Denaturation of double-stranded nucleic acids

Details Denaturation of double-stranded nucleic acids Denaturation of double-stranded nucleic acids

1999-05-05

2001-03-27

Zitomer, Stephanie (Department: 1655)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S091100, C435S091200, C536S025420

Reexamination Certificate

active

06207423

ABSTRACT:

The present invention relates to methods of denaturation of double-stranded nucleic acids and to further processes utilising single-stranded or partially single-stranded nucleic acids produced by such denaturation.
Double-stranded DNA (deoxyribonucleic acid) and RNA/RNA (ribonucleic acid) and DNA/RNA complexes in the familiar double helical configuration are stable molecules that in vitro require aggressive conditions to separate the complementary strands of the nucleic acid. Methods that are commonly employed for strand separation require the use of high temperatures of at last 60° C. and often 100° C. for extended periods of five minutes or more or use an alkaline pH of 11 or higher. Other methods include the use of helicase enzymes such as Rep protein of
E.coli
that can catalyse the unwinding of DNA in an unknown way, or binding proteins such as 32-protein of
E.coli
phage T4 that act to stabilise the single-stranded form of DNA. The denatured single-stranded DNA produced by such known processes of heat or the use of alkali is used commonly for hybridisation studies or is subjected to amplification procedures. We have now discovered that the exposure of double-stranded nucleic acids to certain solid surfaces can produce a rapid denaturation or partial denaturation sufficient to allow the resulting wholly or partially single-stranded nucleic acids to take part in hybridisation or amplification procedures.
Belotserkovskii and Johnston, “Science” vol. 271 1996 have disclosed observing some unspecified degree of denaturation of DNA sequences containing multi-repeats of CA or of GA upon incubation in polypropylene macrophage tubes. The denaturation observed appears to be partial only and at a low level despite incubation over several hours. The mechanism by which denaturation occurs is not disclosed. The phenomenon is not sufficiently pronounced to make the single-stranded DNA produced useable in a practicable manner. Also, in a response to the article, Gaillard and Strauss question whether denaturation is responsible for the observed phenomena.
The present invention now provides a method of denaturing or partially denaturing a double-stranded nucleic acid, comprising contacting a liquid containing said double-stranded nucleic acid with a surface having denaturation activity sufficient to produce said denaturation within a period of not more than one hour.
The denaturation normally takes place within a period of not more than 5 minutes, usually much less, e.g. within a period of not more than 1 minute. A few seconds, e.g. about 10 seconds or less is in fact usually sufficient.
The surface may be chosen to have an acidity or basicity sufficient to produce said denaturation.
The invention includes a method of conducting a nucleic acid hybridisation procedure comprising denaturing a double stranded nucleic acid by a method described to produce at least partially single stranded nucleic acid and hybridising a second nucleic acid with the single stranded nucleic acid so produced.
The invention further includes a method of conducting a nucleic acid amplification procedure requiring single stranded nucleic acid, comprising denaturing a double stranded nucleic acid by a method as described to produce at least partially single stranded nucleic acid and conducting said amplification using said single stranded nucleic acid so produced.
Preferably, the surface is acidic. Most acidic ion exchange materials are not sufficiently acidic to produce the desired effect. It is therefore preferred to employ a strongly acidic surface such as may be obtained where acid groups are attached to an electron withdrawing polymer structure. Suitably, the polymer structure comprises fluorine substituted carbon atoms and is for instance a perfluorinated polymer backbone. Preferably, the polymer is a tetra-fluoroethylene-perfluoro-2-(fluorosulphonylethoxy) propylvinyl ether. Suitable such materials are commercially available under the Trade Mark NAFION, e.g. NAFION 117. NAFION is available as beads or as membranes or in solution for the casting of membranes. Due to the electron withdrawing effect of the perfluorocarbon backbone, the acidic character of the sulphonic acid groups can be comparable to that of 100% H
2
SO
4
and trifluoromethane-sulphonic acid in trifluoroacetic acid anhydride solution.
Other materials likely to produce the effect are other polysulphonates, sulphonated resins such as DAIS585 a sulphonated styrene/ethylene-butylene/styrene triblock copolymer, or activated polymers such as nylon treated with H
2
SO
4
and rinsed in distilled water.
The wholly or partially single-stranded nucleic acid produced may be utilised in any desired manner. In particular, it may be reacted in a sequence specific manner with complementary nucleic acid. Such an action may take place in a hybridisation assay or in an amplification procedure. Many procedures are now known for amplifying nucleic acids. All the known procedures require that a double-stranded template or target nucleic acid is initially denatured to wholly or partially single-stranded form. There may be a need to carry out such denaturation repeatedly during the course of the amplification procedure but in some amplification procedures denaturation is required only once at the beginning of the process. The practice of such amplification procedures falls within the scope of this invention if any one denaturation event is conducted by method as described above. Examples of nucleic acid amplification procedures within the scope of the invention include the polymerase chain reaction (PCR) as described in U.S. Pat. No. 4,683,202 and many other publications. It may be a variant of the classical or standard PCR process, e.g. the so-called “inverted” or “inverse” PCR process, an asymmetric PCR process or the so-called “anchored” PCR process. The amplification may be a ligase chain reaction (LCR) as described in Barany Proc. Natl. Acad. Sci. USA, Vol. 88, pp 189-193 - 1991. It may be a Nasba or 3SR process as described in Biotechnology, Vol. 13, June 1995 by Sooknanan et al or in Proc. Natl. Acad, Sci. USA, Vol. 87, pp 1874-1978, March 1990, Biochemistry by Guatelli et al. It may be used in the preparation of samples for branched DNA signal amplification (bDNA) as described in Biotechnology, Vol. 12, September 1994, pp 926-928 by Urdea. The denaturation technique of the invention may also be used in sample preparation for the strand displacement assay (SDA) as described in Proc. Natl. Acad. Sci. USA, Vol. 89, pp 392-396, Applied Biological Sciences, January 1992 by Walker et al and elsewhere. The denaturation technique according to the invention may be used equally in amplification techniques which depend upon amplifying the amount of nucleic acid and techniques which rely upon conducting a hybridisation to a probe providing an amplified detectable signal, e.g. bDNA.
The nucleic acid to be denatured may be dissolved in water or other suitable solvent with or without other ingredients such as buffer materials. A sample of the nucleic acid may be placed in contact with the active surface to produce denaturation in a variety of ways. The active surface may be the surface of a membrane which may be a free thin layer or may be a layer on a solid support. The nucleic sample may be placed upon the membrane for a few seconds to produce denaturation and may then be removed. Where the active surface is provided by polymer beads, a sample of the nucleic acid may be run through a column of the beads suitable to provide a retention time of a few seconds so as to produce denaturation. The NAFION membrane may be applied as a coating on a surface such as the interior of a tube through which the liquid is run. For instance, the interior of a pipette tip may be coated, the liquid may be briefly drawn up into the pipette and then expelled to transform nucleic acid with liquid into single-stranded form.
If DNA is exposed to a NAFION membrane for a period in excess of a few seconds, adsorption of the DNA on to the NAFION surface occurs and the DNA may be damaged. It is known

Affiliated with

Also associated with

Rating

Denaturation of double-stranded nucleic acids does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Denaturation of double-stranded nucleic acids, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Denaturation of double-stranded nucleic acids will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2538795