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WO1999036571A2 - Procede de detection de sequences nucleotidiques - Google Patents

Procede de detection de sequences nucleotidiques Download PDF

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Publication number
WO1999036571A2
WO1999036571A2 PCT/EP1999/000161 EP9900161W WO9936571A2 WO 1999036571 A2 WO1999036571 A2 WO 1999036571A2 EP 9900161 W EP9900161 W EP 9900161W WO 9936571 A2 WO9936571 A2 WO 9936571A2
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WO
WIPO (PCT)
Prior art keywords
nucleic acid
stranded
oligonucleotides
product
detection
Prior art date
Application number
PCT/EP1999/000161
Other languages
English (en)
Other versions
WO1999036571A3 (fr
Inventor
Holger Klapproth
Hubert S. Bernauer
Original Assignee
Biochip Technologies Gmbh
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Biochip Technologies Gmbh filed Critical Biochip Technologies Gmbh
Priority to JP2000540272A priority Critical patent/JP2002517981A/ja
Priority to CA002318371A priority patent/CA2318371A1/fr
Priority to EP99907365A priority patent/EP1047794A2/fr
Publication of WO1999036571A2 publication Critical patent/WO1999036571A2/fr
Publication of WO1999036571A3 publication Critical patent/WO1999036571A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • C12Q1/6855—Ligating adaptors

