CA2031026A1 - Method of immobilizing nucleic acid on a solid surface for use in nucleic acid hybridization assays - Google Patents
Method of immobilizing nucleic acid on a solid surface for use in nucleic acid hybridization assaysInfo
- Publication number
- CA2031026A1 CA2031026A1 CA002031026A CA2031026A CA2031026A1 CA 2031026 A1 CA2031026 A1 CA 2031026A1 CA 002031026 A CA002031026 A CA 002031026A CA 2031026 A CA2031026 A CA 2031026A CA 2031026 A1 CA2031026 A1 CA 2031026A1
- Authority
- CA
- Canada
- Prior art keywords
- nucleic acid
- solid surface
- variable portion
- modified
- strand
- Prior art date
- Legal status (The legal status 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 status listed.)
- Abandoned
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/81—Packaged device or kit
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/807—Apparatus included in process claim, e.g. physical support structures
- Y10S436/808—Automated or kit
Abstract
J&J-35 (ORD 80) PATENT
Abstract Methods of immobilizing nucleic acid on a solid surface for us in nucleic acid hybridization assays is disclosed. The methods of the invention comprise reacting a modified nucleic acid strand comprising a variable portion and an anchor portion wherein the variable portion comprises a nucleotide sequence having a selected base sequence and the anchor portion comprises at least one nucleotide base modified with a primary amine function or nucleotide base equivalent having a primary amine function and reacting the modified nucleic acid strand with a free aldehyde group of the solid surface in the presence of a reducing agent to form complexes of the modified nucleic acid strand and at least a portion of the free aldehyde groups on the solid surface.
Abstract Methods of immobilizing nucleic acid on a solid surface for us in nucleic acid hybridization assays is disclosed. The methods of the invention comprise reacting a modified nucleic acid strand comprising a variable portion and an anchor portion wherein the variable portion comprises a nucleotide sequence having a selected base sequence and the anchor portion comprises at least one nucleotide base modified with a primary amine function or nucleotide base equivalent having a primary amine function and reacting the modified nucleic acid strand with a free aldehyde group of the solid surface in the presence of a reducing agent to form complexes of the modified nucleic acid strand and at least a portion of the free aldehyde groups on the solid surface.
Description
~ 3 r ~
J&J-35 (O~D 80~ PATENT
Metho~ Of Immobilizi~g ~uclei¢ A¢i~ On A 801~lia Burfzlce For Use ID. Nucleio Ac~ aybriai~at1on As~a~rs Field of th~ I~v~tio~l The present invention relates to methods for immobilizing nucleic acids to solid surfaces for nucleic acid hybridization lo assays.
Bac~groun~ of tha In~e~tio~
Nucleic acid hybridization assays have proven useful in detecting the presence of microorganisms in biological samples (tissue, blood, urine, saliva, etc.) to diagnose infection and for detecting the presence in a mixture of a nucleic acid sequence of interest.
Nucleic acid hybridization assays are generally performed by immobilizing the test nucleic acid strand on a solid surface. A
mixture containing the labeled complementary nucleic acid strand is contacted with the immobilized nucleic acid strand under conditions allowing hybridization of the two complementary strands.
The hybridization of the two strands is then detected by chemical or other means by detecting the label on the probe strand.
Alternatively, the nucleic acids are detected by sandwich hybridization techniques in which one set of sequences complementary to the target sequence lS immobilized onto a solid surface. The surface is then contacted with the targer DNA and a . - ' " ' ' 2 ~
J&~-35 (ORD 80) ~T~N~
labeled probe complementary to the target at a place different than the one attached to the solid ~ur~ace. Under appropriate conditions the labeled probe ~s retained on the paper via the target DNA and detected by chemical or physical methods directly or indirectly.
Nucleic acids are typically immobil~zed on solid surfaces such as cellulose or nylon by physical contact with the surface~ and the nucleic acids are bound to the surface through weak non-covalent bonds. Because of the nonspecific binding of nucleic acid to these surfaces, it is difficult to orient the nucleic acid so that it does not interact with the solid surface with the por~ions of the nucleic acid that are needed to hybridize with the test sequences.
Thus greater amounts of the nucleic acid are needed to ensure a sufficient amount of free sequences to bind to the test nucleic acid. This inefficient use of nucleic acid can be e~pensive and can be limiting if only small quantities of the nucleic acid are available.
Additionally, the inability to accurately place the nucleic acid on the solid surface can hinder the effectiveness of the assay when small amounts of the nucleic acid sequence of interest are present, since it can be difficult to distinguish the nucleic acid present from backqround reactions.
Further, it would be desirable to be able to test for ~ore than one pathogen in a single assay, or to test for ~ore than one nucleic acid sequence in a single microorganis~ in the same assay.
2 ~
J~J-35 (O~D 80) PATENT
Uncertain placement of nucleic acids on the ~olid surface is a drawback to the development of assays of this kind.
8ummary of t~e I~Ye~tiOn The methods of the invention provide methods of immobilizing nucleic acid to a solid surface having a free aldehyde group for use in nucleic acid hybridization assays. In the methods of the invention a modified nucleic acid strand comprising a variable portion and an anchor portion wherein the variable portion comprises a nucleotide sequence having a selected base sequence and the anchor portion comprises at least one nucleotide base modified with a primary amine function or nucleotide base equivalent having a primary amine function is reacted with the free aldehyde group of the solid surface in the presence of a reducing agent to form complexes of the modified nucleic acid strand and at least a portion of the free aldehyde yroups.
Accordingly, the invention also provides solid surfaces for performance of nucleic acid hybridization assays comprising a solid surface prepared in ~ccordance with the ~ethods of the invention. The invention further provides kits for performance of nucleic acid hybridization assays comprising a solid surface of the invention and at least one labeled nucleic acid probe, the solid surface and the probe selected to form a detection system for a target strand of nucleic acid.
The met~ods of the invention provide an efficient procedure for immobilizing nucleic acids, especially oligonucleotides, on to 2 ~ 2 ~
J~J-3s (ORD 803 ~ATENT
solid surfaces without affecting ~heir ability ~o hybridize to complementary nucleic acid. The method is fast, economical and efficient. On flat surfaces such as paper or membranes, the oligonucleotides can be applied in zones alone, ~ogether with, or alongside other oligonucleotides or proteins fiuch as enzymes or antibodies. The immobilization occurs via synthetlc anchors provid~d a~ the ~ermini of ~he oligonucleotide being immobilized.
The ~ethods and solid surfaces of the invention provide specific orientation of the irmobilized nucleic acid by the anchor portion so that the entire or substantially all of the variable portion is free to hybridize with test nucleic acid. This improvement over prlor methods of immobilizing nucleic acids on solid surfaces for nucleic acid hybridization provided by the methods and solid surfaces of the invention allows more efficient use of the immobilized nucleic acid. Smaller quantities are needed as non-specific binding of the nucleic acid to the solid support is eliminated through the binding of the anchor portion with the solid support.
The methods of the invention make it possible to place the immobilized nucleic acid in pre-determined positions on the surface, facilitating detection of hybridized test nucleic acid by allowing it to be more easily distinguished from bacXground reactions. Additionally, controlled placement of the immobilized nucleic acid allows the immobilization of more than one type of .25 nucleic acid sequence in precise locations on the solid ~urface, 2~
J~J-3s ~ORD 801 PA~ENT
so that ~ore than one type of ~icroorganism or nucleic acid sequence of lnterest can be detected in the same assay.
The solid surfaces o~ the invention have the advantage of providing a light, or paper colored, background which gives a good contrast for hybridization assays per~ormed on the solid surface.
