Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-04-05
2004-08-17
Fredman, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S023100, C536S024100, C536S024310, C536S024320, C536S024330
Reexamination Certificate
active
06777183
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a process for allele discrimination employing primer extension using an exonuclease deficient polymerase to distinguish matched 3′-ends from mismatched 3′-ends of hybridized primer and target oligonucleotides.
BACKGROUND OF THE INVENTION
Many diseases are known which have a genetic basis in their etiology and result from the occurrence of mutations in gene sequences present in the genomes of different organisms, especially animals, including humans, afflicted with such diseases. Consequently, methods for detecting slight genetic differences, as small as one nucleotide (called single nucleotide polymorphisms or SNPs), between the genome of a healthy individual and that of a person afflicted with a genetic defect can prove highly valuable in elucidating the nature and causes of such condition. More importantly, obtaining valuable information about such conditions is greatly enhanced if a sensitive process is available for determining small genetic defects, such as a difference of one nucleotide at a particular location in the genome.
A genome is composed of different loci which are themselves composed of one or more genes, which genes may contain variations, so-called alleles, for each system. For example, the immunoglobulin superfamily, which includes, inter alia, the T-cell receptor, the immunoglobulin and the HLA (or human leukocyte antigen) systems, is characterized by the presence of large sequence variations (called polymorphisms). Defects in the immune response, which are due to diverse variations in one or more of the gene arrangements of such systems, may result in disease. Conversely, diseases like cystic fibrosis show varying and complex genetic variations in DNA sequence. Genetic variation may therefore be linked to diseases and their symptoms. Identification of the associated alleles, especially differences in those alleles, may be important in determining the risk of a disease associated with genetic markers or in detecting variations in genes that result in some other malady. Further, the delineation of slight genetic differences can be readily utilized for the diagnosis (even treatment) of certain diseases, as well as furthering efforts toward prevention by identifying persons having the greatest risk of a particular disease. The latter is a critical factor in those situations where early treatment is possible and the development of the disease can be retarded.
Several methods for detecting specific nucleotide variations and genetic polymorphisms in nucleic acids are known. For example, some methods comprise amplifying nucleic acid sequences having nucleotide variations, mutations and polymorphisms, with subsequent detection thereof using allele specific oligonucleotide sequences and a dot blot. This process utilizes allele-specific oligonucleotide sequences that have to be very specific for the nucleotide variation to be detected and offers numerous primer sequences for use therein depending on the DNA sequence to be studied. [See, for example, EP-A-237,362]
R. K. Saiki et al.,
Proc. Natl. Acad. Sci. USA
. 86, 6230-6234 (1989) as well as WO 89/11548 both disclose use of immobilized sequence-specific oligonucleotides. WO 89/11547 discloses methods for determining genotypes having different alleles in the HLA-DP loci. This latter method operates by hybridizing nucleic acid samples with a series of probes which are specific for various segments.
U.S. Pat. No. 5,912,148 discloses a polymerase chain reaction (PCR) method as well as an oligonucleotide ligase assay (OLA) procedure for analyzing complex genetic systems in a single reaction vessel (also see other methods cited therein). This method seeks to determine the products of the OLA reaction using various OLA and PCR probes.
U.S. Pat. No. 5,759,771 discloses a method for determining genotypes by comparing the nucleotide sequences of members of a gene system that flank the polymorphic segments of a particular genetic locus. Here, the compared sequences contain conserved sequences used to amplify the strongly conserved segments from different sources. These are then compared as a means of establishing genotype.
U.S. Pat. No. 5,710,028 discloses a method of simultaneous determination of the identity of nucleotide bases at specific locations in nucleic acids of interest but relies on the use of extension blocking agents, commonly dideoxy-nucleoside triphosphates, to prevent extension in cases where there is a particular nucleotide present at a given location within the target sequence (the latter acting as a template). A similar process is used in U.S. Pat. No. 6,013,431.
Nucleic acid sequence analysis has become important in many research, medical, and industrial fields and a host of methods have been described in the literature. Heretofore, many of these approaches have been motivated by the development of various methods for amplifying target nucleic acids, e.g. polymerase chain reaction (PCR) of U.S. Pat. No. 5,137,806, ligation chain reaction (LCR), and the like, as well as rolling circle amplification (RCA) (See, for example, U.S. Pat. No. 5,854,033; Lizardi et al,
Nature Genetics
, 19, 225-232 (1998). Such amplification techniques are certainly useful as the basis for developing sensitive and specific diagnostic assays but in some cases these methods may be fairly complex and involved, especially when the system to be analyzed is a complex one, such as a complex genetic system, for example, the highly variable cystic fibrosis locus. Because it may be difficult to identify the amplified product in such systems, post-amplification manipulations may often be necessary, especially in cases other than RCA. One approach used to avoid these problems is that of the oligonucleotide ligation assay (OLA). [U.S. Pat. No. 4,883,750] Here, oligonucleotides are prepared that are complementary to adjacent regions of a target sequence and are capable of hybridizing to the target so that they lie end-to-end and can be ligated when no mismatches occur at or near the contiguous ends. Whenever mismatches occur, ligation is precluded. The result is a set of oligonucleotide pairs that are perfect complements of all the allelic variants of interest at a given locus. By carefully selecting the labeling method, a wide range of alleles can be specifically identified in a single assay. However, such assays can be complicated. [Nickerson et al.,
Proc. Natl. Acad. Sci. USA
87:8923-8927 (1990)]
Other methods for allele discrimination have relied on template dependent ligation of two adjacent short oligonucleotides. One such oligonucleotide consists of a reverse polarity oligonucleotide containing a primer for RCA and a short target specific sequence terminating at an allele- specific 3′-end residue. A second oligonucleotide is immobilized on a glass slide and anneals next to the target specific oligonucleotide sequence. Template dependent and allele-specific ligation anchors the RCA platform to the slides. Following RCA, products are detected by standard fluorescent and immunochemical techniques. The use of allele specific primers annealing to different circles allows simultaneous detection of various alleles (called multiplexing). Such methods rely on a ligation step as the allele discrimination event. (see Lizardi et al, supra)
A different method employs RCA using padlock probes to detect mutations in cytological samples. However, padlock probes are not always advantageous due to steric hindrance and topological constraints on DNA targets. Such procedures also rely on a ligation step. [see: Nilsson et al, Padlock Probes: Circularizing Oligonucleotides for Localized DNA Detection,
Science
, 265, 2085-2088 (1994)]
One approach to simplifying these procedures is to eliminate some of the steps, thereby simplifying and speeding the overall procedure. For example, such procedures have the disadvantage of relying on DNA ligation as the allele discrimination step.
The method according to the present invention overcomes these pro
Fredman Jeffrey
Grant Alan J.
Molecular Staging Inc.
Olstein Elliot M.
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