Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-09-29
2002-02-12
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C436S094000
Reexamination Certificate
active
06346386
ABSTRACT:
BACKGROUND
1. The Field of the Invention
This invention relates to a method of identifying sequence alterations in a DNA fragment. More particularly the invention relates to a solution-based method of scanning for single base pair substitutions using fluorescence melting profiles and a double-stranded DNA binding dye.
2. Technical Background
Many human diseases and disorders are associated with genetic alterations. Many diseases can be attributed to a change or a mutation in a single gene. The diseases causes by genetic mutations may be diagnosed based on detection of a mutation within the genome of an individual. With early detection and the proper diagnosis, many of these disease may be treated. However, many of the currently available methods of detection are costly and labor intensive. Because of the labor involved the methods are subject to human error and run the risk of false results.
Several types of mutations can occur in DNA. A point mutation occurs when a single base is changed to one of the three other bases. A deletion occurs where one or more bases are deleted from a gene. An insertion occurs where new bases are inserted at a particular point in a nucleic acid sequence adding additional length to the sequence.
Large insertions or deletions can be readily detected within a DNA sample. Because of the small degree of molecular change, the point mutation is the most difficult type of mutation to screen for and detect. However, a number of diseases including some types of cancer are known to be caused by point mutations.
Methods that permit the detection of single base changes in specific regions of the genome have enjoyed tremendous utility in the field of genetics by facilitating genetic linkage analysis and the identification of mutations with specific disease associations, including those involved in the development and evolution of neoplasia. R. Wallace et al.,
Science
249:181-6 (1990); R. M. Cawthon et al.,
Cell
62:193-201 (1990); R. A. Flavell et al.,
Cell
15:25-41 (1978); A. P. Feinberg et al.,
Science
220:1175-1177 (1983); J. L. Bos et al.,
Nature
327:293-297 (1987); Hollstein et al.,
Science
253:49-53 (1991).
Single base alterations have been detected using a variety of methods. Some methods rely on the abolition or creation of novel restriction enzyme sites for example, restriction fragment length polymorphism analysis. Other methods use the polymerase chain reaction (PCR) and subsequent distinction of base mismatches by oligonucleotide hybridization. Single base changes have also been detected by the differences in the conformational or melting temperature characteristics of the mutated and wild-type sequences for example, single strand conformation polymorphism analysis and denaturation gradient gel electrophoresis. A. R. Wyman & R. White,
Proc Natl Acad Sci USA
77:6754-6758 (1980); K. Mullis et al.,
Cold Spring Harb Symp Quant Biol
51:263-273 (1986); R. K. Saiki et al.,
Science
239:487-91 (1988); A. Neri et al.,
Proc Natl Acad Sci USA
85:9268-9272 (1988); M. Orita et al.,
Proc Natl Acad Sci USA
86:2766-70 (1989). All of these methods require multiple steps including gel electrophoretic separation and/or radioisotopic detection of the sequence variants. The use of radioisotopes creates safety concerns that increase the cost and limit the utility of these methods.
Recently, fluorescence-based technologies have been used for the detection of specific nucleic acid sequences. R. Higuchi et al.,
Biotechnology
(N Y) 11:1026-30 (1993). These assays have typically exploited one of several fluorescence chemistries. C. T. Wittwer et al.,
Biotechniques
22:130-8 (1997). Some of these assays are non-specific methods and incorporate a double stranded DNA (dsDNA) binding dye such as SYBR® Green I into the amplification reaction. The specificity of product detection is entirely dependent on the inherent specificity of the amplification conditions. Subtle changes such as point mutations are difficult to detect using non-specific dsDNA binding dyes.
Other fluorescence-based assays are sequence-specific. These methods are probe-based and incorporate oligonucleotides that hybridize to a sequence within the amplified sample sequence. This method provides an additional parameter for verification of product identity. The specificity of the hybridization interaction has been further exploited for the identification of single nucleotide polymorphisms by virtue of the fact that the single base mismatches within the hybridization probe to DNA target hybrids exhibit lower melting temperatures than perfectly complementary strands. However, the detection of single base changes using probe-based methods is limited to very short segments of DNA of approximately 20 base pairs or less. The probe-based methods would require several probes in multiple separate reactions to scan a region of more than 100 bases. The cost of such probe-based assays is relatively high. Thus, the probe-based methods are unsuitable for mutational scanning of larger regions of DNA.
Unstacking of long helical DNA fragments has been shown in denaturing gel-based systems to occur in a succession of segments or discrete cooperative units referred to as domains. The melting temperatures (Tms) of these domains are principally determined by the base composition and precise DNA sequence. Single base differences in otherwise homologous DNA fragments can be discriminated provided that the differences are located within the lowest melting domain, and this domain is clearly separated from the onset of duplex to single strands dissociation. Both of these conditions can be satisfactorily achieved by the attachment of a higher melting section or a GC-rich sequence to the DNA fragment of interest, thus rendering the detection of virtually all single base changes possible. These principles have been exploited in such gel-based approaches as denaturation gradient gel electrophoresis and related methods. However, the gel-based systems require a significant amount of time, and are not readily adaptable to automation.
In light of the foregoing, it would be a significant advancement in the art to provide a solution-based method for detecting changes in a DNA sequence capable of screening DNA segments of greater than 20 base pairs. It would be an additional advancement if the method were capable of distinguishing even subtle single base changes from the wild-type. It would be an additional advancement if the method used a double-stranded DNA binding dye for detecting the mutation. It would be an additional advancement if the method were rapid and not labor intensive. It would be a further advancement if the method produced accurate and reproducible results. It would be an additional advancement if the method were adaptable to automation.
Such a method is disclosed herein.
BRIEF SUMMARY OF THE INVENTION
The present invention relates to a solution-based method for determining whether a DNA sequence is identical to a wild-type sequence. The alteration may be a point mutation such as a transversion or transition or other mutation such as an insertion or deletion. The method uses a dsDNA binding dye and fluorescent melting profiles to detect a mutation within a DNA segment of interest.
In a presently preferred embodiment, a sample of DNA suspected of containing a mutation is obtained. The sequence to be analyzed for the presence of an alteration is amplified to obtain an adequate amount of the sequence. A GC-rich segment is attached to the 5′ end of the sequence of interest. This GC-clamp creates two melting domains, a higher domain associated with the GC-clamp and a lower domain associated with the DNA segment of interest. The GC-clamp may be of any length or sequence that confers a significantly higher melting temperature than that estimated for the sequence of interest.
In a presently preferred embodiment of the invention, a double stranded DNA (dsDNA) binding dye such as SYBR® Green I (Molecular Probes, Eugene, Oreg.), ethidium bromide, or YO-PRO-I® (Molecular Probes, Eugene, Oreg.) is included in an ampl
ARUP Institue
Horlick Kenneth R.
Madson & Metcalf
Spiegler Alexander H.
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