Detaile~ De0cr~ption o~ th~ I~e~tio~
In the methods of the invention nucleic scid is immobilized on a solid surface through covalen~ bonding. The methods o~ the invention produce a solid surface ha~ing nucleic acid, DNA or RNA, bound thereon in a predetermined pattern that is suitable for use in nucleic acid hybridization assays.
In preferre~ embodiments, ~ ~r RNA to be immobilized is adapted for ~mmobilization by firs~ attaching at least one nucleotide base modified with a primary a~ine function or nucleotide base equivalent having a primary ~mine ~unction at either of the termini of the DNA or RNA to form modified nucleic acid strands. The ~odified nucleic acid strand thus formed comprises a variable portion and an anchor portion wheréin the variable portion comprises a nucleotide sequence having a selected base sequence and the anchor portion comprises at least one nucleotide base modified with a primary amine function or nucleotide base equivalent having a primary amine Punction attached at either ter~inus of the variable portion.
The mod~fied nucleic acid strands to be ~mmobilized and used as for capturing or hybridizing with complementary nucleic acids are 2 ~ 2 ~
~ 35 ~ORD ~0) ~aT~T
preferably prepared by chemical synthesis using a DNA synthesizer and commercially available reagents. The base sequence of the variable portion of the modified nucleic acid strand is selected in accordance with the organism it is desired to de~ec~, or other purpose for the hybridization assay. The variable portion is preferably prepared by chemical synth2sis, however, it can also be prepared from natural cellular or reco~binant sources using recombinant techniques. The variable portion may be derived from human, bacterial, viral, fungal or other sources. If the variable portion is prepared from cellular or recombinant sources, synthesis of the variable portion will not be necessary. The variable portion may also be a homopolymer, such as oligo thymidine (poly thymidine or poly T) or poly adenine (~oly A). The variable portion i6 preferably from about two to about 1,000 nucleotide bases in length, mor2 preferably from about 15 to about 100 nucleotide bases in length.
The anchor portion is added to the variable portion. The anchor portion may be added to either the 3' or 5' terminus of the variable portion. The anc~or portion is preferably comprised of at least one nucleotide base modified with a primary amine function or nucleotide base equivalent having a primary amine function more preferably of from two to about ten nucleotide bases or nucleotide base equivalents, and most preferably of from about five to about eight nucleotide bases or nucleotide base equivalent~. Suitable nucleotide bases include cytosine modified at the 4 position of the 2~3~
J&J-3s (0~D 80) PA~NT
pyrimidine ring, adenine ~odified at the 6 position of the purine ring, and uridlne modified to contain a primary amino function.
Examples of suitable modified nucleotide bases include 5-amino(12)-2'-deoxyuridine-5'-triphosphate (Behring Diagnostics, ~a Jolla, California), 5-(3-aminoallyl)-2'-deoxyuridine 5'-triphosphate, 5-t3-aminoallyl)uridine 5'triphosphate, and N6-~[6 aminohexyl~-carbamoylmethyl)-adenosine 5'triphosphate (Sigma Chemical Co, St.
Louis, Missouri). Other nucleotide bases modifed to contain a primary amine function may also be suitable for use in the anchor portion. A preferred amino-modified nucleotide base is cytosine modified at the 4 position of the pyrimidine ring. Nucleotide base equivalents include phosphoramidites, phosphonates that can be modified with primary amines to give phosphoramidates, and other compounds having, or capable of being modified to contain, primary amine functions that can be substituted for nucleotide bases in a nucleic acid sequence or that can be added at the 3' or 5' ends of a nucleic acid sequence.
The anchor portion is attached to the variable portion by covalent bonding through the synthetic process using a DNA
synthesizer or through any conventional msans for ligation of nucleic acids; for example, ligase enzymes such as T~ DNA ligase and ~. ÇQli ligase; chemical means (Z.A. Shabarova, M~S.
IvanovsXaya and M.G. Isaguilantis, FEBS Letters, vol. 154, number 2, April 1983); and photoligation means ("DNA Assay Using Template-~5 Directed Photoligation' n San Diego Section of the American 2 ~
JfiJ-35 l0RD 30) ~AT~NT
Association of Chemical Industry-AMOCO Technology Company, P.O. Box 400 Naperville, IL 6056S). Phosphoramidites may be added to the variable portion using conventional phosphoramidite chemistry.
Phosphonates that can be modified with primary amines to give phosphoramidatas may be attached to the variable portion according to the method of Froehler, Tetrahedron Letters 27t46): 5575-5578 (1983).
In a preferred embodiment of the invention, the anchor portion of the modified nucleic acid strand comprises cytosine modified at the 4 position of the pyridine ring with a primary amine function.
The preferred anchor portion is prepared by chemical synthesis in which cytosine is attached to the variable portion by covalent bondinq. The cytosines of the anchor portion are then subjected to bisulfite catalyz~d transamination reaction with a diamine. ~he transamination reaction results in modification of the pri~ary amine group at the 4 position of the cytosine pyrimidine ring with the diamine which has a primary amine substituent. Diamines havin~
from about two to about ten carbon atoms, preferably from about five to about seven carbon atoms, are suitable for use in the invention. In preferred embodiments hexanediamine is employed.
~ransamination of cytosine at the exocyclic animo qroup at the 4 position on the pyrimidine ring ~ay be accomplished by conventional transamination methods, such as the method described herein.
If the variable portion of the modified nucleic acid strand contains cytosine, it will be necessary to protect the first 2 ~
J~J-35 (O~D 80) ~ATRN~
variable portion during transamination, ~o that any cytosines present in this portion are not modi~led, thus interfering with later hybridization with the test nucleic acid sequence.
Protection of cytosine in ~he variable portion can readily be provided by hy~ridizing a complementa~y sequence to this portion of the modified nucleic acid ~trand, taking care to exclude the anchor portion. The complementary protective strand may be prepared by chemical synthesis, or purified from natural or recombinant sources using any convenient meansO It is important that only the anchor portion cytosine molecules are single stranded and that the other cytosines in the variable portion are protected from modification by transamination by hydrogen bonding with the complementary 6trand. The complementary strand is removed from the modified nucleic acid strand before a hybridization assay is performed. Denaturing of the protective strand may be accomplished by any conventional means such as alkaline conditions or elevated temperatures and removal may be accomplished by any suitable purification technique, such as chromatography methods and the liXe, or by rinsing the ~olid surface if removal of the complementary protectiva strand takes place after immobili~ation of the modified nucleic acid strand. The complementary protective strand may be removed after the modification ~tep, or after any subsequent ~tep of the methods of the invention.
The modified nucleic acid strand is applied to a ~ol~d ~urface containing aldehyde groups, in the presence of a reducing agent 2 ~
PAT~NT
variable portion during transamination, 50 that any cytosines present in this portion are not modified, thus interfering with later hybridization with the test nucleic acid sequence.
Protection of cytosine in the variable portion can readily be provided by hybridizing a complementary sequence to thi~ portion of the modified nucleic acid ~trand, taking care to exclude the anchor portion. The complementary protective strand may be prepared ~y chemical synthesis, or purified from natural or recombinant sources using any convenient means. It is important that only the anchor portion cytosine molecules are single stranded and that the other cytosines in the variable portion are protected from modification by transamination by hydrogen bonding with the complementary strand. The complementary strand is removed from the modified nucleic acid ~trand before a hybridization assay is performed. Denaturing of the protective strand may be accomplished by any conventional means such as alkaline conditions or elevated temperatures and removal may be accomplished by any suitable purification technique, such as chromatoqraphy methcds and the like, or by rinsing the solid surface if removal of the complementary protective strand takes place after immobilization of the modified nucleic acid strand. The complementary protective strand may be removed after the modification step, or after any subsequent step of the methods of the invention.
