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1.发明授权
公开(公告)号：US6043070A
公开(公告)日：2000-03-28
申请号：US924031
申请日：1997-08-29
Applicant: Nicole M. Ellis , Robert G. Kuimelis , Cheryl R. Heiner , Katherine D. Lazaruk , Patric Sean Walsh
Inventor： Nicole M. Ellis , Robert G. Kuimelis , Cheryl R. Heiner , Katherine D. Lazaruk , Patric Sean Walsh
CPC classification number: C12Q1/686
Abstract: The invention relates to novel chimeric phosphoramidate oligonucleotides and their use in primer-extension methods such as DNA sequencing and nucleic acid amplification. The subject chimeric phosphoramidate oligonucleotides have both N3'-phosphoramidate linkages and phosphodiester linkages. The invention includes methods of primer extension using the subject chimeric oligonucleotides as primers. Primer extension methods of interest include nucleic acid amplification reactions, e.g. PCR, and polynucleotide sequencing reactions. In the primer extension methods of the invention, a chimeric phosphoramidate oligonucleotide primer is annealed to a polynucleotide template. After annealing, the chimeric oligonucleotide primer is extended by joining a nucleotide to the 3' end of the primer by a DNA polymerase catalyzed reaction. Other embodiments of the invention include methods of primer extension using phosphoramidate linkage containing polynucleotide templates. Such methods include annealing an oligonucleotide primer (chimeric or otherwise) to a polynucleotide template comprising at least one phosphoramidate linkage. Nucleotides are added to the primer in a DNA polymerase catalyzed reaction. Primer extension takes place across one or more of the phosphoramidate linkages in the template. The invention includes compositions comprising a chimeric phosphoramidate oligo-nucleotide of the invention and a divalent cation. Divalent cations serve to increase the binding affinity between the chimeric oligonucleotides of the invention and a polynucleotide template. Additionally, divalent cations may be used to stabilize the phosphoramidate linkages of the subject chimeric oligonucleotides against hydrolysis at elevated temperatures, such as the temperatures used in PCR and cycle sequencing. The subject compositions may further comprise a thermostable DNA polymerase such as Taq DNA polymerase.
Abstract translation: 本发明涉及新型嵌合氨基磷酸酯寡核苷酸及其在引物延伸方法如DNA测序和核酸扩增中的应用。 本发明的嵌合氨基磷酸酯寡核苷酸具有N 3'-氨基磷酸酯键和磷酸二酯键。 本发明包括使用本发明的嵌合寡核苷酸作为引物的引物延伸方法。 引物引物扩增方法包括核酸扩增反应，例如 PCR和多核苷酸测序反应。 在本发明的引物延伸方法中，将嵌合氨基磷酸酯寡核苷酸引物与多核苷酸模板退火。 退火后，通过DNA聚合酶催化反应将核苷酸连接到引物的3'末端来延伸嵌合寡核苷酸引物。 本发明的其它实施方案包括使用含有氨基磷酸酯键的多核苷酸模板引物延伸的方法。 这样的方法包括将寡核苷酸引物（嵌合或其它）退火至包含至少一个氨基磷酸酯键的多核苷酸模板。 在DNA聚合酶催化反应中将核苷酸加入到引物中。 引物延伸在模板中的一个或多个氨基磷酸酯键上进行。 本发明包括包含本发明的嵌合氨基磷酸酯寡核苷酸和二价阳离子的组合物。 二价阳离子用于增加本发明的嵌合寡核苷酸与多核苷酸模板之间的结合亲和力。 此外，二价阳离子可以用于稳定本发明嵌合寡核苷酸的氨基磷酸酯键与升高的温度例如在PCR和循环测序中使用的温度下的水解。 本发明组合物还可以包含热稳定的DNA聚合酶如Taq DNA聚合酶。
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公开(公告)号：US20100203525A1
公开(公告)日：2010-08-12
申请号：US12614318
申请日：2009-11-06
Applicant: Kenneth J. Livak , Junko Stevens , Katherine D. Lazaruk , Janet S. Ziegle , Lily Y. Wong
Inventor： Kenneth J. Livak , Junko Stevens , Katherine D. Lazaruk , Janet S. Ziegle , Lily Y. Wong
CPC classification number: G16B20/00 , C12Q1/6827 , C12Q1/6851 , C12Q2600/156 , C12Q2563/107 , C12Q2545/101 , C12Q2565/101 , C12Q2563/173 , C12Q2565/1015
Abstract: Methods of detecting a candidate genetic anomaly such as a candidate duplication in a genome are disclosed. The methods comprise quantifying fluorogenic assays for alleles of a genetic locus from a plurality of individual genomes, identifying ranges of fluorescent intensities indicative of individual genomes homozygous for a first allele, homozygous for a second allele, or heterozygous for both alleles, and identifying individual genomes in which the fluorescence intensities are outside the range of intensities indicative of homozygosity or heterozygosity for the genetic locus.