Definitions

  • the present invention relates to a novel method for the detection of a nucleic acid sequence within a nucleic acid molecule.
  • the method of the invention relies on the combination of nucleic acid protection, ligation of oligonucleotides to the protected nucleic acid molecules and amplification of the ligation products.
  • the detection of the amplified products is advantageously effected by converting the same to the single-stranded form and hybridizing the single-stranded form thereof to an array of single-stranded nucleic acid molecules of at least partially predetermined sequence fixed to a solid support.
  • the solid support is preferably a chip. Detection of hybridized molecules can be effected according to conventional methods.
  • the present invention additionally relates to a kit for carrying out the method of the invention.
  • PCR PCR, RFLP analysis or Southern blotting in combination with nucleic acid hybridization.
  • these methods are, however, still rather laborious or allow a detection of only one nucleic acid sequence at a time.
  • the classic PCR method as a rule, requires specific primers for each DNA sequence that is to be analyzed.
  • An improvement of the classic PCR is represented by the so-called multiplex PCR which has, however, turned out not to be a very robust technique.
  • oligonucleotide primer pairs as used for PCR show different efficiencies for priming and amplifying a DNA sequence.
  • the technical problem underlying the present invention was to develop a method that allows for the easy detection of specific nucleic acid sequences within the sample and which further can be carried out at a rather low cost.
  • the solution to said technical problem is achieved by providing the embodiments characterized in the claims.
  • the present invention relates to a method for the detection of a nucleic acid sequence within a nucleic acid molecule comprising the steps of
  • step (b) removing non-hybridized nucleic acid from the product of step (a);
  • step (c) converting the hybrid obtained in step (b) into single-stranded form
  • step (d) ligating oligonucleotides to the single-stranded nucleic acid obtained in step (c);
  • step (c') ligating oligonucleotides to the hybrid obtained in step (b);
  • step (d') converting the ligation product of step (c') into single-stranded form; (e) carrying out an amplification reaction with the product of step (d) or (d') using primers that hybridize to said oligonucleotides or transcribing the product of step (d) or (c); and
  • step (f) detecting the product of step (e).
  • the method of the invention allows, by using only one pair of e.g. complementary oligonucleotides and two primers (subsequently also termed “universal primers") hybridizing thereto, the amplification and subsequent detection of virtually any nucleic acid sequence that forms a double-stranded nucleic acid hybrid with a chosen probe. It is particularly preferred that one and the same oligonucleotide is used for ligation to the 5'-end as well as the 3'-end and also as the primer. Examples of such oligonucleotides are oligonucleotides comprising palindromic sequences. Thus, optionally, one oligonucleotide is sufficient for all steps to be carried out in the method of the invention.
  • nucleic acid molecule comprises also any feasible derivative of a nucleic acid to which a nucleic acid probe may hybridize.
  • nucleic acid probe may be any derivative of a nucleic acid capable of hybridizing to said nucleic acid molecule.
  • the primers employed in the method of the invention may not be identical with the oligonucleotides used for ligation. Nevertheless, advantageously the same primer may be used for hybridization to the 3'-end and the 5'-end of the target oligonucleotide, depending on the sequence of said target.
  • oligonucleotides or pairs of oligonucleotides with different nucleic acid sequences may be employed and manipulated according to conventional methods for ligation either to the 5'- or 3'- ends of the amplified nucleic acid molecules (see, for example, Sambrook et al, "Molecular Cloning, A Laboratorial Manual” CSH Press, Cold Spring Harbor, (1989)).
  • the sample may comprise more than one different nucleic acid molecule, such as a pool of different nucleic acid molecules.
  • the sample may be obtained from any biological or synthetic source.
  • the sample is taken from a natural source such as blood, serum, stool, sputum, tissue etc.
  • a natural source such as blood, serum, stool, sputum, tissue etc.
  • the method of the invention may be used for the detection of genetic disorders indicative of human or animal diseases.
  • the method of the invention envisages,, with respect to the timing of the ligation of the oligonucleotides, two alternatives.
  • the oligonucleotides are ligated to single-stranded protected nucleic acid molecules after removal of the protecting strand or they are, preferably in double stranded form, ligated to the double-stranded hybridization product. In both cases, non- hybridized single-stranded nucleic acid is removed prior to the ligation.
  • either single-stranded or double-stranded oligonucleotides may be ligated to the product according to the first and to the second alternative.
  • the first alternative it is to be noted that the protecting strands to be removed may be either the strand that was originally present in the sample or the single-stranded probe.