The modified nucleic acid strand i6 appl;ed to a ~olid surface containing aldehyde groups, in the presence of a reducing agent _ g _ J&J-35 SORD 80~ PAT~N~
such as sodium cyano~orohydride. ~ ~ixture o~ ~he modified nucleic acid strand and reducing agent in a liquid such as water or phosphate buffer having a pH in the range of from about 6.0 to about 8.S, preferably about 7.6, is applied to the aldehyde surface by spraying, soaking or any convenient method. The concentration of modified nucleic acid strand in the mixture should be great enough to ensure detection in a nucleic acid hybridization assay.
Generally, a concentration of modified nucleic acid strand in the range of 50 p moles to 150 p moles per square centimeter of solid surface, preferably a concentration of about 100 p moles per square centimeter will given an easily detectable result under assay conditions. The actual concentration used, however, will depend on the method of application and the surface to be coated, more concentrated solution is preferred ~o as to avoid the lateral movement of the solution on the surface.
Solid surfaces Ruita~le for use in the methods of the invention are those containing a free aldehyde group, or which can be modified to contain a free aldehyde group. For example, cellulose paper can be modified by limited oxidation with periodate to contain free aldehyde groups. Cellulose paper having free aldehyde groups may al60 be purchased from commercial sources such as Sterogene Biochemicals, San Gabriel, California.
The ~odified nucleic acid ~trand may be positioned on the solid surface by any suitable method. ~or application ~o the solid surface, the modified nucleic acid strand will typically be in a 2 0 3 ~ J ~
J~J-35 ~o~D 80) PA~
liquid solution wh~ch may be applied to the solid surface by any convenient ~ethod including, manual application of the l~quid solution, sprayîng, or by dipp~ng the 601id surface into the solution containing the modified nucleic acid strand. The modiPied nucleic acid strand may be applied to the ~olld surface in any desired pa~tern or combination of pattern~. More than one type of DNA sequence can be positioned on the solid surface; for instance a nucleic acid sequence unique to microorganism A and a nucleic acid sequence unique to micr~organism B can be immobilized in discrete stripes onto the solid surface to detect microorganisms A and B in the same hybridization assay by using appropriately labeled probes.
The modified nucleic acid strand and reducing agent is allowed to incubate with the solid surface for a length of time sufficient for the reaction between the nucleic acid and diamine to take place. Depending on the reducing agent and transamination method used this time will be approximately two hours. At the end of the incubation period, the solid surface is washed with water or a buffer such as phosphate buffered saline (PBS) to remove all excess reagents and finally washed with a buffer such as PBS. The ~olid surface is then dried by any convenient method, such as blotting between filter paper, or over a desiccant.
To reduce back~round interference in a hybridization assay, aldehyde groups on the solid surface that did not react with the modified nucleic acid strand may be optionally modified by reaction 2~3:~2.~
J~J-35 ~oaD 80) PAT~N~
with an amino acid, such as ~-amino caproic acid in the presence of a reducing agent. This step will convert the remaining aldehyde groups to acidic functions by reaction with the acid, 80 that the aldehyde groups will not be available to bind with nucleic acid or other reagents in nucleic acid hybridization assays and also introduces a negative charge on the surface, which aids in keeping background interference lo~. Conversion of unreacted aldehyde groups may readily be accomplished after immobilization of the modified nucleic acid ~trand by applying a mixture of 0.1 aminocaproic acid and 0.1 ~ sodium cyanoborohydride to ~he solid surface. The mixture may be applied to the solid surface by soa~ing the solid surface in a solution containing ~-amino caproic acid and sodium cyanoborohydride, spraying or any other &uitable method. After approximately one-half to two ~ours the paper is washed with water few times to remove all reagents and ~inally with phosphate buffered ~aline (PBS) and dried and stored.
The kits of the invention comprise a olid surface of the invention and at least one labeled nucleic acid probe. The solid surface and the labeled nucleic acid probe are selected to form a detection system for target nucleic acid. For example, to detect nucleic acid sequence A fro~ microorganism A, a solid surface of the invention having the variable portion of the modified nucleic acid strand complementary to at least a portion of nucleic acid sequence A would be provided in the kit. A labeled nucleic acid probe complmentary to a different portion of nucleic acid 6equence ~J-35 (O~D B9~ ~ATRNT
A would then be provided for detection o~ nucleic acid sequence A
hybridized to the solid surface.
Nucleic acid sequences use~ul ~n the labeled nucleic acid probes are readily prepared by any conventional method such as organic synthesis, reco~binant DNA techniques or isolation from genomic DNA. However, these sequences are particularly amenable to organic synthesis usin~ techniques known in the art such as techniques utilizing a nucle~c acid s~nthesizer and commercially available reagents.
The labeled nucleic acid probes may ~e labelad by conventional radioisotopic labeling, chemical labeling, immunogenic labeling, or a labal with light scattering effect, and the like. Suitable methods to detect ~uch labels are ficintillation counting, autoradiography, fluorescence ~easurement, calori~etric measurement, or light emission measurement.
Thus, the labeling may comprise a radiolabel ~e.g. l'C, 32p, 'H, and the like), an enzyme (e.g., peroxidase, alkaline or acid phosphatase, and the like), a bacterial label~ a fluorescent label, an antibody (which may be used in a double antibody syste~), an antigen (to be used with a labeled antibody), a small molecule such as biotin ~to be used with an avidin, streptavidin, or antibiotin system), a latex particle (to be used in a buoyancy or latex agglut~nation ~ystem), an electron dense compound such as ferritin (to be used with electron microscopy), or a light ~cattering particle such as colloidal gold, or any combination~ or - 2~3 1~3?.~
J~J 35 ~o~D 803 2AT~NT
permutations o~ the foregoing.
For example, if the labeling portion of the probe is an antigen, a signal can be generated by complexing said antigen with an antibody/enzyme conjugate, ~ollowed by addition of an enzyme substrate. If this portion wera an antibody, signal can be generated by complexing anti-antibody or an Fc binding protein such as Protein A therewith, when ~uch second antibody or Protein A have been con~ugated to an enzyme.
For reasons of ease and safety in the handling of the probe, it is preferred that it be chemically labeled, especially enzymatically or immunologically. In more preferred embodiments, the chemical label of choice is a hapten such as biotin, iminobiotin, fluorescein and the like.
Among the preferred labeling systems that may be mentioned are those based on the biotin/strepavidin system. This system can be incorporated into the probe by a variety of means. For example, the probe can be covalently attached to biotin via a cytochrome c bridge (~anning et al, Biochemistry, 16: 1364-1370 (1977), anninq et al, Chromosoma, S3: 107-117 (1975), Sod~a. A., Nucleic Acids Research, S: 385-401 (197~)), or the biotin can be covalently incorporated into specific nucleotide residues ( nqe~. P~Ro ~
Pxoceedings of one National Academy o~ Sciences, USA, 7~: 6633-6637 (1981), or the biotin can be attached to a polynucleotide by means of a diamine (e.g~, pentane diamine) bridge (BroXer, T.~. et al, Nucleic Acids Research ~: 363-384 (1978)). Interaction of the 2~3~0~
J~J~35 ~ORD 80) ~AT2NT
biotin molecules with avidin, ~treptavidin or antibiotin antibodies i6 then carried out~ whereln the avidin, streptavidin or the antibodies are conjugated to such signalling components as latex particles (Sodi3. A.. et al, supra, or Mann~na. et al Chromosoma, supra.) ferritin (~Q~EI ~Ye~ a fluorogen such as fluorescein, an enzyme, secondary antibodies, magnetic particles, or the like.
