placed at -70 degrees C for 15 mins to several hours before being centrifuged at top speed in an eppendorf table top centrifuge for 10-15 mins at 4 degrees C. (Ref. 1). Oh pellet, sweet pellet *********************** Even though claims of 100% recovery are sometimes made, pessimistic netters feel this is over-estimated and in practice they typically expect to lose up to 50% of their DNA upon precipitation, especially if the DNA is less than 200 bp long or of low concentration. They therefore feel the necessity of doing everything possible to prevent such losses. Although the small amount of DNA used for most molecular biology experiments (less than 2 ug) would cause the DNA sample to be invisible to the naked eye, some netters say that seeing a pellet of DNA in the bottom of the microcentrifuge tube can be a real psychological boost along the way when several steps of DNA manipulations are to be performed in combination, especially when the cleaning up process involves a precipitation at the end of each step. Most researchers would probably agree that a pellet is a positive sign that things are going well, and can even provoke a sigh of relief that, after rinsed with 70% ethanol, the DNA pellet has not been accidentally washed out of the tube and lost down the sink. To see a pellet when precipitating very small amounts of DNA, a co-precipitant or carrier can be a real advantage. The type of carrier to be added will depend on what the DNA is to be used for after precipitation, and the following tips should help you in selecting an appropriate one. Spermine or tRNA. Some people add 0.1 vols of 100 mM spermine to precipitate DNA or use a final concentration of 50 ug ml-1 bacterial or yeast transfer RNA as carrier [2,3]. However, spermine does not precipitate DNA below 60 bp long and can be tricky to remove later. Transfer RNA can also be a real problem. In past discussions, one netter reported having a problem when doing RNase protection assays - extra protected bands were routinely seen on gels when bacterial and/or yeast tRNA carrier was used for precipitation of RNA transcripts. Other netters agree that tRNA is to be avoided, or else extra care should be taken when studies are performed with any type of hybridization reaction, because isolation of DNA or RNA by precipitation with tRNA carrier could cause false positives due to the carryover of contaminating nucleic acids. Linear polyacrylamide (LPA) carrier can easily be made by polymerizing a 5% w/v acrylamide solution in 40 mM Tris, 20 mM sodium acetate, 1 mM EDTA (pH 7.8), together with 0.01 vols of 10% ammonium persulphate and 0.001 vols TEMED. When the solution becomes viscous (15-30 mins) the polymer is precipitated with 2.5 vols of ethanol and centrifuged for 5 mins when it forms into a clot. The pelleted LPA is dried and 20 vols of sterile water added, then left overnight to swell. Afterwards, the LPA stock is mixed by pipetting. 10 ul of a 1 x LPA (0.25%) or 2 ul of a 5 x (1.25%) LPA stock is added to DNA samples in 100 to 400 ul and 2.5 volumes of ethanol added for precipitation [4]. Netters generally use 1 ul of 0.25% LPA and 0.1 vols of 3 M sodium acetate or 0.20-0.25 vols of 10 M ammonium acetate and 2-2.5 vols of absolute ethanol for volumes of DNA solution up to 50 ul. They also advise that the chilling step is unnecessary and that it can generally be disregarded. One person wrote that routinely placing the mixed samples on ice for 15-30 mins, followed by centrifugation for 30 mins at 4 degrees C is more than sufficient in most cases, and if more than 100 ng ul-1 of DNA is present, then there is no need for chilling at all and the precipitated DNA can be held at room temperature [5]. David A. Johnston (daj@nhm.ac.uk) wrote that he tested various amounts of plasmid DNA ranging from 600 ng to 1 ng, and determined by comparison on an ethidium bromide-stained agarose gel that there was no apparent loss of DNA when as little as 4 ng were used for precipitation. As this amount of DNA is very close to the minimum that can be detected using ethidium bromide agarose gel electrophoresis, it is likely that even smaller amounts can be recovered. In a study using more sensitive radioactive labelling, it has been reported that 20 bp of DNA in the 20 pg range can be recovered efficiently when using LPA as carrier [4]. In that study, Fig. 1 shows that less than 20 bp DNA is lost by this method, which is desirable if unwanted primers, added linker DNA or unincorporated nucleotides are to be removed, but not so if that is the size of DNA you want to recover. By contrast, when glycogen is used as carrier, an oligonucleotide as short as 8 bp can be recovered, albeit at less than about 50% efficiency. David Johnston also wrote that he routinely uses 5-15 ul of 0.25% LPA without adjusting the salt concentration or adding ammonium acetate, and that the precipitated DNA worked well for fluorescent cycle sequencing. Netters agree that the use of LPA is less likely to cause problems when the DNA is to be used in other enzyme reactions as well. However, the use of LPA does have some drawbacks. Some netters warned that the polymerization efficiency of the acrylamide will probably not exceed 95%, and there will certainly be some leftover monomers in solution, which might have to be removed by dialysis. The monomeric form of acrylamide is known to be a cumulative neurotoxin. However, researchers running gels day-to-day are familiar with the handling of acrylamide, know the risks involved and take the proper precautions. Therefore, the dialysis step would add to the prep time and not necessarily be an advantage. Someone else wrote that when using LPA for precipitations, the pelleted DNA and added acrylamide does not seem to stick to the side of a polypropylene centrifuge tube as well as pure DNA does, and therefore, the use of LPA is more likely to cause loss of the pellet. One person reported losing half of his DNA pellets when LPA was used as carrier, and yet none were lost when the DNA was precipitated