Fluorescence polarization in nucleic acid analysis

Details Fluorescence polarization in nucleic acid analysis Fluorescence polarization in nucleic acid analysis

2000-11-07

2002-08-27

Siew, Jeffrey (Department: 1656)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S006120, C435S091100, C435S091410, C536S022100, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330

Reexamination Certificate

active

06440707

ABSTRACT:

TECHNICAL FIELD OF THE INVENTION
This invention relates to new diagnostic methods and diagnostic kits for the analysis of DNA using template-directed primer extension and highly sensitive detection by fluorescence polarization. This invention further relates to methods of identifying single nucleotide polymorphisms (SNPs) as well as other DNA sequence variations. Also, this invention relates broadly to the fields of biotechnology, molecular genetics and biomedicine.
BACKGROUND OF THE INVENTION
DNA analysis is becoming increasingly important in the diagnosis of hereditary diseases, detection of infectious agents, tissue typing for histocompatability, identification of individuals in forensic and paternity testing, and monitoring the genetic makeup of plants and animals in agricultural research (Alford, R. L., et al.,
Curr Opn. Biotechnol
(1994) 5:29-33). In addition, DNA analysis is crucial in large-scale genetic studies to identify susceptibility alleles associated with common diseases involving multiple genetic and environmental factors (Risch, N., et al.,
Science
(1996) 273:1516-1517). Recently, attention is focused on single nucleotide polymorphisms (SNPs), the most common DNA sequence variation found in mammalian genomes (Cooper, D. N., et al.,
Hum Genet
(1985) 69:201-205). While most of the SNPs do not give rise to detectable phenotypes, a significant fraction of them are disease-causing mutations responsible for genetic diseases. As the DNA sequence of the human genome is completely elucidated, large-scale DNA analysis will play a crucial role in determining the relationship between genotype (DNA sequence) and phenotype (disease and health) (Cooper, D. N., et al.,
Hum Genet
(1988) 78:299-312). Although some assays have considerable promise for high throughput, the recently developed DNA diagnostic methods, including the high-density chip arrays for allele-specific hybridization analysis (Pease, A. C., et al.,
Proc Natl Acad Sci USA
(1994) 91:5022-5026; Yershov, G., et al.,
Proc Natl Acad Sci USA
(1996) 93:4913-4918), Wang, D. G., et al.,
Science
(1998) 280:1077-1081, the homogeneous 5′-nuclease allele-specific oligonucleotide cleavage assay (TaqMan ASO, Livak, K. J., et al.,
Nat Genet
(1995) 9:341-342), Whitcombe, D., et al.,
Clin Chem
(1998) 44:918-923 a homogeneous fluorescence assay for PCR amplifications: its application to real-time, single-tube genotyping, the homogeneous template-directed dye-terminator incorporation (TDI) assay (Chen, X., et al.,
Nucleic Acids Res
(1997) 25:347-353; Chen, X., et al.,
Proc Natl Acad Sci USA
(1997) 94:10756-10761) the homogeneous dye-labeled oligonucleotide ligation (DOL) assay (Chen, X. et al. Genome Research (1998) 8: 549-556.), and the homogeneous molecular beacon ASO assay (Tyagi, S. et al. Nature Biotechnology (1998) 16: 49-53), all require specialty reagents and expensive detection instrumentation.
All the DNA diagnostic methods listed above involve amplification of target sequences to increase the sensitivity and specificity of the assays through polymerase chain reaction (PCR) or other similar amplification technologies. For example, one of the best known amplification methods is the polymerase chain reaction (referred to as PCR) which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159. Briefly, in PCR, two primer sequences are prepared which are complementary to regions on opposite complementary strands of the target sequence. An excess of deoxynucleoside triphosphates are added to a reaction mixture along with a thermostable DNA polymerase (e.g., Taq polymerase). If the target sequence is present in a sample, the primers will bind to the target and the polymerase will cause the primers to be extended along the target sequence by adding on nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primers reaction products will dissociate from the target to become new targets. The excess primers will bind to the target and to the reaction products and the process is repeated. Other technologies result in amplification of a target sequence by strand displacement. These techniques include an enzymatic “nicking” or preferential cleavage of one of two strands present in a double-stranded DNA recognition site and the separation or detection of amplified products that include the target site, which is presented in U.S. Pat. Nos. 5,270,184. and 5,455,166 and each of which is hereby incorporated by reference herein.
Still other techniques employ the use of a fluorescently labeled primer and detect fluorescence or fluorescence polarization after the primer is hybridized to the target region, which is presented in U.S. Pat. No. 5,593,867. In U.S. Pat. No. 5,641,633, double-stranded DNA binding protein is also used to further preserve the hybridization of the fluorescently labeled primer to the target site. These methods require the use of fluorescently labeled primers and their detection while hybridized to the target site.
Template-directed primer extension is a dideoxy chain terminating DNA sequencing protocol designed to ascertain the nature of the one base immediately 3′ to the sequencing primer that is annealed to the target DNA immediately upstream from the polymorphic site. In the presence of DNA polymerase and the appropriate dideoxyribonucleoside triphosphate (ddNTP), the primer is extended specifically by one base as dictated by the target DNA sequence at the polymorphic site. By determining which ddNTP is incorporated, the allele(s) present in the target DNA can be inferred. This genotyping method has been widely used in many different formats and proven to be highly sensitive and specific (Syvanen, A.-C et al, Genomics (1990) 8: 684-692; Syvanen, A.-C. and Landegren, U. Human Mutation (1994) 3: 172-179).
Fluorescence polarization (FP) is based on the observation that when a fluorescent molecule is excited by plane-polarized light, it emits polarized fluorescent light into a fixed plane if the molecules remain stationary between excitation and emission (FIG.
1
). Because the molecule rotates and tumbles in space during the fluorescent decay time, however, FP is not observed fully by an external detector. The observed FP of a fluorescent species is described by the Perrin equation and is related to the ratio of the rotational relaxation time and the fluorescent lifetime. If the temperature and viscosity are held constant the rotational relaxation time is proportional to the molecular volume of the fluorescent species. If local rotational motion of the fluorophore is minimal the FP is directly proportional to the molecular weight. In other words, when a fluorescent molecule and its conjugate are large (with high molecular weight), it rotates and tumbles slowly in solution compared to the fluorescent lifetime and FP is preserved. If the molecule is small (with low molecular weight), it rotates and tumbles faster and FP is largely lost (depolarized). The FP phenomenon has been used to study protein-DNA and protein-protein interactions (Dunkak, K. S. et al., Anal. Biochem. (1996) 243: 234-244; Heyduk, T. et al. Methods Enzymol. (1996) 274: 492-503; Wu, P. et al., Anal. Biochem. (1997) 249: 29-36), DNA detection by strand displacement amplification (Walker, G. T. et al., Nucleic Acids Res. (1996) 24: 348-353), and in genotyping by hybridization (Gibson, N. J. et al., Clin. Chem. (1997) 43: 1336-1341). More than 50 fluorescence polarization immunoassays (FPIA) are currently commercially available, many of which are routinely used in clinical laboratories for the measurement of therapeutics, metabolites, and drugs of abuse in biological fluids (Checovich, W. J., et al.,
Nature
(1995) 375:254-256).
FP is expressed as the ratio of fluorescence detected in the vertical and horizontal axes and is therefore independent of the total fluorescence intensity. This is a clear advantage over other fluorescence detection methods in that as long as the fluorescence is above detection limits of the instrument used, FP is a reliable measure. The FP di

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