Detaile~ Descriptlo~ o~ Pref~rre~ ~m~o~e~t~
~ransaminat~on of t~e ~o~ied nu~leio ac~ ~tra~0 Dried nucl~ic acids are dissolved in a transam;nation mix, each milliliter of the transamination mix containing 560 mg hexanediamine hydrochloride, 23 mg 4-morpholineethane sulfonic acid (MES), and 100 mg sodium metabisulfite adjusted to pH6. The mixture is then set aside at room temperature for twenty four to seventy two hours. Aft~r this time the pH is raised to 8.5 for two hours and then lowered to 7.0~ The transaminated nucleic acid is then isolated by gel filtration on Sephadex G-50.
Preparation of Al~ehy~e Paper The aldehyde paper used may be prepared by limited oxidation of cellulose paper by periodate or can be purchased from commerci~l sources such as Sterogene Biochemicals, San Gabriel, California.
The paper is cut into appropriate ~ize.
Immobllizat~on 0~ ~ucleio Acias on ~o Al~e~y~e Paper.
The transaminated nucleic acid is dissolved in phosphate buffer pH 7.5 and sodium cyanoborohydride is added to make the concentration of sodium cyanoborohydride ~.1 molar. This solu~ion 2~3~2 ~
~J-35 ~0RD 80) - p~
is then applied to the paper at desired locations and concentration manually or mechanically. After application o~ the transa~ination mix, the paper is incubated in a humidity chambsr for approximately two hours. After this time the whole paper is soaked in a solution of 1.0 molar aminocaproic acid and 0.1 molar cyanoborohydride for thirty minutes. The paper is then washed with 0.5 molar ~odium chloride for fifteen minutes followed by two washing~ with phosphate buffered saline (PBS) for thirty minutes each. The paper is dried between two sheets of filter paper and stored in the dark over a desiccant such as Drierite.
Character~zat~on Of P~per The presence of immobilized nucleic acids on the paper is shown by exposin~ this paper to labeled DNA complementary to the nucleic acids immobilized on the paper. After washing the label is detected on the region where the nucleic acid was immobilized.
Example Tran~i~atio~ An Oligo~uoleoti~e 5.6 grams of hexanediamine dihydrochloride were put in~o a 50 ml screw cap tube and dissolved in 0.231 grams of 4-morpholineethane sulfonic acid (MES) in 500 ul 10M NaOH. The vol~me is then brought up to 9.5 mls with warm H20. The tube is then shaken until all the solid is dissolved.
1.0 grams o~ NaS205 is added to the screw cap tube and shaken.
The pH is adjusted to 6.0 with concentrated HCl. The ~olution is '~ ~ 3 ~
J&J- 35 ~ORD 80) PA~BN~
allowed to stand for thirty minutes, and the pH is checked and readjusted to 6.0 by adding more HCl i~ necessary.
2 ml of the solution is added to 100 - 200 nmoles o~ dried oligonucleotide in a test tube. The tube is then covered with parafilm and shake at room temperature for three days. After three days, the pH of the solution is brought up to 8.3 with NaOH and incubated for two hours. The pH i~ then reduced to 7.0 with HCl and incubated for thirty minutes.
The transaminated ollgo is purified over the a Sephadex G~50 column using lOmM triethyl ammonium bicarbonate (TEAB) as the buffer.
Attachment of Oligo~usleoti~2s ~o Al~e~y~e Cellulo~e P~per ~8i~g ~ ~ech~n~a~l ~prayer The transaminated modified nucleic acid strands were immobilized onto aldehyde paper by spraying them onto the aldehyde paper with a sprayer (CAMAG ~inomat IV Machine, CAMAG Scientific, Inc., Wrightsville Beach, North Carolina) according to the manufacturer's instructions.
Aldehyde paper (BioBind C, Sterogene Biochemicals, San Gabriel, California) was cut to an appropriate size (185 mm X -15mm is a suitable size) and aligned on the sprayer.
The following reagents were combined:
a. ~ransaminated oligonucleotide at appropriate amount (For a paper 185mm long, an oligonucleotide concentration of 100 2 1~
J&JD35 (ORD 80~ ~ATBN~
pmoles/cm2, and assuming that the l~ne the oligonucleotide will be on is 2mm wide, use 370 pmoles of oligonucleotide.) b. 5 ul 2M KHPO~pH 7~5 c. H20 to 90 ul 10 ul 1~0 ~ NaCNBH, was then added to ~he oligonucleotide ~ixture for a final volume of 100 ul. The mixture was then place~ into a syringe and inserted into the spraying ~achine.
The transaminated ~odlfied nucleic acid strand was sprayed onto the aldehyde paper in 2 m~ wide stripes. When spraying was donel the aldehyde paper was removed from the sprayer and incubated in a humidity chamber ~or two hours at room temperature.
The aldehyde paper was then placed in lM amino caproic acid ~ O.1 M ~odium cyanoborohydride for thirty ~inutes at room temperature and afterwards washed for fifteen minutes at room temperature in 0.5M NaCl. The paper was subsequently washed twice for thirty minutes each time at room temperature in 1 volume of PBS. The paper was dried between two pieces of filter paper and stored in the dark on the presence of a desiccant (Drierite).
Cbasacteri~at~on Qf oliqo fl~ ~er The paper is cut into strips about 5mM wide and one of the strips is put in a test tube containing a poly A sequence containing approximately 10% biotinylated uridine residues. This biotin containing poly A rises up by capillary action. After all the solution is drawn up, the strip is transferred to another tube containing 200 ~1 of streptavidin gold. After a ~ew minutes, one 2~3-~2~
J&J-35 50RD 803 ~AT~NT
sees a dark reddish brown band at the location where oligo dT was immobilized .
J&J-35 (O~D 80~ PATENT
Metho~ Of Immobilizi~g ~uclei¢ A¢i~ On A 801~lia Burfzlce For Use ID. Nucleio Ac~ aybriai~at1on As~a~rs Field of th~ I~v~tio~l The present invention relates to methods for immobilizing nucleic acids to solid surfaces for nucleic acid hybridization lo assays.
Bac~groun~ of tha In~e~tio~
Nucleic acid hybridization assays have proven useful in detecting the presence of microorganisms in biological samples (tissue, blood, urine, saliva, etc.) to diagnose infection and for detecting the presence in a mixture of a nucleic acid sequence of interest.
Nucleic acid hybridization assays are generally performed by immobilizing the test nucleic acid strand on a solid surface. A
mixture containing the labeled complementary nucleic acid strand is contacted with the immobilized nucleic acid strand under conditions allowing hybridization of the two complementary strands.
The hybridization of the two strands is then detected by chemical or other means by detecting the label on the probe strand.
Alternatively, the nucleic acids are detected by sandwich hybridization techniques in which one set of sequences complementary to the target sequence lS immobilized onto a solid surface. The surface is then contacted with the targer DNA and a . - ' " ' ' 2 ~
J&~-35 (ORD 80) ~T~N~
labeled probe complementary to the target at a place different than the one attached to the solid ~ur~ace. Under appropriate conditions the labeled probe ~s retained on the paper via the target DNA and detected by chemical or physical methods directly or indirectly.
Nucleic acids are typically immobil~zed on solid surfaces such as cellulose or nylon by physical contact with the surface~ and the nucleic acids are bound to the surface through weak non-covalent bonds. Because of the nonspecific binding of nucleic acid to these surfaces, it is difficult to orient the nucleic acid so that it does not interact with the solid surface with the por~ions of the nucleic acid that are needed to hybridize with the test sequences.
Thus greater amounts of the nucleic acid are needed to ensure a sufficient amount of free sequences to bind to the test nucleic acid. This inefficient use of nucleic acid can be e~pensive and can be limiting if only small quantities of the nucleic acid are available.
