Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-08-01
2002-11-26
Ketter, James (Department: 1636)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C210S656000
Reexamination Certificate
active
06486309
ABSTRACT:
FIELD OF THE INVENTION
The present application is directed to a method of separating and detecting small sequence variations in macromolecules, such a polynucleotides having a single nucleotide alteration in a DNA sequence.
BACKGROUND OF THE INVENTION
Several prior methods have been available for the detection of small changes or polymorphisms (PMs) in polynucleotide sequences, such as a single nucleotide mutation within a double stranded DNA (dsDNA) molecule. Restriction fragment length polymorphism (RFLP) analysis is one method of polymorphism detection but it can only detect those polymorphisms which contain an altered enzymatic reaction site. It is not suitable for the high sensitivity detection of unknown PMs.
By other methods, short sequences of complementary polynucleotides can be used as hybridization probes which allow one to distinguish specific known complementary sequences from sequences not complementary to the probe. However, these methods cannot be used to detect unknown polymorphisms in unknown locations within a polynucleotide sequence without first creating all possible combinatorial sequences. The use of combinatorial methods greatly increases the complexity of the detection of polymorphisms, since highly specialized chemical processes and costly instrumentation are required.
Some effective prior methods used to detect small changes in polynucleotide species such as DNA sequences have been based on the differing mobilities of the variant species, or their cleavage products in electrophoretic separations. These techniques may be used for separation of variant species of DNA. Single-stranded conformation polymorphism (SSCP) analysis relies on changes in the mobility of denatured DNA molecules due to the formation of secondary structures during electrophoresis. This technique requires analyzing a sample under several conditions to achieve optimum sensitivity, but only about 70% of polymorphisms can be detected this way.
Other techniques rely on detection of a heteroduplex formation in dsDNA. Denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) have much greater sensitivity than SSCP, but typically require that the sample contain so called “GC clamps” to produce reliable detection under chemical or thermal denaturing conditions. The incorporation of these GC clamps into the sample molecules increases the complexity and cost of sample preparation.
In other techniques the heteroduplex is detected by chemical or enzymatic cleavage at the polymorphism, followed by electrophoretic separation of the cleavage products. The chemical methods often require highly toxic reagents, and multiple reaction steps. The enzymatic methods typically require exotic proprietary enzymes and are difficult to reproducibily perform.
For the electrophoretic methods described above, slab gel electrophoresis is the most commonly used. Slab gel electrophoreis suffers from the drawback of being relatively slow, and attaining poor detection and quantitation of the samples. It is also quite labor intensive. Capillary electrophoresis is faster and not as labor intensive as slab gel electrophoresis, but reproducibility and quantitation are generally poor due to variation in capillary performance. Finally, all of the electrophoretic methods use polymeric matrices which complicate recovery of the identified variant species as the subsequent analysis, often performed by mass spectrometry.
More recently, an improved method of polymorphism detection utilizes liquid chromatography under denaturing conditions to detect polymorphisms in heteroduplex species dsDNA species. See U.S. Pat. No. 5,795,976. The technique, denaturating high performance liquid chromatography (DHPLC), requires that the sample be separated using optimized gradient reversed-phased liquid chromatography methods. Both the separation solvent composition gradient and the operating temperatures must be optimized since they are highly specific to a particular DNA fragment sequence. Homoduplex species can be distinguished from heteroduplex species by the altered chromatographic retention under optimized conditions.
Temperature gradient programming in liquid chromatography has been known but only to utilize the general effect of temperature in reducing retention, thereby enhancing elution from a column. For example, Djordjevic, et al. Anal. Chem., 70 (9)1921-25 (1998) disclose the analysis of oligonucleotides using a temperature programmed ion pair reversed phased method. The molecules are, however, single stranded nucleic acids. They are separated by the reduction of retention that occurs with increasing temperature. There is no showing of a conformational change induced by increasing the temperature nor detection of the extent of hybridization, since the samples are single stranded.
Jarrett, et al. J. Chrom., 508, 279-87 (1990) disclose the preparation of a chromatography affinity phase to which an oligothymidylic acid (DT)18 is covalently bonded. The affinity phase is used to separate single stranded oligonucleotides by temperature gradient elution. The sample is retained on the column by on-column hybridization, then by increasing the temperature there is successive displacement of longer complementary probes from the stationary phase based upon the affinity of the complementary pair DNA strands. The disadvantage of the technique is that it requires the synthesis of a unique phase for each target sample.
The present invention provides a method for separating macromolecules which does not suffer from such disadvantages and which is particularly useful for separating single nucleotide variants of DNA. The invention does not require chemical or enzymatic cleavage reactions, modification of sample DNA sequences with GC clamps, preparation of combinatorial probes, or extensive optimization of liquid chromatography gradient conditions and temperatures. The invention provides an easily automated high speed sensitive method which allows recovery of the analyzed dsDNA sample free from polymeric contaminants.
SUMMARY OF THE INVENTION
A liquid chromatographic method is provided for separating macromolecules such as polynucleotides which undergo a temperature sensitive structural denaturation by means of a temporal or spatial thermal gradient to produce differential chromatographic mobility of polynucleotide molecules on interaction with a stationary phase. The method is particularly adapted for the separation of variant DNA polymorphisms. A mixture of polynucleotide molecules is applied in a mobile phase to a stationary reversed phased support. The polynucleotide molecules remain undenatured or can be at least partially denatured. The mixture is then eluted with a mobile phase while exposing the polynucleotides to a temperature gradient effective to at least partially denature the polynucleotides, which results in separation. This temperature gradient may be formed either as a temporal temperature gradient applied to the entire stationary phase or as a spatial temperature gradient in the stationary phase through which the polynucleotides migrate.
REFERENCES:
patent: 5795976 (1998-08-01), Oefner et al.
patent: 2002/0042060 (2002-04-01), Races et al.
Article by Djordjevic, et al. entitled “HPLC Separation of Oligo nucleotides in Isocratic and Temperature-Programming Mode”, published in Anal. Chem. 1998, 70, pp. 1921-1925.
Article by Goss, et al. entitled “High-Performance Affinity Chromatography of DNA”, published in Journal of Chromatography, 508 (1990), pp. 279-287.
Article by Guthridge, et al. entitled “High-Performance Liquid Chromatography of Amino Acids, Peptides, Proteins and Polynucleotides”, published in the Journal of Chromatography, 476 (1989), pp. 445-453.
Article by Masson, et al. entitled “High-Performance Affinity Chromatography of DNA”, published in Journal of Chromatography, 600 (1992), pp. 221-228.
Article by Goss, et al., entitled “High-Performance Affinity Chromatography of Messenger RNA”, published in Journal of Chromatography, 588 (1991), pp. 157-164.
Article by Paul Molander, et
Gerber Reinhold E.
Hatch Richard G.
Ketter James
Varian Inc.
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