  • the oligonucleotides As regards the nature and nucleotide sequence of the oligonucleotides the following should be noted: if said oligonucleotides are ligated as double-stranded complementary nucleic acid molecules, it is advantageous to ligate them first to the 5'-end and subsequently to the 3'-end of the protected nucleic acid molecules, or vice versa.
  • the double-stranded oligonucleotides comprise a recognition and cleavage site for a restriction endonuclease.
  • At least one of the oligonucleotides to be ligated to the protected or protecting nucleotide sequence comprises a promoter for a polymerase, preferably a RNA polymerase.
  • the corresponding transcript can be produced and visualized according to standard protocols.
  • the person skilled in the art can determine if strictly complementary sequences or sequences with a higher or lower degree of homology are to be detected.
  • the setting of conditions is well within the skill of the artisan and to be determined according to protocols described, for example, in Sambrook, loc. cit. or Hames and Higgins, "Nucleic acid hybridization, a practical approach", IRL Press, Oxford (1985).
  • the detection of only specifically hybridizing sequences will usually require stringent hybridization and washing conditions such as O.lxSSC, 0.1% SDS at 65° C.
  • Non- stringent hybridization conditions for the detection of homologous and not exactly complementary sequences may be set at ⁇ xSSC, 1% SDS at 65°C.
  • the length of the probe and the composition of the nucleic acid to be determined constitute further parameters of the hybridization conditions.
  • the person skilled in the art is in the position to practice them by reverting, for example, to conventional protocols.
  • the removal of single-stranded nucleic acid that did not form a hybridization product with the oligo- or polynucleotide used as a probe as well as of probes that did not hybridize to any target nucleic acid sequence can be effected according to protocols described, for example, in Sambrook et al, loc. cit.
  • single-stranded nucleic acid such as mRNA may be removed by S1 nuclease or mung bean nuclease digestion.
  • Double-stranded nucleic acid such as DNA may be removed by employing protocols using ⁇ -exonuclease. Conventional protocols may also be employed for the ligation step, the conversion of double- stranded nucleic acid into single-stranded nucleic acid or for the amplification which may conveniently be carried out by using PCR technology, strand displacement amplification, linker ligation combined with in vitro transcription or techniques comprising rolling circle amplification if the protector or the protected molecule is a circular molecule.
  • T4-DNA ligase may advantageously be used. Template independent ligation would require a different enzymatic activity such as conferred by T4-RNA ligase.
  • the conversion of double-stranded into single-stranded nucleic acid may be done by base or heat denaturation.
  • the person skilled in the art is further capable of converting the complete double-stranded nucleic acid within a sample into a single-stranded form, if this is desired.
  • Suitable protocols include, alone or in combination, strand displacement reactions by polymerase, chemical or enzymatic degradation of either strand, affinity chromatography if the introduced primer is labeled and chemical denaturation of (hetero-)duplex nucleic acid molecules. This measure will also prevent the formation of double-stranded hybrids within the nucleic acid to be analyzed to which later oligonucleotides would be ligated, thus giving rise to false positive results.
  • Double-stranded template dependent ligation of 5'-primer oligonucleotides may also be performed in a cyclic ligation reaction using thermostable ligases (e.g. Taq Ligase).
  • thermostable ligases e.g. Taq Ligase
  • different types of single-stranded adaptor molecules may be used which are complementary to the amplification primer oligonucleotides and terminally complementary to the protected oligonucleotides.
  • the 5'- and 3'-ends of the adaptor and the first primer oligonucleotide may in this embodiment be connected forming a hairpin loop.
  • reaction vials after each step, it is preferred to carry out at least steps (b) to (e) in one reaction vessel, preferably in an Eppendorf tube.
  • the nucleic acid to be detected and/or the nucleic acid probe is comprised of RNA, DNA or PNA.
  • RNA are rRNA and mRNA, of DNA, cDNA and genomic DNA. Any of the above preferred embodiments may also be (semi)synthetically produced.
  • step (b) it is preferred that said removal is effected by exonuclease activity, such as ⁇ -exonuclease activity optionally in combination with (restriction) endonuclease activity, by affinity chromatography using, for example, antibodies specific for double-stranded nucleic acid coupled to a conventional matrix or by gel-electrophoresis or HPLC.
  • exonuclease activity such as ⁇ -exonuclease activity optionally in combination with (restriction) endonuclease activity
  • affinity chromatography using, for example, antibodies specific for double-stranded nucleic acid coupled to a conventional matrix or by gel-electrophoresis or HPLC.
  • any one or the combination of the above mentioned nuc'ease activities are combined with affinity chromatography, gel-electrophoresis ' and/or HPLC in order to remove non- hybridized nucleic acid in step (b).
  • the phrase "removing non-hybridized nucleic acid” evidently also comprises the removal of single-stranded regions of said nucleic acid that have not formed a nucleic acid hybrid with said one or more nucleic acid probes.