Additionally, the inability to accurately place the nucleic acid on the solid surface can hinder the effectiveness of the assay when small amounts of the nucleic acid sequence of interest are present, since it can be difficult to distinguish the nucleic acid present from backqround reactions.
Further, it would be desirable to be able to test for ~ore than one pathogen in a single assay, or to test for ~ore than one nucleic acid sequence in a single microorganis~ in the same assay.
2 ~
J~J-35 (O~D 80) PATENT
Uncertain placement of nucleic acids on the ~olid surface is a drawback to the development of assays of this kind.
8ummary of t~e I~Ye~tiOn The methods of the invention provide methods of immobilizing nucleic acid to a solid surface having a free aldehyde group for use in nucleic acid hybridization assays. In the methods of the invention a modified nucleic acid strand comprising a variable portion and an anchor portion wherein the variable portion comprises a nucleotide sequence having a selected base sequence and the anchor portion comprises at least one nucleotide base modified with a primary amine function or nucleotide base equivalent having a primary amine function is reacted with the free aldehyde group of the solid surface in the presence of a reducing agent to form complexes of the modified nucleic acid strand and at least a portion of the free aldehyde yroups.
Accordingly, the invention also provides solid surfaces for performance of nucleic acid hybridization assays comprising a solid surface prepared in ~ccordance with the ~ethods of the invention. The invention further provides kits for performance of nucleic acid hybridization assays comprising a solid surface of the invention and at least one labeled nucleic acid probe, the solid surface and the probe selected to form a detection system for a target strand of nucleic acid.
The met~ods of the invention provide an efficient procedure for immobilizing nucleic acids, especially oligonucleotides, on to 2 ~ 2 ~
J~J-3s (ORD 803 ~ATENT
solid surfaces without affecting ~heir ability ~o hybridize to complementary nucleic acid. The method is fast, economical and efficient. On flat surfaces such as paper or membranes, the oligonucleotides can be applied in zones alone, ~ogether with, or alongside other oligonucleotides or proteins fiuch as enzymes or antibodies. The immobilization occurs via synthetlc anchors provid~d a~ the ~ermini of ~he oligonucleotide being immobilized.
The ~ethods and solid surfaces of the invention provide specific orientation of the irmobilized nucleic acid by the anchor portion so that the entire or substantially all of the variable portion is free to hybridize with test nucleic acid. This improvement over prlor methods of immobilizing nucleic acids on solid surfaces for nucleic acid hybridization provided by the methods and solid surfaces of the invention allows more efficient use of the immobilized nucleic acid. Smaller quantities are needed as non-specific binding of the nucleic acid to the solid support is eliminated through the binding of the anchor portion with the solid support.
The methods of the invention make it possible to place the immobilized nucleic acid in pre-determined positions on the surface, facilitating detection of hybridized test nucleic acid by allowing it to be more easily distinguished from bacXground reactions. Additionally, controlled placement of the immobilized nucleic acid allows the immobilization of more than one type of .25 nucleic acid sequence in precise locations on the solid ~urface, 2~
J~J-3s ~ORD 801 PA~ENT
so that ~ore than one type of ~icroorganism or nucleic acid sequence of lnterest can be detected in the same assay.
The solid surfaces o~ the invention have the advantage of providing a light, or paper colored, background which gives a good contrast for hybridization assays per~ormed on the solid surface.
Detaile~ De0cr~ption o~ th~ I~e~tio~
In the methods of the invention nucleic scid is immobilized on a solid surface through covalen~ bonding. The methods o~ the invention produce a solid surface ha~ing nucleic acid, DNA or RNA, bound thereon in a predetermined pattern that is suitable for use in nucleic acid hybridization assays.
In preferre~ embodiments, ~ ~r RNA to be immobilized is adapted for ~mmobilization by firs~ attaching at least one nucleotide base modified with a primary a~ine function or nucleotide base equivalent having a primary ~mine ~unction at either of the termini of the DNA or RNA to form modified nucleic acid strands. The ~odified nucleic acid strand thus formed comprises a variable portion and an anchor portion wheréin the variable portion comprises a nucleotide sequence having a selected base sequence and the anchor portion comprises at least one nucleotide base modified with a primary amine function or nucleotide base equivalent having a primary amine Punction attached at either ter~inus of the variable portion.
The mod~fied nucleic acid strands to be ~mmobilized and used as for capturing or hybridizing with complementary nucleic acids are 2 ~ 2 ~
~ 35 ~ORD ~0) ~aT~T
preferably prepared by chemical synthesis using a DNA synthesizer and commercially available reagents. The base sequence of the variable portion of the modified nucleic acid strand is selected in accordance with the organism it is desired to de~ec~, or other purpose for the hybridization assay. The variable portion is preferably prepared by chemical synth2sis, however, it can also be prepared from natural cellular or reco~binant sources using recombinant techniques. The variable portion may be derived from human, bacterial, viral, fungal or other sources. If the variable portion is prepared from cellular or recombinant sources, synthesis of the variable portion will not be necessary. The variable portion may also be a homopolymer, such as oligo thymidine (poly thymidine or poly T) or poly adenine (~oly A). The variable portion i6 preferably from about two to about 1,000 nucleotide bases in length, mor2 preferably from about 15 to about 100 nucleotide bases in length.
The anchor portion is added to the variable portion. The anchor portion may be added to either the 3' or 5' terminus of the variable portion. The anc~or portion is preferably comprised of at least one nucleotide base modified with a primary amine function or nucleotide base equivalent having a primary amine function more preferably of from two to about ten nucleotide bases or nucleotide base equivalents, and most preferably of from about five to about eight nucleotide bases or nucleotide base equivalent~. Suitable nucleotide bases include cytosine modified at the 4 position of the 2~3~
J&J-3s (0~D 80) PA~NT
pyrimidine ring, adenine ~odified at the 6 position of the purine ring, and uridlne modified to contain a primary amino function.
Examples of suitable modified nucleotide bases include 5-amino(12)-2'-deoxyuridine-5'-triphosphate (Behring Diagnostics, ~a Jolla, California), 5-(3-aminoallyl)-2'-deoxyuridine 5'-triphosphate, 5-t3-aminoallyl)uridine 5'triphosphate, and N6-~[6 aminohexyl~-carbamoylmethyl)-adenosine 5'triphosphate (Sigma Chemical Co, St.
Louis, Missouri). Other nucleotide bases modifed to contain a primary amine function may also be suitable for use in the anchor portion. A preferred amino-modified nucleotide base is cytosine modified at the 4 position of the pyrimidine ring. Nucleotide base equivalents include phosphoramidites, phosphonates that can be modified with primary amines to give phosphoramidates, and other compounds having, or capable of being modified to contain, primary amine functions that can be substituted for nucleotide bases in a nucleic acid sequence or that can be added at the 3' or 5' ends of a nucleic acid sequence.
The anchor portion is attached to the variable portion by covalent bonding through the synthetic process using a DNA
synthesizer or through any conventional msans for ligation of nucleic acids; for example, ligase enzymes such as T~ DNA ligase and ~. ÇQli ligase; chemical means (Z.A. Shabarova, M~S.
IvanovsXaya and M.G. Isaguilantis, FEBS Letters, vol. 154, number 2, April 1983); and photoligation means ("DNA Assay Using Template-~5 Directed Photoligation' n San Diego Section of the American 2 ~
JfiJ-35 l0RD 30) ~AT~NT
Association of Chemical Industry-AMOCO Technology Company, P.O. Box 400 Naperville, IL 6056S). Phosphoramidites may be added to the variable portion using conventional phosphoramidite chemistry.
Phosphonates that can be modified with primary amines to give phosphoramidatas may be attached to the variable portion according to the method of Froehler, Tetrahedron Letters 27t46): 5575-5578 (1983).