Abstract translation: 公开了检测候选遗传异常（例如基因组中候选重复）的方法。 所述方法包括定量来自多个单个基因组的遗传基因座的等位基因的荧光测定，鉴定指示第一等位基因纯合的单个基因组的荧光强度的范围，对于第二等位基因纯合，或对于两个等位基因是杂合的，并鉴定个体基因组 其中荧光强度在表示遗传基因座的纯合性或杂合性的强度范围之外。
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3.发明授权Phosphoramidate-phosphodiester oligonucleotide chimera utilized as primers 有权氨基磷酸酯 - 磷酸二酯寡核苷酸嵌合体用作引物
公开(公告)号：US6030813A
公开(公告)日：2000-02-29
申请号：US271060
申请日：1999-03-16
Applicant: Nicole M. Ellis , Robert G. Kuimelis , Cheryl R. Heiner , Katherine D. Lazaruk , Patric Sean Walsh
Inventor： Nicole M. Ellis , Robert G. Kuimelis , Cheryl R. Heiner , Katherine D. Lazaruk , Patric Sean Walsh
CPC classification number: C12Q1/686
Abstract: The invention relates to novel chimeric phosphoramidate oligonucleotides and their use in primer-extension methods such as DNA sequencing and nucleic acid amplification. The subject chimeric phosphoramidate oligonucleotides have both N3'-phosphoramidate linkages and phosphodiester linkages. The invention includes methods of primer extension using the subject chimeric oligonucleotides as primers. Primer extension methods of interest include nucleic acid amplification reactions, e.g. PCR, and polynucleotide sequencing reactions. In the primer extension methods of the invention, a chimeric phosphoramidate oligonucleotide primer is annealed to a polynucleotide template. After annealing, the chimeric oligonucleotide primer is extended by joining a nucleotide to the 3' end of the primer by a DNA polymerase catalyzed reaction. Other embodiments of the invention include methods of primer extension using phosphoramidate linkage containing polynucleotide templates. Such methods include annealing an oligonucleotide primer (chimeric or otherwise) to a polynucleotide template comprising at least one phosphoramidate linkage. Nucleotides are added to the primer in a DNA polymerase catalyzed reaction. Primer extension takes place across one or more of the phosphoramidate linkages in the template. The invention includes compositions comprising a chimeric phosphoramidate oligo-nucleotide of the invention and a divalent cation. Divalent cations serve to increase the binding affinity between the chimeric oligonucleotides of the invention and a polynucleotide template. Additionally, divalent cations may be used to stabilize the phosphoramidate linkages of the subject chimeric oligonucleotides against hydrolysis at elevated temperatures, such as the temperatures used in PCR and cycle sequencing. The subject compositions may further comprise a thermostable DNA polymerase such as Taq DNA polymerase.
Abstract translation: 本发明涉及新型嵌合氨基磷酸酯寡核苷酸及其在引物延伸方法如DNA测序和核酸扩增中的应用。 本发明的嵌合氨基磷酸酯寡核苷酸具有N 3'-氨基磷酸酯键和磷酸二酯键。 本发明包括使用本发明的嵌合寡核苷酸作为引物的引物延伸方法。 引物引物扩增方法包括核酸扩增反应，例如 PCR和多核苷酸测序反应。 在本发明的引物延伸方法中，将嵌合氨基磷酸酯寡核苷酸引物与多核苷酸模板退火。 退火后，通过DNA聚合酶催化反应将核苷酸连接到引物的3'末端来延伸嵌合寡核苷酸引物。 本发明的其它实施方案包括使用含有氨基磷酸酯键的多核苷酸模板引物延伸的方法。 这样的方法包括将寡核苷酸引物（嵌合或其它）退火至包含至少一个氨基磷酸酯键的多核苷酸模板。 在DNA聚合酶催化反应中将核苷酸加入到引物中。 引物延伸在模板中的一个或多个氨基磷酸酯键上进行。 本发明包括包含本发明的嵌合氨基磷酸酯寡核苷酸和二价阳离子的组合物。 二价阳离子用于增加本发明的嵌合寡核苷酸与多核苷酸模板之间的结合亲和力。 此外，二价阳离子可以用于稳定本发明嵌合寡核苷酸的氨基磷酸酯键与升高的温度例如在PCR和循环测序中使用的温度下的水解。 本发明组合物还可以包含热稳定的DNA聚合酶如Taq DNA聚合酶。
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