  • the oligonucleotides used for ligation are masked at their 3'-ends. Masking the 3'- or ⁇ '-ends of said oligonucleotides in this embodiment of the invention is required if template independent ligation method as with T4-RNA-ligase is used. For example, one would use one primer which has no 5'-phosphate and a normal 3'- hydroxy group which is ligated to the 5'-phosphate group of the nucleic acid to be analyzed thereby forming a phosphodiesterbond.
  • a 3'-blocked oligonucleotide e.g. an oligonucleotide carrying an amino block
  • 3'- as well as 5'- primer oligonucleotides are able to form a dimer.
  • the dimers have a 5-hydroxy group and a 3'-blocked end unable to form polymers of higher order.
  • the primer ligation steps to the 3'-end and to the 5'-end have, in these embodiments, therefore to be carried out sequentially in separate reactions to avoid primer-dimer formations.
  • the oligonucleotides used for ligation are designed to form, upon dimer formation, a restriction site for an endonuclease which can subsequently be cleaved.
  • Thermostable restriction enzymes can be used in a PCR amplification reaction for cleaving the primer dimers.
  • the following is an example of this embodiment.
  • the 5'-oligomer to be ligated to the 5'-end of the protected nucleic acid molecule has the sequence
  • X denotes the amino block.
  • Tthl restriction site is emphasized by bold letters.
  • Final amplification may be effected using single-primer oligonucleotides of the following sequence:
  • Cleavage with Tthl should be effected early, i.e. once the first double-strands have been formed.
  • labeled nucleotides are incorporated into the amplification product.
  • This embodiment of the method of the invention has the advantage that the amplification product is directly visible in detection step (f) without the need for an additional detectable means.
  • the label is a radioactive label, a fluorochrome, a bioluminescent label, a chemiluminescent label, a hapten, an enzyme such as horseradish peroxidase or a chelator for the detection of bound metals.
  • step (f) For detecting the amplification product in step (f), the following detailed protocol is particularly preferred:
  • step (f ) converting the amplified product of step (e) into single-stranded form
  • step (f ) contacting the single-stranded nucleic acid molecules obtained in step (f ) with an array of single-stranded nucleic acid molecules with at least partially predetermined sequences attached to a solid support under conditions that allow the formation of hybrids between said single-stranded nucleic acid molecules and nucleic acid molecules with said at least partially predetermined sequence to occur;
  • step (f ) detecting the formation of hybrids formed in step (f ').
  • the conversion of the amplified product into single-stranded form can be effected by conventional protocols that have been outlined above.
  • the hybridization conditions in step (f ) will be determined along the various schemes delineated herein above.
  • the single-stranded nucleic acid molecules arranged in array form on a solid support may have a fully or partially predetermined sequence. They may be of natural, synthetic or semi-synthetic origin. Attachment to the solid support may be effected according to conventional protocols, for example, via a biotin-avidin bridge.
  • the detection of hybrids in step (f") can be carried out by a variety of means, usually depending on the labeling that is employed. For example, if the amplification in step (e) is effected by using fluorochrome-labeled oligonucleotides, said detection may be by visual means, for example with a correspondingly equipped microscope. Other options include capillary and gel electrophoresis, HPLC, mass spectroscopy, nucleic acid hybridization and nucleic acid sequencing. One preferred embodiment also applicable to the other embodiments of the method referred to above, provides for the detection by using an anti-double-stranded DNA antibody.
  • Said antibody may either be detectably labeled or it may be detected using, for example, a tertiary antibody that is detectably labeled.
  • Another preferred embodiment employs for detection is hybridization with a detectably labelled probe. Any detection method discussed above may be assisted by computer technology.
  • the array referred to above may be any two- or three dimensional arrangement of molecules.
  • the array is in grid form.
  • said solid support is a chip.
  • this chip consists of inorganic substances (glass, silica and others) or of organic substances (for example polymers) or is a hybrid consisting of different substances or layers of different substances (plastic coated glass, for example, silane coated silica).
  • the oligonucleotide bound to this chip could be directly bound by covalent binding or indirectly via hapten interactions (e.g. a biotin-avidin bridge).
  • hapten interactions e.g. a biotin-avidin bridge
  • step (b') cleaving a mismatch contained in the hybrid obtained in step (b).
  • the step characterizing this embodiment may be carried out prior to or after any of steps (c), (c'), (d), and (d'). Cleaving of this matches is conveniently done by the employment of appropriate enzymes such as CleavaseTM (Boehringer Mannheim). Upon cleavage of one or optionally both of the hybridized strands, an amplification product will not form any longer.