In a preferred embodiment of the invention, the anchor portion of the modified nucleic acid strand comprises cytosine modified at the 4 position of the pyridine ring with a primary amine function.
The preferred anchor portion is prepared by chemical synthesis in which cytosine is attached to the variable portion by covalent bondinq. The cytosines of the anchor portion are then subjected to bisulfite catalyz~d transamination reaction with a diamine. ~he transamination reaction results in modification of the pri~ary amine group at the 4 position of the cytosine pyrimidine ring with the diamine which has a primary amine substituent. Diamines havin~
from about two to about ten carbon atoms, preferably from about five to about seven carbon atoms, are suitable for use in the invention. In preferred embodiments hexanediamine is employed.
~ransamination of cytosine at the exocyclic animo qroup at the 4 position on the pyrimidine ring ~ay be accomplished by conventional transamination methods, such as the method described herein.
If the variable portion of the modified nucleic acid strand contains cytosine, it will be necessary to protect the first 2 ~
J~J-35 (O~D 80) ~ATRN~
variable portion during transamination, ~o that any cytosines present in this portion are not modi~led, thus interfering with later hybridization with the test nucleic acid sequence.
Protection of cytosine in ~he variable portion can readily be provided by hy~ridizing a complementa~y sequence to this portion of the modified nucleic acid ~trand, taking care to exclude the anchor portion. The complementary protective strand may be prepared by chemical synthesis, or purified from natural or recombinant sources using any convenient meansO It is important that only the anchor portion cytosine molecules are single stranded and that the other cytosines in the variable portion are protected from modification by transamination by hydrogen bonding with the complementary 6trand. The complementary strand is removed from the modified nucleic acid strand before a hybridization assay is performed. Denaturing of the protective strand may be accomplished by any conventional means such as alkaline conditions or elevated temperatures and removal may be accomplished by any suitable purification technique, such as chromatography methods and the liXe, or by rinsing the ~olid surface if removal of the complementary protectiva strand takes place after immobili~ation of the modified nucleic acid strand. The complementary protective strand may be removed after the modification ~tep, or after any subsequent ~tep of the methods of the invention.
The modified nucleic acid strand is applied to a ~ol~d ~urface containing aldehyde groups, in the presence of a reducing agent 2 ~
PAT~NT
variable portion during transamination, 50 that any cytosines present in this portion are not modified, thus interfering with later hybridization with the test nucleic acid sequence.
Protection of cytosine in the variable portion can readily be provided by hybridizing a complementary sequence to thi~ portion of the modified nucleic acid ~trand, taking care to exclude the anchor portion. The complementary protective strand may be prepared ~y chemical synthesis, or purified from natural or recombinant sources using any convenient means. It is important that only the anchor portion cytosine molecules are single stranded and that the other cytosines in the variable portion are protected from modification by transamination by hydrogen bonding with the complementary strand. The complementary strand is removed from the modified nucleic acid ~trand before a hybridization assay is performed. Denaturing of the protective strand may be accomplished by any conventional means such as alkaline conditions or elevated temperatures and removal may be accomplished by any suitable purification technique, such as chromatoqraphy methcds and the like, or by rinsing the solid surface if removal of the complementary protective strand takes place after immobilization of the modified nucleic acid strand. The complementary protective strand may be removed after the modification step, or after any subsequent step of the methods of the invention.
The modified nucleic acid strand i6 appl;ed to a ~olid surface containing aldehyde groups, in the presence of a reducing agent _ g _ J&J-35 SORD 80~ PAT~N~
such as sodium cyano~orohydride. ~ ~ixture o~ ~he modified nucleic acid strand and reducing agent in a liquid such as water or phosphate buffer having a pH in the range of from about 6.0 to about 8.S, preferably about 7.6, is applied to the aldehyde surface by spraying, soaking or any convenient method. The concentration of modified nucleic acid strand in the mixture should be great enough to ensure detection in a nucleic acid hybridization assay.
Generally, a concentration of modified nucleic acid strand in the range of 50 p moles to 150 p moles per square centimeter of solid surface, preferably a concentration of about 100 p moles per square centimeter will given an easily detectable result under assay conditions. The actual concentration used, however, will depend on the method of application and the surface to be coated, more concentrated solution is preferred ~o as to avoid the lateral movement of the solution on the surface.
Solid surfaces Ruita~le for use in the methods of the invention are those containing a free aldehyde group, or which can be modified to contain a free aldehyde group. For example, cellulose paper can be modified by limited oxidation with periodate to contain free aldehyde groups. Cellulose paper having free aldehyde groups may al60 be purchased from commercial sources such as Sterogene Biochemicals, San Gabriel, California.
The ~odified nucleic acid ~trand may be positioned on the solid surface by any suitable method. ~or application ~o the solid surface, the modified nucleic acid strand will typically be in a 2 0 3 ~ J ~
J~J-35 ~o~D 80) PA~
liquid solution wh~ch may be applied to the solid surface by any convenient ~ethod including, manual application of the l~quid solution, sprayîng, or by dipp~ng the 601id surface into the solution containing the modified nucleic acid strand. The modiPied nucleic acid strand may be applied to the ~olld surface in any desired pa~tern or combination of pattern~. More than one type of DNA sequence can be positioned on the solid surface; for instance a nucleic acid sequence unique to microorganism A and a nucleic acid sequence unique to micr~organism B can be immobilized in discrete stripes onto the solid surface to detect microorganisms A and B in the same hybridization assay by using appropriately labeled probes.
The modified nucleic acid strand and reducing agent is allowed to incubate with the solid surface for a length of time sufficient for the reaction between the nucleic acid and diamine to take place. Depending on the reducing agent and transamination method used this time will be approximately two hours. At the end of the incubation period, the solid surface is washed with water or a buffer such as phosphate buffered saline (PBS) to remove all excess reagents and finally washed with a buffer such as PBS. The ~olid surface is then dried by any convenient method, such as blotting between filter paper, or over a desiccant.
To reduce back~round interference in a hybridization assay, aldehyde groups on the solid surface that did not react with the modified nucleic acid strand may be optionally modified by reaction 2~3:~2.~
J~J-35 ~oaD 80) PAT~N~
with an amino acid, such as ~-amino caproic acid in the presence of a reducing agent. This step will convert the remaining aldehyde groups to acidic functions by reaction with the acid, 80 that the aldehyde groups will not be available to bind with nucleic acid or other reagents in nucleic acid hybridization assays and also introduces a negative charge on the surface, which aids in keeping background interference lo~. Conversion of unreacted aldehyde groups may readily be accomplished after immobilization of the modified nucleic acid ~trand by applying a mixture of 0.1 aminocaproic acid and 0.1 ~ sodium cyanoborohydride to ~he solid surface. The mixture may be applied to the solid surface by soa~ing the solid surface in a solution containing ~-amino caproic acid and sodium cyanoborohydride, spraying or any other &uitable method. After approximately one-half to two ~ours the paper is washed with water few times to remove all reagents and ~inally with phosphate buffered ~aline (PBS) and dried and stored.
The kits of the invention comprise a olid surface of the invention and at least one labeled nucleic acid probe. The solid surface and the labeled nucleic acid probe are selected to form a detection system for target nucleic acid. For example, to detect nucleic acid sequence A fro~ microorganism A, a solid surface of the invention having the variable portion of the modified nucleic acid strand complementary to at least a portion of nucleic acid sequence A would be provided in the kit. A labeled nucleic acid probe complmentary to a different portion of nucleic acid 6equence ~J-35 (O~D B9~ ~ATRNT
A would then be provided for detection o~ nucleic acid sequence A
hybridized to the solid surface.
