Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-04-01
2002-03-26
Riley, Jezia (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S022100, C536S023100, C536S024300, C536S025300
Reexamination Certificate
active
06361940
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to compositions and methods for hybridization of oligonucleotides, and more specifically to certain solutions and/or oligonucleotide analogues which may increase hybridization and priming specificity.
BACKGROUND OF THE INVENTION
The detection of diseases is increasingly important in prevention and treatments. While multifactorial diseases are difficult to devise genetic tests for, more than 200 known human disorders are caused by a defect in a single gene, often a change of a single amino acid residue (Olsen,
Biotechnology: An industry comes of age
, National Academic Press, 1986). Many of these mutations result in an altered amino acid that causes a disease state.
Sensitive mutation detection techniques offer extraordinary possibilities for mutation screening. For example, analyses may be performed even before the implantation of a fertilized egg (Holding and Monk,
Lancet
3:532, 1989). Increasingly efficient genetic tests may also enable screening for oncogenic mutations in cells exfoliated from the respiratory tract or the bladder in connection with health checkups (Sidransky et al.,
Science
252:706, 1991). Also, when an unknown gene causes a genetic disease, methods to monitor DNA sequence variants are useful to study the inheritance of disease through genetic linkage analysis. However, detecting and diagnosing mutations in individual genes poses technological and economic challenges. Several different approaches have been pursued, but none are both efficient and inexpensive enough for truly widescale application.
Mutations involving a single nucleotide can be identified in a sample by physical, chemical, or enzymatic means. Generally, methods for mutation detection may be divided into scanning techniques, which are suitable to identify previously unknown mutations, and techniques designed to detect, distinguish, or quantitate known sequence variants.
Several scanning techniques for detection of mutations have been developed on the observation that heteroduplexes of mismatched complementary DNA strands exhibit an abnormal behavior, especially when denatured. This phenomenon is exploited in denaturing and temperature gradient gel electrophoresis (DGGE and TGGE, respectively) methods. Duplexes mismatched in even a single nucleotide position can partially denature, resulting in retarded migration, when electrophoresed in an increasingly denaturing gradient gel (Myers et al.,
Nature
313:495, 1985; Abrams et al.,
Genomics
7:463, 1990; Henco et al.,
Nucl. Acids Res.
18:6733, 1990). Although mutations may be detected, no information is obtained regarding the precise location of a mutation. Mutant forms must be further isolated and subjected to DNA sequence analysis.
Alternatively, a heteroduplex of an RNA probe and a target strand may be cleaved by RNase A at a position where the two strands are not properly paired. The site of cleavage can then be determined by electrophoresis of the denatured probe. However, some mutations may escape detection because not all mismatches are efficiently cleaved by RNase A.
Mismatched bases in a duplex are also susceptible to chemical modification. Such modification can render the strands susceptible to cleavage at the site of the mismatch or cause a polymerase to stop in a subsequent extension reaction. The chemical cleavage technique allows identification of a mutation in target sequences of up to 2 kb and it provides information on the approximate location of mismatched nucleotide(s) (Cotton et al.,
PNAS USA
85:4397, 1988; Ganguly et al.,
Nucl. Acids Res.
18:3933, 1991). However, this technique is labor intensive and may not identify the precise location of the mutation.
An alternative strategy for detecting a mutation in a DNA strand is by substituting (during synthesis) one of the normal nucleotides with a modified nucleotide, thus altering the molecular weight or other physical parameter of the product. A strand with an increased or decreased number of this modified nucleotide relative to the wild-type sequence exhibits altered electrophoretic mobility (Naylor et al.,
Lancet
337:635, 1991). This technique detects the presence of a mutation, but does not provide the location.
Two other strategies visualize mutations in a DNA segment by altered gel migration. In the single-strand conformation polymorphism technique (SSCP), mutations cause denatured strands to adopt different secondary structures, thereby influencing mobility during native gel electrophoresis. Heteroduplex DNA molecules, containing internal mismatches, can also be separated from correctly matched molecules by electrophoresis (Orita,
Genomics
5:874, 1989; Keen,
Trends Genet.
7:5, 1991). As with the techniques discussed above, the presence of a mutation may be determined but not the location. As well, many of these techniques do not distinguish between a single and multiple mutations.
All of the above-mentioned techniques indicate the presence of a mutation in a limited segment of DNA and some of them allow approximate localization within the segment. However, sequence analysis is still required to unravel the effect of the mutation on the coding potential of the segment. Sequence analysis is a powerful tool, allowing, for example, screening for the same mutation in individuals of an affected family, monitoring disease progression in the case of malignant disease, or for detecting residual malignant cells in bone marrow before autologous transplantation. Despite these advantages, the procedure is unlikely to be adopted as a routine diagnostic method because of the high expense involved.
A large number of other techniques have been developed to analyze known sequence variants. Automation and economy are very important considerations for implementation of these types of analyses. In this regard, none of the alternative techniques discussed below combine economy and automation with the required specificity.
A number of strategies for nucleotide sequence distinction all depend on enzymes to identify sequence differences (Saiki,
PNAS USA
86:6230, 1989; Zhang,
Nucl. Acids Res.
19:3929, 1991).
For example, restriction enzymes recognize sequences of about 4-8 nucleotides. Based on an average G+C content, approximately half of the nucleotide positions in a DNA segment can be monitored with a panel of 100 restriction enzymes. As an alternative, artificial restriction enzyme recognition sequences may be created around a variable position by using partially mismatched PCR primers. With this technique, either the mutant or the wild-type sequence alone may be recognized and cleaved by a restriction enzyme after amplification (Chen et al.,
Anal. Biochem.
195:51, 1991; Levi et al.,
Cancer Res.
51:3497, 1991).
Another method exploits the property that an oligonucleotide primer that is mismatched to a target sequence at the 3′ penultimate position exhibits a reduced capacity to serve as a primer in PCR. However, some 3′ mismatches, notably G-T, are less inhibitory than others, thus limiting its usefulness. In attempts to improve this technique, additional mismatches are incorporated into the primer at the third position from the 3′ end. This results in two mismatched positions in the three 3′ nucleotides of the primer hybridizing with one allelic variant, and one mismatch in the third position in from the 3′ end when the primer hybridizes to the other allelic variant (Newton et al.,
Nucl. Acids Res.
17:2503, 1989). For this technique to be successful, it is necessary to define amplification conditions that significantly disfavor amplification in the presence of a 1 bp (basepair) mismatch. In fact, this technique is rarely successful (see, e.g., Sininsky,
J. Nucl. Acids Res.,
1990).
DNA polymerases have also been used to distinguish allelic sequence variants by determining which nucleotide is added to an oligonucleotide primer immediately upstream of a variable position in the target strand. Based on this approach, a ligation assay has been developed. In this method, two olig
Garrison Lori K.
Ness Jeffrey Van
Tabone John C.
QIAGEN Genomics, Inc.
Riley Jezia
Seed Intellectual Property Law Group PLLC
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