Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-11-21
2002-11-05
Siew, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S022100, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330, C702S019000, C702S020000
Reexamination Certificate
active
06475737
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to a method of automatically selecting oligonucleotide hybridization probes for detecting a mutation causing a mismatch in a DNA duplex.
BACKGROUND OF THE INVENTION
The hybridization of oligonucleotide hybridization probes is a technique with widespread application in molecular biology. It has been used for the detection of immobilized nucleic acids in northern or southern blotting and for allele specific oligonueleotide hybridization. Provided stringency conditions are met, only a perfect complementary hybridization probe will basepair with the DNA strand, observed. The method can therefore be used for mutation detection when the presence of a mismatch caused by a base exchange disrupts the Watson-Crick pairing and destabilizes the partial duplex formed by the hybridization probe and the DNA strand observed.
A known possible tool for monitoring the hybridization of a labeled hybridization probe to a DNA strand to be observed in an homogenous assay is the LightCycler (Trademark, manufactured by Roche Molecular Biochemicals). Three different wavelengths are available for the optical detection of oligonucleotide hybridization when an appropriate fluorescence resonance energy transfer (FRET) pair is formed by adjacent hybridization of two dye labeled hybridization probes with the DNA strand to be observed. One of the two hybridization probes is called the anchor probe while the other probe which is directly sensitive to the mutation to be detected is called the detection probe. The monitored fluorescence signal is proportional to the amount of the hybridized hybridization probe pairs. The temperature at which a certain percentage (normally 50%) of the hybridization probe pairs is hybridized or in a so-called annealed state is called the melting point Tm of the hybridization probe pair. This melting point can also be regarded as the melting point of only the detection probe as the melting point of the anchor probe is typically set to be considerably higher than that of the detection probe.
For mutation detection the Tm shift caused by the mutation should be as high as possible to ensure good discrimination of heterozygotes.
General guidelines for the construction of hybridization probes for use in both, quantification and mutation detection, have been given [1], but these depend on an accurate estimation of DNA melting temperatures.
The melting temperature of short (<20 bp) oligonucleotides is often estimated with the Wallace/Ikatura rule Tm=2° C. (A-T bp)+4° C. (G-C bp). Although this approximation assumes a salt concentration of 0.9M NaCl, typical for dot blots and other hybridizations, it also works well for PCR applications which are not very sensitive to different Tms. However, for hybridization probes a more accurate estimation of Tm is required.
From U.S. Pat. No. 5,556,749 it is known to automatically select oligonucleotide hybridization probes taking their respective melting points into consideration. However, this known method proves not to be accurate.
Therefore, it is an object of this invention to provide a method of automatically selecting oligonucleotide hybridization probes for detecting a mutation causing a mismatch in a DNA duplex by a sufficient accurate estimation of their Tm.
This object is achieved by a method according to claim 1.
Advantageous embodiments of this method are outlined in the subclaims 2 to 10.
The nearest-neighbor (n-n) model is based on thermodynamic calculations and gives the most precise prediction of oligonucleotide stability. This model assumes that the thermodynamic parameters for a given pair depend only on the identity of its adjacent pair and that these nearest neighbor parameters are pairwise additive. The stability of a given oligonucleotide sequence is a function of two basic sources, the sum of the interstrand H bonding between Watson-Crick paired bases and the intrastrand base stacking. The application of this model for calculation of DNA duplex stability will be detailed in the methods section of this chapter. The derivation of the used formula E can be found in the literature [2] and will not be given here. Several data sets are now available that describe the ten n-n pairs that occur in double stranded oligonucleotide DNA [3-6]. The contribution of a mismatch to the duplex stability depends on the location and orientation and on the neighboring bases as well [3]. The destabilizing effect of the 48 possible single mismatches can also be taken into account if the n-n data of the respective mismatches are used for the calculation of the melting temperature [3; 7-10]. Oligonucleotides with repetitive sequences or strings of A-T base pairs may deviate from the n-n model as well as molecules which do not melt in a two state (all or nothing) manner (11). Hybridization conditions (ionic strength and probe concentration) also influence the Tm and must be considered. It could be proved that the n-n model is able to predict the melting points observed with hybridization probe assays with a standard error of less than 1° C. The n-n model is most beneficial for the thermodynamic predictions of oligonucleotide DNA. In longer DNA strands, interactions that are independent of the neighboring bases become increasingly important. This reduces the utility of the n-n model for the prediction of longer (>150 bp) DNA duplexes.