Nucleic acid sequences use~ul ~n the labeled nucleic acid probes are readily prepared by any conventional method such as organic synthesis, reco~binant DNA techniques or isolation from genomic DNA. However, these sequences are particularly amenable to organic synthesis usin~ techniques known in the art such as techniques utilizing a nucle~c acid s~nthesizer and commercially available reagents.
The labeled nucleic acid probes may ~e labelad by conventional radioisotopic labeling, chemical labeling, immunogenic labeling, or a labal with light scattering effect, and the like. Suitable methods to detect ~uch labels are ficintillation counting, autoradiography, fluorescence ~easurement, calori~etric measurement, or light emission measurement.
Thus, the labeling may comprise a radiolabel ~e.g. l'C, 32p, 'H, and the like), an enzyme (e.g., peroxidase, alkaline or acid phosphatase, and the like), a bacterial label~ a fluorescent label, an antibody (which may be used in a double antibody syste~), an antigen (to be used with a labeled antibody), a small molecule such as biotin ~to be used with an avidin, streptavidin, or antibiotin system), a latex particle (to be used in a buoyancy or latex agglut~nation ~ystem), an electron dense compound such as ferritin (to be used with electron microscopy), or a light ~cattering particle such as colloidal gold, or any combination~ or - 2~3 1~3?.~
J~J 35 ~o~D 803 2AT~NT
permutations o~ the foregoing.
For example, if the labeling portion of the probe is an antigen, a signal can be generated by complexing said antigen with an antibody/enzyme conjugate, ~ollowed by addition of an enzyme substrate. If this portion wera an antibody, signal can be generated by complexing anti-antibody or an Fc binding protein such as Protein A therewith, when ~uch second antibody or Protein A have been con~ugated to an enzyme.
For reasons of ease and safety in the handling of the probe, it is preferred that it be chemically labeled, especially enzymatically or immunologically. In more preferred embodiments, the chemical label of choice is a hapten such as biotin, iminobiotin, fluorescein and the like.
Among the preferred labeling systems that may be mentioned are those based on the biotin/strepavidin system. This system can be incorporated into the probe by a variety of means. For example, the probe can be covalently attached to biotin via a cytochrome c bridge (~anning et al, Biochemistry, 16: 1364-1370 (1977), anninq et al, Chromosoma, S3: 107-117 (1975), Sod~a. A., Nucleic Acids Research, S: 385-401 (197~)), or the biotin can be covalently incorporated into specific nucleotide residues ( nqe~. P~Ro ~
Pxoceedings of one National Academy o~ Sciences, USA, 7~: 6633-6637 (1981), or the biotin can be attached to a polynucleotide by means of a diamine (e.g~, pentane diamine) bridge (BroXer, T.~. et al, Nucleic Acids Research ~: 363-384 (1978)). Interaction of the 2~3~0~
J~J~35 ~ORD 80) ~AT2NT
biotin molecules with avidin, ~treptavidin or antibiotin antibodies i6 then carried out~ whereln the avidin, streptavidin or the antibodies are conjugated to such signalling components as latex particles (Sodi3. A.. et al, supra, or Mann~na. et al Chromosoma, supra.) ferritin (~Q~EI ~Ye~ a fluorogen such as fluorescein, an enzyme, secondary antibodies, magnetic particles, or the like.
Detaile~ Descriptlo~ o~ Pref~rre~ ~m~o~e~t~
~ransaminat~on of t~e ~o~ied nu~leio ac~ ~tra~0 Dried nucl~ic acids are dissolved in a transam;nation mix, each milliliter of the transamination mix containing 560 mg hexanediamine hydrochloride, 23 mg 4-morpholineethane sulfonic acid (MES), and 100 mg sodium metabisulfite adjusted to pH6. The mixture is then set aside at room temperature for twenty four to seventy two hours. Aft~r this time the pH is raised to 8.5 for two hours and then lowered to 7.0~ The transaminated nucleic acid is then isolated by gel filtration on Sephadex G-50.
Preparation of Al~ehy~e Paper The aldehyde paper used may be prepared by limited oxidation of cellulose paper by periodate or can be purchased from commerci~l sources such as Sterogene Biochemicals, San Gabriel, California.
The paper is cut into appropriate ~ize.
Immobllizat~on 0~ ~ucleio Acias on ~o Al~e~y~e Paper.
The transaminated nucleic acid is dissolved in phosphate buffer pH 7.5 and sodium cyanoborohydride is added to make the concentration of sodium cyanoborohydride ~.1 molar. This solu~ion 2~3~2 ~
~J-35 ~0RD 80) - p~
is then applied to the paper at desired locations and concentration manually or mechanically. After application o~ the transa~ination mix, the paper is incubated in a humidity chambsr for approximately two hours. After this time the whole paper is soaked in a solution of 1.0 molar aminocaproic acid and 0.1 molar cyanoborohydride for thirty minutes. The paper is then washed with 0.5 molar ~odium chloride for fifteen minutes followed by two washing~ with phosphate buffered saline (PBS) for thirty minutes each. The paper is dried between two sheets of filter paper and stored in the dark over a desiccant such as Drierite.
Character~zat~on Of P~per The presence of immobilized nucleic acids on the paper is shown by exposin~ this paper to labeled DNA complementary to the nucleic acids immobilized on the paper. After washing the label is detected on the region where the nucleic acid was immobilized.
Example Tran~i~atio~ An Oligo~uoleoti~e 5.6 grams of hexanediamine dihydrochloride were put in~o a 50 ml screw cap tube and dissolved in 0.231 grams of 4-morpholineethane sulfonic acid (MES) in 500 ul 10M NaOH. The vol~me is then brought up to 9.5 mls with warm H20. The tube is then shaken until all the solid is dissolved.
1.0 grams o~ NaS205 is added to the screw cap tube and shaken.
The pH is adjusted to 6.0 with concentrated HCl. The ~olution is '~ ~ 3 ~
J&J- 35 ~ORD 80) PA~BN~
allowed to stand for thirty minutes, and the pH is checked and readjusted to 6.0 by adding more HCl i~ necessary.
2 ml of the solution is added to 100 - 200 nmoles o~ dried oligonucleotide in a test tube. The tube is then covered with parafilm and shake at room temperature for three days. After three days, the pH of the solution is brought up to 8.3 with NaOH and incubated for two hours. The pH i~ then reduced to 7.0 with HCl and incubated for thirty minutes.
The transaminated ollgo is purified over the a Sephadex G~50 column using lOmM triethyl ammonium bicarbonate (TEAB) as the buffer.
Attachment of Oligo~usleoti~2s ~o Al~e~y~e Cellulo~e P~per ~8i~g ~ ~ech~n~a~l ~prayer The transaminated modified nucleic acid strands were immobilized onto aldehyde paper by spraying them onto the aldehyde paper with a sprayer (CAMAG ~inomat IV Machine, CAMAG Scientific, Inc., Wrightsville Beach, North Carolina) according to the manufacturer's instructions.
Aldehyde paper (BioBind C, Sterogene Biochemicals, San Gabriel, California) was cut to an appropriate size (185 mm X -15mm is a suitable size) and aligned on the sprayer.
The following reagents were combined:
a. ~ransaminated oligonucleotide at appropriate amount (For a paper 185mm long, an oligonucleotide concentration of 100 2 1~
J&JD35 (ORD 80~ ~ATBN~
pmoles/cm2, and assuming that the l~ne the oligonucleotide will be on is 2mm wide, use 370 pmoles of oligonucleotide.) b. 5 ul 2M KHPO~pH 7~5 c. H20 to 90 ul 10 ul 1~0 ~ NaCNBH, was then added to ~he oligonucleotide ~ixture for a final volume of 100 ul. The mixture was then place~ into a syringe and inserted into the spraying ~achine.
