Mehdad

It is well appreciated that Mg2+ stabilizes duplex DNA 80 to 100 fold to as much as 140-fold (1). How ever Promega (2) and other online calculators of primer Tm do not take into account the effects of magnesium on helix stability. I personally ran a multiplex PCR which its annealing temperature is close to Tm calculated without magnesium effect. I used Wetmore and Sninsky (1995) N-N formula with a little change in [salt] calculation, [salt]= [K+] + [NH4].

Now my question is, do we need to consider magnesium effect for Tm calculation of primers in PCR? since doing so raises Tm about 5-10 oC?

[PLEASE SUPPORT YOUR ANSWERS WITH PUBLISHED ARTICLES IF POSSIBLE]

References

(1) Nakano S, Fujimoto M, Hara H, Sugimoto N. Nucleic acid duplex stability: influence of base composition on cation effects. Nucleic Acids Res 1999;27:2957–65.

(2) http://www.promega.com/biomath/calc11.htm#salt_Desc

(3) Wetmur, J.G., and J.J. Sninsky (1995). Nucleic acid hybridization and unconven- tional bases. In PCR Strategies (M.A. Innis, D.H. Gelfand, and J.J. Sninsky, eds.). Acade mic Press, San Diego, CA, pp. 69–83.

Thanks

Sure thing. After further reading it's clear that the level and type of damage to DNA depends on many factors including:

 

• Whether the DNA is isolated or in cells
• Strand length
• Whether cryoprotectants are used
• Duration and number of freeze-thaw cycles

It seems as though ice crystals are more likely to cause damage to genomic DNA in cells. Free DNA in suspension is less likely to be damaged by ice, but the medium is important. There are also damaging effects due to dehydration of cells and free radical production if whole cells are freeze-thawed. Here are some of the articles I read:

 

 

• Anchordoquy, Thomas J., Lorinda G. Girouard, John F. Carpenter, and David J. Kroll. 1998. Stability of lipid/DNA complexes during agitation and freeze-thawing. Journal of Pharmaceutical Sciences 87, no. 9: 1046-1051. doi:10.1021/js9801891.
• Calcott, Peter H. 1986. Cryopreservation of Microorganisms. Critical Reviews in Biotechnology 4, no. 3: 279-297. doi:10.3109/07388558609150797.
• Calcott, Peter H., and Anne M. Gargett. 1981. Mutagenicity of freezing and thawing. FEMS Microbiology Letters 10, no. 2: 151-155. doi:10.1111/j.1574-6968.1981.tb06227.x.
• Cox, C.S., and R.J. Heckly. 1973. Effects of oxygen upon freeze-dried and freeze-thawed bacteria: viability and free radical studies. Canadian Journal of Microbiology 19, no. 2: 189-194. doi:10.1139/m73-029.
• Grecz, Nicholas, Teri L. Hammer, Christie J. Robnett, and Mel D. Long. 1980. Freeze-thaw injury: Evidence for Double strand breaks in DNA. Biochemical and Biophysical Research Communications 93, no. 4 (April 29): 1110-1113. doi:10.1016/0006-291X(80)90603-8.
• Krajden, Mel, James M. Minor, Oretta Rifkin, and Lorraine Comanor. 1999. Effect of Multiple Freeze-Thaw Cycles on Hepatitis B Virus DNA and Hepatitis C Virus RNA Quantification as Measured with Branched-DNA Technology. J. Clin. Microbiol. 37, no. 6 (June 1): 1683-1686.
• MAZUR, P. 1984. FREEZING OF LIVING CELLS - MECHANISMS AND IMPLICATIONS. AMERICAN JOURNAL OF PHYSIOLOGY 247, no. 3: C125-C142.
• Ross, K S, N E Haites, and K F Kelly. 1990. Repeated freezing and thawing of peripheral blood and DNA in suspension: effects on DNA yield and integrity. Journal of Medical Genetics 27, no. 9: 569 -570. doi:10.1136/jmg.27.9.569.
• Seutin, Gilles, Bradley N. White, and Peter T. Boag. 1991. Preservation of avian blood and tissue samples for DNA analyses. Canadian Journal of Zoology 69, no. 1: 82-90. doi:10.1139/z91-013.
• Sleight, Sean C., and Richard E. Lenski. 2007. Evolutionary Adaptation to Freeze‐Thaw‐Growth Cycles in Escherichia coli. Physiological and Biochemical Zoology 80, no. 4 (July 1): 370-385.
• Zilli, L., R. Schiavone, V. Zonno, C. Storelli, and S. Vilella. 2003. Evaluation of DNA damage in Dicentrarchus labrax sperm following cryopreservation. Cryobiology 47, no. 3 (December): 227-235. doi:10.1016/j.cryobiol.2003.10.002.

ummm thanks, could you support your explanation with some published articles if there's any?

If you have long DNA strands, repeated freeze-thaw cycles can create ice crystals which can shear the strands. Usually if working with DNA which has been frozen in a -80°C you would take out the amount you need and keep it at 4°C until it was used up, rather than keep refreezing the same sample.

 

In practice though, it takes quite a few freeze-thaw cycles before a DNA sample gets significantly damaged.

ummm thanks, could you support your explanation with some published articles if there's any?

Depends on the size, really. However, genomic DNA even from bacteria (a few megabases) often show degradation effects from as little as 2-3 freeze-thaw cycles.

What is the effect of multiple freeze-thaw cycles on genomic DNA?

Hi, name's Mehdad, I'm a human and so happy to be ooh and btw I don't like the way we are living.