  • This specific embodiment of the invention will find wide range of applications in the demonstration of the presence of specific mRNA sequences or of mutations in a genomic sequences related, for example, to cancer. Thus, it is known Ras sequences contributing to the formation of tumorous cells carry one specific mutation in their coding sequence.
  • a protecting oligonucleotide that is exactly complementary to the mutated sequence will result in the amplification of that mutated sequence. Accordingly, cancerous cells can be identified in the sample by applying the method of the invention. If the protecting oligomer is hybridized only to wildtype sequences, a mismatch will be formed and, upon cleavage, no amplification product will be produced. Accordingly, the lack of appearance of an amplification product is indicative of the absence of tumorous cells.
  • the basic teaching underlying this embodiment can, of course, be applied to the person skilled in the art to a wide variety of purposes.
  • the kit of the invention further comprises, besides the universal primer oligonucleotide to be used for optionally both backward and forward priming as well as optionally for ligation to the protected target nucleic acid molecule, hybridization buffer(s), nuclease(s) such as nuclease S1 or mung bean nuclease, nuclease buffers, ligase(s) and ligation buffer(s), DNA polymerase(s) and polymerase buffer(s) suitable for the amplification of the ligation products and/or a control nucleic acid the antisense sequence of which is also bound to the above referenced matrix.
  • the components of the kit of the invention may be packaged in containers such as vials, optionally in buffers and/or solutions. If appropriate, one or more of said components may be packaged in one and the same container.
  • the kit may be advantageously used for carrying out the method of the invention.
  • Figure 1 Schematic overview of one preferred embodiment of the method of the invention. Nucleic acids are protected by a protector molecule from degradation with nucleases. After removal of degradation products primers are ligated to each end of the protected nucleic acids and the protected nucleic acids are amplified by a universal PCR (one primer pair) for all protected nucleic acid fragments. Subsequently, the amplification products are visualized by various methods.
  • Figure 2 One further embodiment of the invention drawn schematically and comprising the amplification of nucleic acid fragments after the protection reaction using matrix bound protector molecules.
  • Figure 3 Universal PCR for the amplification of nucleic acid fragments after the protection reaction.
  • a primer oligonucleotide is ligated to the 5'- end of the protected molecule.
  • an other oligonucleotide is ligated to the 3'-end of the protected DNA- molecule.
  • a primer oligonucleotide is ligated to the 5'- end of the protected molecule.
  • a oligonucleotide complementary to the 5'-oligonucleotide is ligated to the protected DNA-molecule.
  • Insulin specific mRNA was prepared from freshly obtained pancreatic tissue according to standard procedures. The mRNA was dissolved in hybridization buffer and insulin specific oligonucleotides of the following sequence:
  • hybridization was carried out under constant agitation of the hybridization mixture for 16 hours at 65°C. Subsequently; the hybridization solution was centrifuged and unhybridized RNA was removed by washing the pellet and then repeating the centrifugation and washing steps three times. Single-stranded nucleic acid was subsequently removed by using either S1 nuclease or mung bean nuclease and incubating the mixture at 37°C for an hour. Afterwards, the nucleases were removed by alternative washing steps with conventional washing buffers and centrifugation. The remaining hybrids were denatured by treatment either under heating conditions or with low salt buffer.
  • the protecting nucleic acid was removed by centrifugation. The supernatant was transferred to a new reaction vessel. To the supernatant conventional ligation buffer was added. The first oligonucleotide was added and ligated for one hour with T4-RNA ligase. Not ligated oligonucleotide was removed by conventional washing steps. Subsequently, the second primer was ligated using the same ligation conditions as above. Again, the not ligated oligonucleotide was removed by conventional washing steps. The ligated nucleic acid was precipitated by adding ethanol and salt using conventional conditions. To the precipitated nucleic acid a conventional RT-PCR buffer mix was added.
  • the nucleic acid was amplified employing 30 PCR cycles in the presence of fluorescence labeled primer oligonucleotides complementary to the oligonucleotides ligated to the target sequence.
  • the amplified product was analyzed with a ⁇ -lmager (Molecular Dynamics).
  • Tumor cell lines containing an amplification in chromosome 3 (3q27-3q28) were analyzed for the overexpression of the following candidate oncogenes: BCL6, DLGH, DVL3, E1F4G, HSERM, HSFGF12, HSLPP, HUMP, HUMSOMI, RFC4.
  • 5' phosphorylated single-stranded DNA protectors complementary to the mRNA transcribed from said oncogenes of different distinguishable length in polyacrylamide gels were constructed using Genbank data and conventional oligonucleotide synthesis techniques.
  • RNA from the tumor cell lines was prepared by acidic guanidinethiocyanate/phenol/chloroform extraction.
  • RNAse free DNAse I DNA specific nuclease treatment

