Process for detecting and quantifying nucleic acid molecules

Details Process for detecting and quantifying nucleic acid molecules Process for detecting and quantifying nucleic acid molecules

1998-12-02

2001-10-23

Horlick, Kenneth R. (Department: 1656)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S007100, C435S091200, C435S091100, C436S063000, C436S094000, C436S501000, C536S023100, C536S024310, C536S024320, C536S024330, C210S656000

Reexamination Certificate

active

06306592

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a method of identifying and quantifying nucleic acid molecules within a population of nucleic acid molecules of identical, similar, or different sequence, by chemically reacting unpaired or incorrectly paired nucleotides with a carbodiimide compound and forming heteroduplexes, which may then be quantified.
BACKGROUND OF THE INVENTION
An important field of molecular biology relates to the revealing of sequence variations in mixtures of largely homologous nucleic acids. The sequence comparison between DNA molecules for identifying variations not only adds to what is known about the molecular bases of phenotypic differences, for example hereditary diseases, but also permits continuous monitoring of NA populations, for example virus populations, during an infection. NA population is to be understood as meaning a plurality of NA molecules with an identical, the same or a different sequence. Furthermore, the sequence comparison also serves as a quality assurance characteristic when producing genetically engineered, bacterial or viral products or for detecting the occurrence of minute quantities of differing sequences in a population of homologous sequences.
The prior art knows several methods for tracking down sequence variations. Arguably the most laborious method is direct sequencing (Sanger F., Nicklen S., Coulson A. R., 1977, Proc. Natl. Acad. Sci. USA 74, 5463 et seq.; Maxam A. M., Gilbert W., 1977, Proc. Natl. Acad. Sci. USA 74, 560-564). This method does not allow a statistically significant number of individuals of an NA population to be tested for the occurrence of mutations. The application of indirect hybridization methods such as Southern (Southern E. M., 1975, J. Mol. Biol. 98, 503-517) or Northern (Alwine J. C., Kemp D. J., Stark G. R., 1977, Proc. Natl. Acad. Sci. USA 74, 5350-5354) only allow massive quantitative variations to be detected. Methods such as the ribonuclease protection assays (D. J. Freeman, A. S. Juan, 1981, J. Gen. Virol. 57, 103-117; E. Winter et al., 1985, PNAS 82, 7575-7579) for ribonucleic acid (RNA)-RNA heteroduplexes or for RNA-deoxyribonucleic acid (DNA) heteroduplexes (R. M. Myers et al., 1985, Science 230 , 1242-1246) are slightly more sensitive.
Denaturing gradient gel electrophoresis (DGGE) has been made markedly more sensitive in recent years by employing “polymerase chain reaction”(PCR) technology and by using specific primers which facilitate separation in the gradient gel (V. C. Sheffield et al., 1992, Biofedback 12, 386-387). To separate the reaction products, even the differing sequences must be present in reasonable quantities. A further disadvantage of this method is the fact that, after separation and detection of a mutant, the site of the mutation cannot be specified, so that further identification reactions, for example sequencing, are subsequently required.
“Chemical cleavage reactions” using hydroxylamine and osmium tetroxide have the disadvantage that a large number of experimental manipulations with toxic chemicals and complex procedures are required (R. G. H. Cotton, 1989, Biochemistry 263, 1-10). In addition, they only work if substantial amounts of mutants are present. Finally, only certain mutations can be identified using this method.
A further prior-art method is based on the “single strand conformation polymorphism” (SSCP) reaction. Both this method and DGGE are carried out ((M Urita et al., 1989, PNAS 86, 2766-2770). A disadvantage of the SSCP reaction is that it leads to the identification of wrongly-positive samples. Moreover, this method fails in at least 10% of all cases if large amounts of mutant molecules are present.
Other prior-art methods only allow testing for the presence of a specific mutation, i.e. the verification of the presence, or absence, of an individual nucleotide (MAPREC: Chumakov K. M., Powers L. B., Noonan K. F., Roninson L. B., Levenbook I. S., 1991, Proc. Natl. Acad. Sci. USA 88, 199-203).
Methods in which carbodiimide is used have hitherto not proved popular in practice because this substance is difficult to handle and the methods lack sensitivity (D. F. Novack, 1986, Proc. Natl. Acad. Sci. USA, 83, 586 590; A. Ganguly, 1991, J. Biol. Chem. 266, 1235-1240; Offenlegungsschrift [Published Specification] DE 36 29 190 A1, A. Ganguly and D. J. Prockop, 1993, Nucl. Acids Res. 18 No. 13, 3933-3939).
A further method known from the prior art (WO 93/02216) is the method for detecting “mismatch” in heteroduplexes. A “mismatch-binding protein” is used, which is bound by first antibodies. These first antibodies, in turn, are recognized by second antibodies. Again, the method is complicated to carry out and can only be employed within limits.
In total, the lack of sensitivity relative to the minimum amount of mutants present within an NA population is the main disadvantage of the methods known from the prior art. Also, quantification of the NA molecules revealed is not possible. A further problem of the known methods is their unduly high failure rate.
It is an object of the present invention to provide a method, a device and a composition of means which overcome the disadvantages of the prior art.
SUMMARY OF THE INVENTION
Described is a method of detecting and quantifying nucleic acid (NA) molecules within a population (I) of NA molecules of identical, similar or different sequence, in which
a) the single-stranded or duplex ribonucleic acid (RNA) molecules or single-stranded deoxyribonucleic acid (DNA) molecules contained in a population (I) are converted into a population (II) of duplex DNA molecules,
b) the population (II) of duplex DNA molecules is subjected to denaturation and subsequent renaturation, so that a mixture of homo- and heteroduplex DNA molecules is present,
c) heteroduplexes formed in the population (II) where unpaired or incorrectly paired nucleotides are present are reacted chemically with a carbodiimide compound, and
d) the resulting carbodiimide reaction products are characterized and quantified, characterized in that
a chromatographic purification method is carried out between each of the steps a to d.


REFERENCES:
patent: 4794075 (1988-12-01), Ford et al.
patent: 5217863 (1993-06-01), Cotton et al.
patent: 5623049 (1997-04-01), Lobberding et al.
patent: 5795976 (1998-08-01), Oefner et al.
patent: 5874212 (1999-02-01), Prockop et al.
patent: WO93/02216 (1993-02-01), None
patent: WO 95/06652 (1995-03-01), None
Smooker et al Mutation Research vol. 288 pp. 65-77, 1993.*
Ganguly et al NAR vol. 18, No. 13 pp. 3933-3939, 1990.*
Babon et al NAR vol. 23, No. 24 pp. 5082-5084, 1995.

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