It is well known in this art that a nucleic acid duplex which comprises two single nucleic acid strands specifically or non-specifically bound together may be separated when subjected to harsh processing conditions such as heating to an elevated temperature.
Specifically, certain DNA purification techniques require or usefully may include a heating step. It is known that in such techniques it is difficult to purify double stranded DNA, for example, from blood or other biological samples because heating causes denaturing of the double stranded DNA to form single stranded DNA. This creates a problem because many downstream DNA processing techniques, such as Southern blotting, may only be carried out usefully using a certain quantity of double stranded DNA. Accordingly, it is often necessary with some existing DNA purification techniques to return to an individual patient in order to collect further samples so that a sufficient quantity of double stranded DNA is recovered in order to be able to perform downstream analysis of the DNA.
Various forces affecting the conformation of DNA and, in particular, the stability of the double-helix structure are known. These are discussed in general terms in reference 1. Electrostatic forces attributable to ionisation, base-stacking forces, hydrogen bonding and hydrophobic forces are mentioned.
The effect of salts of 1:1 electrolytes on the thermal denaturation temperature of DNA secondary structure at neutral pH is discussed in reference 2. A lowering of the thermal denaturation temperature is observed. This effect is attributed mainly to the presence of the anions: CCl3COO−, CF3COO−, CNS− and ClO4−.
The formation of heat labile interstrand crosslinks formed when cyanomorpholinoadriamycin is reacted with DNA is the subject of reference 3. Interstrand crosslinking was detected as a resistance of DNA to separate under normal DNA denaturing conditions. The interstrand crosslinks had a midpoint melting temperature of 70° C. Clearly, these crosslinks do not inhibit heat denaturation of DNA.
A distinction is drawn in reference 4 between the problems of thermodegradation and thermodenaturation of covalently closed DNA in hyperthermic conditions. A reduction in thermodenaturation due to stabilising of the DNA primary structure in the presence of K+ or Mg2+ is reported.
A study of the effect of physiological concentrations of KCl (50-500 mM) and MgCl2 (1-25 mM) on the chemical stability of double-stranded and single-stranded DNA at temperatures typical for hyperthermophiles is reported in reference 5. It is proposed that these two salts act to protect both double-stranded and single-stranded DNA against heat induced cleavage by inhibiting depurination. This effect is rationalised for double-stranded DNA by the proposal that these salts stabilise the double helix.
The effects of cation counter ion on nucleic acid duplex stability are the subject of reference 6. It is suggested that nucleic acid stability may be accounted for by nearest-neighbour interactions. A passive effect of cations bound to a double helix on nucleic acid duplex stability is suggested. It is suggested that there are preferential base pairs or nearest-neighbour base pairs for the counter ion bonding. Various physiological salt concentrations were studied.
A physiological stabilisation of DNA by a prokaryotic histone-like protein is reported in reference 7. It is stated that DNA associated with a protein which closely resembles the histones of eukaryotes is more stable than free DNA against thermal denaturation by about 40° C. The protein is tightly bound to the DNA.
Reference 8 reviews the factors that might contribute to the stability of the genomes of hypothermophiles. Extrinsic factors such as the intracellular ionic environment and so-called histone-like DNA binding proteins are discussed. It is mentioned that circular DNA molecules are intrinsically more resistant to heat denaturation than are linear DNA molecules. The role of DNA binding proteins in eukaryotes and prokaryotes in genome compaction and thermostabilisation is suggested.
A specific histone from a family of archaeal histones that bind and compact DNA molecules into nucleosome-like structures is the subject of reference 9. It is stated that the DNA-binding and compacting activities of the protein are resistant to heat inactivation, for example at 95° C. for 5 minutes. This feature is suggested as a mechanism to resist heat denaturation of DNA.
Reference 10 relates to protecting DNA from thermal denaturation using the non-specific binding protein Sso7d of the hyperthermophilic archaeon Sulfolobus solfataricus. The authors report on promotion of renaturation of complementary DNA strands at temperatures above the melting point of the duplex by the non-specific DNA-binding protein.
In reference 11 the effect of ethanol concentration on the thermal stability and structure of aggregated DNA in an ethanol-water solution is discussed. It is observed that with increasing ethanol concentration the melting point of DNA decreases. It is stated that at a critical ethanol concentration aggregation of the DNA molecules sets in and that aggregated DNA molecules are thermally more stable than dissolved ones. The effect of the nature of the counter ion on the stability of aggregated DNA is considered to some extent.
In view of the above, there still remains a problem to provide a simple, cost-effective method of inhibiting denaturation such as heat denaturation of a nucleic acid duplex or, more specifically, a double-stranded DNA.
Accordingly, the present invention aims to at least partially address the above problem.
USPTO Backgrounds
USPTO Backgrounds