Effect of multiple freeze-thaw cycles on genomic DNA

[Mehdad]

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

[Blahah]

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.

Edited February 22, 2011 by Blahah

[CharonY]

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.

[Mehdad]

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.

[Blahah]

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.

Edited February 23, 2011 by Blahah

[CharonY]

Also, this can be easily monitored in a simple experiment in which you monitor for sheared products. I gave a course on capillary electrophoresis and with increase of the cycle (and everything kept constant), detection (in form of noise) of unspecific signals appeared.

But almost everyone has seen sheared genomic DNA in agarose gels, too.

[Mehdad]

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.