The transaminated ~odlfied nucleic acid strand was sprayed onto the aldehyde paper in 2 m~ wide stripes. When spraying was donel the aldehyde paper was removed from the sprayer and incubated in a humidity chamber ~or two hours at room temperature.
The aldehyde paper was then placed in lM amino caproic acid ~ O.1 M ~odium cyanoborohydride for thirty ~inutes at room temperature and afterwards washed for fifteen minutes at room temperature in 0.5M NaCl. The paper was subsequently washed twice for thirty minutes each time at room temperature in 1 volume of PBS. The paper was dried between two pieces of filter paper and stored in the dark on the presence of a desiccant (Drierite).
Cbasacteri~at~on Qf oliqo fl~ ~er The paper is cut into strips about 5mM wide and one of the strips is put in a test tube containing a poly A sequence containing approximately 10% biotinylated uridine residues. This biotin containing poly A rises up by capillary action. After all the solution is drawn up, the strip is transferred to another tube containing 200 ~1 of streptavidin gold. After a ~ew minutes, one 2~3-~2~
J&J-35 50RD 803 ~AT~NT
sees a dark reddish brown band at the location where oligo dT was immobilized .
Claims (23)
1. A method of immobilizing nucleic acid to a solid surface having a free aldehyde group, comprising the steps of:
(a) providing a modified nucleic acid strand comprising a variable portion and an anchor portion wherein said variable portion comprises a nucleotide sequence having a selected base sequence and said anchor portion comprises at least one nucleotide base modified with a primary amine function or nucleotide base equivalent having a primary amine function; and (b) reacting said modified nucleic acid strand with the free aldehyde group of said solid surface in the presence of a reducing agent to form complexes of said modified nucleic acid strand and at least a portion of said free aldehyde.
(a) providing a modified nucleic acid strand comprising a variable portion and an anchor portion wherein said variable portion comprises a nucleotide sequence having a selected base sequence and said anchor portion comprises at least one nucleotide base modified with a primary amine function or nucleotide base equivalent having a primary amine function; and (b) reacting said modified nucleic acid strand with the free aldehyde group of said solid surface in the presence of a reducing agent to form complexes of said modified nucleic acid strand and at least a portion of said free aldehyde.
2. The method of claim 1 wherein said reducing agent is sodium cyanoborohydride.
3. The method of claim 1 wherein said solid surface is cellulose paper having free aldehyde groups on its surfaces.
4. The method of claim 1 wherein said anchor portion comprises from two to about 10 nucleotide bases or nucleotide base equivalents.
5. The method of claim 4 wherein said anchor portion comprises from about five to about eight nucleotide bases or nucleotide base equivalents.
6. The method of claim 1 wherein said nucleotide base or J&J-35 (ORD 80) PATENT
nucleotide base equivalent is cytosine having nitrogen at the exocyclic 4 position of the pyrimidine ring modified with a substituent having a primary amine function.
nucleotide base equivalent is cytosine having nitrogen at the exocyclic 4 position of the pyrimidine ring modified with a substituent having a primary amine function.
7. The method of claim 1 wherein said variable portion is from about two to about 1,000 nucleotide bases in length.
8 . The method of claim 7 wherein said variable portion is from about 15 to about 100 nucleotide bases in length.
9. The method of claim 1 wherein said variable portion of the modified nucleotide strand is a homopolymer.
10. The method of claim 9 wherein said homopolymer is poly thymidine.
11. The method of claim 9 wherein said homopolymer is poly adenine.
12. The method of claim 1 wherein said variable portion is RNA.
13. The method of claim 1 wherein said variable portion is DNA.
14. The method of claim 1 wherein said variable portion is of human origin.
15. The method of claim 1 wherein said variable portion is of bacterial origin.
16. The method of claim 1 wherein said variable portion is of viral origin.
17. The method of claim 1 wherein said variable portion is of fungal origin.
J W-35 (ORD 80) PATENT
J W-35 (ORD 80) PATENT
18. The method of claim 1 further comprising the step of reacting unreacted aldehyde groups from step (b) with a mixture of an amino acid and a reducing agent.
19. The method of claim 18 wherein said amino acid is .alpha.-amino caproic acid.
20. A solid surface for conducting nucleic acid hybridization assays comprising a solid surface prepared in accordance with the method of claim 1.
21. A solid surface for conducting nucleic acid hybridization assays comprising a solid surface prepared in accordance with the method of claim 9.
22. A kit for performing a nucleic hybridization assay comprising, a solid surface of claim 20 and at least one labeled nucleic acid probe, said solid surface and said at least one nucleic acid probe selected to form a detection system for a target strand of nucleic acid.
23. A kit for performing a nucleic hybridization assay comprising, a solid surface of claim 21 and at least one labeled nucleic acid probe, said solid surface and said at least one nucleic acid probe selected to form a detection system for a target strand of nucleic acid.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US444,031 | 1989-11-30 | ||
| US07/444,031 US5215882A (en) | 1989-11-30 | 1989-11-30 | Method of immobilizing nucleic acid on a solid surface for use in nucleic acid hybridization assays |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2031026A1 true CA2031026A1 (en) | 1991-05-31 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002031026A Abandoned CA2031026A1 (en) | 1989-11-30 | 1990-11-28 | Method of immobilizing nucleic acid on a solid surface for use in nucleic acid hybridization assays |
Country Status (7)
| Country | Link |
|---|---|
| US (1) | US5215882A (en) |
| EP (1) | EP0435470B1 (en) |
| AT (1) | ATE147860T1 (en) |
| CA (1) | CA2031026A1 (en) |
| DE (1) | DE69029717T2 (en) |
| GR (1) | GR1001043B (en) |
| HK (1) | HK95097A (en) |
Families Citing this family (81)
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| DE361631C (en) * | 1922-10-17 | Karl Kramb | Row chair for theaters, cinemas and concert halls | |
| US3594037A (en) * | 1970-04-09 | 1971-07-20 | Mc Donnell Douglas Corp | Cabin attendant seat |
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| DE8521459U1 (en) * | 1985-07-25 | 1985-09-19 | Hohenloher Spezialmöbelwerk Schaffitzel GmbH + Co, 7110 Öhringen | Lecture hall chairs |
| EP0276302B1 (en) * | 1986-08-11 | 1993-04-28 | Siska Diagnostics,Inc. | Nucleic acid probe assay methods and compositions |
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1989
- 1989-11-30 US US07/444,031 patent/US5215882A/en not_active Expired - Lifetime
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1990
- 1990-11-28 CA CA002031026A patent/CA2031026A1/en not_active Abandoned
- 1990-11-28 GR GR900100828A patent/GR1001043B/en not_active IP Right Cessation
- 1990-11-29 DE DE69029717T patent/DE69029717T2/en not_active Expired - Lifetime
- 1990-11-29 AT AT90313007T patent/ATE147860T1/en not_active IP Right Cessation
- 1990-11-29 EP EP90313007A patent/EP0435470B1/en not_active Expired - Lifetime
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1997
- 1997-06-26 HK HK95097A patent/HK95097A/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| ATE147860T1 (en) | 1997-02-15 |
| EP0435470A1 (en) | 1991-07-03 |
| GR900100828A (en) | 1992-04-17 |
| DE69029717T2 (en) | 1997-06-05 |
| US5215882A (en) | 1993-06-01 |
| GR1001043B (en) | 1993-03-31 |
| EP0435470B1 (en) | 1997-01-15 |
| DE69029717D1 (en) | 1997-02-27 |
| HK95097A (en) | 1997-08-01 |
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| EEER | Examination request | ||
| FZDE | Discontinued |