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Abstract

L'invention concerne un nouveau procédé de détection d'un séquence nucléotidique au sein d'une molécule d'acide nucléique. Le procédé de l'invention consiste principalement à combiner la protection de l'acide nucléique, la ligature d'oligonucléotides auxdites molécules d'acide nucléique protégées et l'amplification des produits de la ligature. La détection des produits amplifiés s'effectue avantageusement par la conversion de ces derniers, de sorte qu'ils se présentent sous une forme à simple brin, et par l'hybridation de leur forme à simple brin en un réseau de molécules d'acide nucléique simple brin d'au moins une séquence partiellement déterminée, fixée à un support solide. Le support solide est de préférence une pastille. La détection des molécules hybridées peut s'effectuer selon des procédés classiques. L'invention concerne également un kit pour la mise en oeuvre du procédé de l'invention.
PCT/EP1999/000161 1998-01-13 1999-01-13 Procede de detection de sequences nucleotidiques WO1999036571A2 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP2000540272A JP2002517981A (ja) 1998-01-13 1999-01-13 核酸配列を検出するための方法
CA002318371A CA2318371A1 (fr) 1998-01-13 1999-01-13 Procede de detection de sequences nucleotidiques
EP99907365A EP1047794A2 (fr) 1998-01-13 1999-01-13 Procede de detection de sequences nucleotidiques

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP98100485 1998-01-13
EP98100485.6 1998-01-13

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WO1999036571A2 true WO1999036571A2 (fr) 1999-07-22
WO1999036571A3 WO1999036571A3 (fr) 1999-10-21

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US7399589B2 (en) 1998-02-06 2008-07-15 Digene Corporation Immunological detection of RNA:DNA hybrids on microarrays
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US7601497B2 (en) 2000-06-15 2009-10-13 Qiagen Gaithersburg, Inc. Detection of nucleic acids by target-specific hybrid capture method
US8288520B2 (en) 2008-10-27 2012-10-16 Qiagen Gaithersburg, Inc. Fast results hybrid capture assay and system
WO2014153408A1 (fr) * 2013-03-19 2014-09-25 Directed Genomics, Llc Enrichissement en séquences cibles
US9074244B2 (en) 2008-03-11 2015-07-07 Affymetrix, Inc. Array-based translocation and rearrangement assays
US9315807B1 (en) 2013-01-26 2016-04-19 New England Biolabs, Inc. Genome selection and conversion method
US9605303B2 (en) 2010-01-29 2017-03-28 Qiagen Gaithersburg, Inc. Method of determining and confirming the presence of an HPV in a sample
US9689047B2 (en) 2010-01-29 2017-06-27 Qiagen Gaithersburg Inc. Methods and compositions for sequence-specific purification and multiplex analysis of nucleic acids
US9797000B2 (en) 2009-05-01 2017-10-24 Qiagen Gaithersburg Inc. Non-target amplification method for detection of RNA splice-forms in a sample
US9885092B2 (en) 2011-02-24 2018-02-06 Qiagen Gaithersburg Inc. Materials and methods for detection of HPV nucleic acids
US10087481B2 (en) 2013-03-19 2018-10-02 New England Biolabs, Inc. Enrichment of target sequences
US10329600B2 (en) 2007-09-14 2019-06-25 Affymetrix, Inc. Locus specific amplification using array probes

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CA2799200A1 (fr) 2010-05-19 2011-11-24 Qiagen Gaithersburg, Inc. Procedes et compositions pour purification sequence-specifique et analyse multiplex d'acides nucleiques
JP6153866B2 (ja) 2010-05-25 2017-06-28 キアゲン ガイサーズバーグ アイエヌシー． 迅速なハイブリッド捕捉アッセイ、及び関連する戦略的に切断されたプローブ
CN111979303A (zh) 2020-08-11 2020-11-24 上海奕谱生物科技有限公司 一种核酸检测试剂盒、方法及其应用

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