without any carrier. Glycogen. Purified oyster glycogen [6], mussel glycogen [7], molecular biology grade glycogen from Boehringer Mannheim (Cat. No. 901-393) or Sigma glycogen type II (Cat. No. G-8751) can also be used as carrier. Precipitations with glycogen can be accomplished by adding 1 ul of a 20 mg ml-1 stock to DNA solutions up to a volume of 1 ml. Some prefer the use of glycogen as compared to LPA because the pellet is more sticky and they say this prevents its loss. In addition, DNAs less than 20 bp long are recovered more efficiently using glycogen than with LPA. Netters warn, however, that low grades of glycogen might have small amounts of contaminating DNA, and if the recovered DNA is to be used for any type of amplification procedure, including the polymerase chain reaction (PCR), the carrier should be treated with DNase and thoroughly washed beforehand. One netter wrote that using glycogen had reduced the efficiency of a DNA ligation by as much as 30%, but others have not seen any such adverse effects. In addition, glycogen was shown to interfere with a specific DNA-binding protein during a DNA mobility shift experiment, while LPA did not [4]. Pretty in Pink ************** Many companies are now selling carrier solutions or DNA co-precipitants. For example, Supelco advertise GenElute[TM]-LPA (5 mg ml-1 Linear Polyacrylamide) as a better co-precipitant than glycogen or tRNA; LigoChem, Inc. are promoting ProCipitate[TM], a non-toxic polymer bridging network for removal of proteins from DNA samples; and the filter people are even getting into the act with Amicon claiming 99% recovery of DNA by using Micron microconcentrators, alleviating the need for precipitation altogether. Unfortunately, the filter units are far more expensive than the small amount of chemicals needed for precipitation. A relatively new product from Novagen, Inc. called Pellet Paint[TM] seems to be all the rage. The glycogen-based co-precipitant has a colorific moiety bound to it so that when it is precipitated along with DNA, a visible pink pellet can easily be identified within the tube. With patent pending, the paint is claimed to be compatible with restriction digestion, ligations, transformation, manual sequencing, PCR, and many more molecular methods. An additional feature is that, owing to the attached fluorescent dye, the DNA pellet can be visualized under long wavelength UV light (366 nm). This allows the visualization of a very small pellet under UV, but can be rather bothersome if you plan to use the DNA for automated fluorescent sequencing. The coupled fluorophore has an absorbent and fluorescent emission profile similar to rhodamine. The paint has been shown to interfere with the rhodamine tag used for detection of dye-linked primers, dideoxy-terminators used with the Applied BioSystems, Inc. (ABI) automated DNA sequencing instrument and with the detection of the carbocyanine dye Cy5 used with Pharmacia Automated Laser Fluorescent (ALF) sequencing units. One other drawback is that the UV absorption makes quantitating DNA with added paint more complicated. As the fluorophore peaks within the spectral range of DNA, using a spectrophotometer for estimating the amount of DNA in a sample requires some extra manoeuvring, which involves subtracting out any contribution the dye would make to the peak at 260 nm. However, this might not be a problem if a fluorimeter is used, but that has not yet been tested. One consideration is the size of DNA to be precipitated. As Pellet Paint[TM] is glycogen based, DNA less than 20 bp in length will be precipitated and, although they would be recovered somewhat less efficiently than much larger DNA fragments, this makes the paint less than ideal for removing primers and linkers. As far as cost is concerned, some netters feel that glycogen alone can be used as carrier without any added fluorophore, because it is much cheaper and the pellet can still be seen, even when there is no dye attached. Glycogen does seem to be the carrier favored by netters in most cases, because the pellet is less likely to slide out of the tube and it is thought to be less liable to interfere with subsequent enzymatic operations on the nucleic acids than the others. References ********** [1] Wallace, D. M. (1987) Methods Enzymol. 152, 41-48 [2] Gosule, L. C. and Schellman, J. A. (1976) Nature 259, 333-335 [3] Hoopes, B. and McClure, W. R. (1981) Nucleic Acids Res. 20, 5493-5504 [4] Gaillard, C. and Strauss, F. (1990) Nucl. Acids Res. 18, 378 [5] Crouse, J. and Amorese, D. (1987) B.R.L. Focus 9(2), 3-5 [6] Tracy, S. (1981) Prep. Biochem. 11, 251-268 [7] Helms, C. et. al. (1985) DNA 4, 39-49 ******************************************************************************* Any statements made by the author are not meant to advocate the use of a particular commercial product or endorse any company. All opinions are those of the author and do not reflect the opinion of the National Cancer Institute or the National Institutes of Health. Copyright: This manuscript is not copyrighted by Elsevier Publishing Company. However, you may not reproduce any portion for resale or edit the text for redistribution, sale, or otherwise without written permission from the author. You found this at the World Wide Web (WWW) Uniform Resource Locator (URL) ftp://ftp.ncifcrf.gov/pub/methods/TIBS/jun96.txt Any reference to this column must be cited as the following published article: Hengen, P. N. 1996. Methods and reagents - Carriers for precipitating nucleic acids. Trends in Biochemical Sciences 21(6):224-225. ******************************************************************************* * Paul N. Hengen, Ph.D. /--------------------------/* * National Cancer Institute |Internet: pnh@ncifcrf.gov |* * Laboratory of Mathematical Biology | Phone: (301) 846-5581 |* * Frederick Cancer Research and Development Center| FAX: (301) 846-5598 |* * Frederick, Maryland 21702-1201 USA /--------------------------/* *******************************************************************************