Patent•
Detection of specific sequences in nucleic acids
06 Dec 1985-
TL;DR: In this article, the authors proposed a method for diagnosis of genetic abnormalities or other genetic conditions which can be readily automated and is used to determine the presence or absence of a target sequence in a sample of denatured nucleic acid.
Abstract: The invention provides a method for diagnosis of genetic abnormalities or other genetic conditions which can be readily automated. The method is used to determine the presence or absence of a target sequence in a sample of denatured nucleic acid and entails hybridizing the sample with a probe complementary to a diagnostic portion of the target sequence (the diagnostic probe), and with a probe complementary to a nucleotide sequence contiguous with the diagnostic portion (the contiguous probe), under conditions wherein the diagnostic probe remains bound substantially only to the sample nucleic acid containing the target sequence. The diagnostic probe and contiguous probe are then covalently attached to yield a target probe which is complementary to the target sequence, and the probes which are not attached are removed. In the preferred mode, one of the probes is labeled so that the presence or absence of the target sequence can then be tested by melting the sample nucleic acid-target probe duplex, eluting the dissociated target probe, and testing for the label. In another embodiment, the testing is accomplished without first removing probes not covalently attached, by attaching a hook to the probe that is not labeled, so that the labeled target probe may be recovered by catching the hook. In both instances, the presence of both the diagnostic probe and the contiguous probe is required for the label to appear in the assay. The above method is then applied to the detection of genetic diseases.
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Patent•
07 Jun 1990
TL;DR: In this paper, a method and apparatus for preparation of a substrate containing a plurality of sequences is described, where a set of photoremovable groups are attached to a surface of the substrate and selected regions of the surface are exposed to light so as to activate the selected areas.
Abstract: A method and apparatus for preparation of a substrate containing a plurality of sequences. Photoremovable groups are attached to a surface of a substrate. Selected regions of the substrate are exposed to light so as to activate the selected areas. A monomer, also containing a photoremovable group, is provided to the substrate to bind at the selected areas. The process is repeated using a variety of monomers such as amino acids until sequences of a desired length are obtained. Detection methods and apparatus are also disclosed.
1,171 citations
Patent•
11 Aug 1998TL;DR: In this article, the authors present a method for detecting specific nucleic acids in a sample with high specificity and sensitivity, which consists of a DNA ligation operation, an amplification operation, and a detection operation.
Abstract: Disclosed are compositions and a method for of amplifying nucleic acid sequences useful for detecting the presence of molecules of interest. The method is useful for detecting specific nucleic acids in a sample with high specificity and sensitivity. The method also has an inherently low level of background signal. A preferred form of the method consists of a DNA ligation operation, an amplification operation, and a detection operation. The DNA ligation operation circularizes a specially designed nucleic acid probe molecule. This operation is dependent on hybridization of the probe to a target sequence and forms circular probe molecules in proportion to the amount of target sequence present in a sample. The amplification operation is rolling circle replication of the circularized probe. A single round of amplification using rolling circle replication results in a large amplification of the circularized probe sequences. Following rolling circle replication, the amplified probe sequences are detected and quantified using any of the conventional detection systems for nucleic acids such as detection of fluorescent labels, enzyme-linked detection systems, antibody-mediated label detection, and detection of radioactive labels. Because, the amplified product is directly proportional to the amount of target sequence present in a sample, quantitative measurements reliably represent the amount of a target sequence in a sample. Major advantages of this method are that the ligation step can be manipulated to obtain allelic discrimination, the DNA replication step is isothermal, and signals are strictly quantitative because the amplification reaction is linear and is catalyzed by a highly processive enzyme. In multiplex assays, the primer oligonucleotide used for the DNA polymerase reaction can be the same for all probes. Also described are modes of the method in which additional amplification is obtained using a cascade of strand displacement reactions.
824 citations
Patent•
27 May 1997TL;DR: In this paper, the use of a ligase detection reaction coupled to a polymerase chain reaction has been proposed for the detection of nucleic acid sequence differences using coupled ligases detection reaction and polymerases chain reaction.
Abstract: The present invention relates to the detection of nucleic acid sequence differences using coupled ligase detection reaction and polymerase chain reaction. One aspect of the present invention involves use of a ligase detection reaction coupled to a polymerase chain reaction. Another aspect of the present invention relates to the use of a primary polymerase chain reaction coupled to a secondary polymerase chain reaction coupled to a ligase detection reaction. A third aspect of the present invention involves a primary polymerase chain reaction coupled to a secondary polymerase chain reaction. Such coupling of the ligase detection reaction and the polymerase chain reaction permits multiplex detection of nucleic acid sequence differences.
619 citations
Patent•
18 Jun 1991
TL;DR: In this article, the conditions under which oligonucleotides hybridize only with entirely homologous sequences are recognized by simultaneous hybridization of DNA molecules applied as dots and bound onto a filter.