In the following the invention will be described in more detail with regard to preferred embodiments of the new method. Further, a brief description on how thermodynamic data for use in the new method can be calculated will be given. Next, the application of the n-n model to hybridization probe assays on the LightCycler will be validated. At last a general comment will be given on sensitivity and specificity of hybridization probes selected by the new method.
REFERENCES:
patent: 5556749 (1996-09-01), Mitsuhashi et al.
patent: 5856103 (1999-01-01), Gray et al.
patent: PCT/US97/19673 (1997-10-01), None
SantaLucia et al. “Improved Nearest-Neighbor Parameters for Predicting DNA Duplex Stability,” Biochemistry, US, American Chemical Society, Easton, PA, vol. 35, Jan. 1, 1996, pp. 3555-3562; abstract, p. 3561.
Giesen et al. “A formula for thermal stability (Tm) prediction of PNA/DNA duplexes” Nucleic Acids Research, GB, Oxford University Press, Surrey, vol. 26, No. 21, Jan. 1, 1998, pp. 5004-5006.
Doktycz et al. “Optical Melting for 128 Octamer DNA Duplexes, Effects of Base Pair Location and Nearest Neighbors on Thermal Stability,” Journal of Biological Chemistry, US, American Society of Biological Chemists, Baltimore, MD, vol. 270, No. 15, Apr. 14, 1995, pp. 8439-8445.
B. E.Caplin, A. Rasmussen, P.S. Bernard, and C.T. Wittwer (1999) “LightCycler Hybridization Probes,”Biochemica1: 5-8.
D.M. Gray (1997) “Derivation of Nearest-Neighbor Properties from Data on Nucleic Acid Oligomers. I. Simple Sets of Independent Sequences and the Influence of Absent Nearest Neighbors,”Biopolymers42: 783-793.
H.T. Allawi and J. SantaLucia, Jr. (1997) Thermodynamics and NMR of Internal G•T Mismatches in DNA, Biochemistry 36: 10581-10594.
J. Santa Lucia, Jr., H.T. Allawi, and P.A. Seneviratne (1996) “Improved Nearest-Neighbor Parameters for Predicting DNA Duplex Stability,” Biochemistry 35: 3555-3562.
J. SantaLucia, Jr. (1998) “A Unified View of Polymer, Dumbbell, and Oligonucleotide DNA Nearest_neighbor Thermodynamics,” Proc Natl Acad Sci USA 95: 1460-1465.
N. Sugimoto, S. Nakano, M. Yoneyama and K. Honda (1996) “Improved Thermodynamics Parameters and Helix Initiation Factor to Predict Stability of DNA Duplexes,” Nucleic Acids Res 24:4501-4505.
H.T. Allawi and J. SantaLucia, Jr. (1998) “Nearest-Neighbor Thermodynamics of Internal A•C Mismatches in DNA: Sequence Dependence and pH Effects,” Biochemistry 37: 9435-9444.
H.T. Allawi and J. SantaLucia, Jr. (1998) “Thermodynamics of Internal C•T Mismatches in DNA,” Nucleic Acids Res 26: 2694-2701.
H.T. Allawi and J. SantaLucia, Jr. (1998) “Nearest Neighbor Thermodynamic Parameters
Schütz Ekkehard
von Ahsen Nicolas
Siew Jeffrey
Thomas George M.
Thomas Kayden Horstemeyer & Risley
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