Abstract: The conditions under which oligonucleotides hybridize only with entirely homologous sequences are recognized. The sequence of a given DNA fragment is read by the hybridization and assembly of positively hybridizing probes through overlapping portions. By simultaneous hybridization of DNA molecules applied as dots and bound onto a filter, representing single-stranded phage vector with the cloned insert, with about 50,000 to 100,000 groups of probes, the main type of which is (A,T,C,G)(A,T,C,G)N8(A,T,C,G), information for computer determination of a sequence of DNA having the complexity of a mammalian genome are obtained in one step. To obtain a maximally completed sequence, three libraries are cloned into the phage vector, M13, bacteriophage are used: with the 0.5 kb and 7 kbp insert consisting of two sequences, with the average distance in genomic DNA of 100 kbp. For a million bp of genomic DNA, 25,000 subclones of the 0.5 kbp are required as well as 700 subclones 7 kb long and 170 jumping subclones. Subclones of 0.5 kb are applied on a filter in groups of 20 each, so that the total number of samples is 2,120 per million bp. The process can be easily and entirely robotized for factory reading of complex genomic fragments or DNA molecules.
579 citations
Patent•
17 Oct 1988TL;DR: Linear or branched oligonucleotide multimers are useful as amplifiers in biochemical assays which comprise (1) at least one first single-stranded oligonotide unit that is complementary to a singlestranded sequence of interest, and (2) a multiplicity of second single strand-linked oligon nucleotide units that are complementary to the label of interest as mentioned in this paper.
Abstract: Linear or branched oligonucleotide multimers useful as amplifiers in biochemical assays which comprise (1) at least one first single-stranded oligonucleotide unit that is complementary to a single-stranded oligonucleotide sequence of interest, and (2) a multiplicity of second single-stranded oligonucleotide units that are complementary to a single-stranded labeled oligonucleotide. Amplified sandwich nucleic acid hybridizations and immunoassays using the multimers are exemplified.
521 citations
References
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Book•
15 Jan 2001
TL;DR: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years as mentioned in this paper and has been so popular, or so influential, that no other manual has been more widely used and influential.
Abstract: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential. Molecular Cloning, Fourth Edition, by the celebrated founding author Joe Sambrook and new co-author, the distinguished HHMI investigator Michael Green, preserves the highly praised detail and clarity of previous editions and includes specific chapters and protocols commissioned for the book from expert practitioners at Yale, U Mass, Rockefeller University, Texas Tech, Cold Spring Harbor Laboratory, Washington University, and other leading institutions. The theoretical and historical underpinnings of techniques are prominent features of the presentation throughout, information that does much to help trouble-shoot experimental problems. For the fourth edition of this classic work, the content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories. Core chapters from the third edition have been revised to feature current strategies and approaches to the preparation and cloning of nucleic acids, gene transfer, and expression analysis. They are augmented by 12 new chapters which show how DNA, RNA, and proteins should be prepared, evaluated, and manipulated, and how data generation and analysis can be handled. The new content includes methods for studying interactions between cellular components, such as microarrays, next-generation sequencing technologies, RNA interference, and epigenetic analysis using DNA methylation techniques and chromatin immunoprecipitation. To make sense of the wealth of data produced by these techniques, a bioinformatics chapter describes the use of analytical tools for comparing sequences of genes and proteins and identifying common expression patterns among sets of genes. Building on thirty years of trust, reliability, and authority, the fourth edition of Mol
215,169 citations
TL;DR: This paper describes a method of transferring fragments of DNA from agarose gels to cellulose nitrate filters that can be hybridized to radioactive RNA and hybrids detected by radioautography or fluorography.
30,291 citations
TL;DR: Two new methods were used to establish a rapid and highly sensitive prenatal diagnostic test for sickle cell anemia, using primer-mediated enzymatic amplification of specific beta-globin target sequences in genomic DNA, resulting in the exponential increase of target DNA copies.
Abstract: Two new methods were used to establish a rapid and highly sensitive prenatal diagnostic test for sickle cell anemia. The first involves the primer-mediated enzymatic amplification of specific beta-globin target sequences in genomic DNA, resulting in the exponential increase (220,000 times) of target DNA copies. In the second technique, the presence of the beta A and beta S alleles is determined by restriction endonuclease digestion of an end-labeled oligonucleotide probe hybridized in solution to the amplified beta-globin sequences. The beta-globin genotype can be determined in less than 1 day on samples containing significantly less than 1 microgram of genomic DNA.
9,107 citations
1,438 citations
TL;DR: This allele-specific hybridization behavior of oligonucleotides provides a general method for diagnosis of any genetic disease which involves a point mutation in the DNA sequence of a single-copy gene.
Abstract: Two 19-base-long oligonucleotides were synthesized, one complementary to the normal human beta-globin gene (beta A) and one complementary to the sickle cell beta-globin gene (beta S). The nonadecanucleotides were radioactively labeled and used as probes in DNA hybridization. Under appropriate hybridization conditions, these probes can be used to distinguish the beta A gene from the beta S allele. The DNA from individuals homozygous for the normal beta-globin gene (beta A beta A) only hybridized with the beta A specific probe; the DNA from those homozygous for the sickle cell beta-globin gene (beta S beta S) only hybridized with the beta S specific probe. The DNA from heterozygous individuals (beta A beta S) hybridized with both probes. This allele-specific hybridization behavior of oligonucleotides provides a general method for diagnosis of any genetic disease which involves a point mutation in the DNA sequence of a single-copy gene.
658 citations