CA1314794C - Assay for nucleic acid sequence in a sample - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
A method for detecting (i) one or more microorganisms or (ii) nucleic acid sequences from a prokaryotic source or an eukaroytic source in a nucleic acid-containing test sample comprising (a) labeling the nucleic acids in the test sample, (b) contacting, under hybridization conditions, the labeled hybridizable nucleic acid and one or more immobilized hybridizable nucleic acid probes comprising (i) one or more known microorganisms or (ii) sequences from eukaroytic or prokaryotic sources, to form hybridized labeled nucleic acids, and (d) assaying for the hybridized nucleic acids by detecting the label. The method can be used to detect genetic disorders, e.g., sickle-cell anemia.

Description

i~ ~ 31~7~

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is related to Canadian Application S.N. 530,235 filed February 20, 1987.

~CKGROUND OF THE INVENTI~N
Field o~ the Inventlon The present application relates to the detection and identification of microorganisms and the detection and identification of particular prokaryotic or eukaryotic D~A sources in a nucleic acid containing test sample.
Still further, the present invention relates to a method for the lysis of whole cells.
Background Information A. The Detection of Microor~anisms The identification of species of microorqanisms in a sample containing a mixture of microorganisms, by immobilizing the DNA from the sample and subjecting it to hybridization with a labelled specimen of species -speci~ic DNA from a known microorganism and observing whether hybridization occurs between the immobilized DNA
and the labelled specimen, has been disclosed in PCT
patent application No. PCT/US83/01029.
The most efficient and sensitive method of detection of nucleic acids such as D~A after hybridization requires radioactively labeled DNA. The use of autoradiography and enzymes prolongs the assay time and reauires experiellced technicians.
U.S.P. 4,353,535 to Falkow et al describe infectious disease diagnosis using labeled nucleotide probes complementary to nucleic acid coding for a c~lara(~eri.~itlc pa thogen pr oduct.

~3~4794 B. The Detection of Specific Eukaryotic Sequences The identification of specific sequence alteration in an eukaryotic nucleic acid sample by immobllizing the DNA from the sample and subjecting it to hybridization with a labeled oligonucleotide and observing whether hybridization occurs between the immobilized DNA and the labeled probe, has been described-It is known that the expression of a specificgène determines the physical condition of a human being.
Adult hemoglobin is a tetrameric association o~ two alpha and two beta subunits. During embryonic and etal life, the alpha chains are associated with, successively, gamma and delta chains before the adult beta form predominates.
The genes for alpha hemoglobin are located on human chromosome number 16 and tlle genes ~or gamma, delta and beta h~moglobin are tandemly linked on human chromosome ll. Hemoglobinopathies are heritable diseases that are the result of alterations in the structure of one or more of the hemoglobin genes. Many of the mutations have been characterized in considerable molecular detail and can range fronl single bas~ pair changes to wholesale deletion of a gene family. For e~ample, a change in the beta-globin gene coding sequence rom GAG to GTG at the sixth amino acid position produces sickle-beta-globin and a homozygate can have a disease known as slckle cell anemia. Similarly deletion of particular sequences from alpha-globin or beta-globin genes can cause thalassemias.
A recent survey, The ~ew Genetics and Clinical Practice, D. J. Weatherall, The Nuffield Provincial ~ospitals Trust, (1982), chapter 2 describes the frequency and clinical spectrum of genetic diseases.

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Problems associated with genetic defects can be diagnosed by nucleic acid sequence information. The easiest way ~o detect such sequence information is to use the method of hybridization with a specific probe of a known sequence.
U.S.P. ~,395,48~ to Wilson et al describe a method for the direct analysis of sickle cell anemia using a restriction endonuclease assay.
Edward M. I~lbin and Yuet Wai Kan, "A Simple Sensi~ive Prenatal Test for l~ydrops Fetalis Caused By ~-Thalassaemia", The Lancet, January 12, 19~5, pp. 75-77 describes a do-t blot analysis to differentiate between the genotypes of homozygous alpha-thalassemia and those o~ the haemoglobin-~l disease and alpha-thalassemia trait.
The most efficient and sensitive method of detection of nucleic acids, such as VNA, after hybridization requires radioactively labelled DNA. The use of autoradiography and enzymes prolongs the assay time and requires experienced technicians.
Recently, a non-radioactive method of labelling DNA was descrihed by Ward et al, European Patent ApplicatioJl 63,~79. Ward et al, use the method of nick translatioll to introduce biotinylated U (uracil) residues into DNA, replacing T (thymine). The biotin residue is then assayed wi~h antibiotin antibody or an avidin-containing system. The detection in this case is quicker than autoradiography, but the nick translatioll method requires highly skilled personnel. Moreover, biotinylation using biotinylated UTP (uridine triphosphate) works only for thymine-containing polynucleotides. The use of other nucleoside triphosphates is very difficult because the chemical derivatization of A (adenine) or G (guanine) or C
(cytosine) (containing -N~12) with biotin requires the ~s~ills oE trained or~Janic chemi.s~s.

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C. Effect of Nonionic Polymers on liybridization It has been shown by Wetm~lr (Biopolymers, 14, 2517-2524, (1974)) that anionic polymers such as dextran sul~ate increase the rate of DNA-DNA hybridization in solu~ion. It has also been shown that the heterogeneous pllase hybridization rate can also be increased by dextran sulfate. ~.S. Patent 4,302,204 discloses the effect of charged polysaccharides on the rate of nucleic acid hybridization.
Recently Amasino (_nalytical Biochemistry, l_ , 304-307 (1986)) has shown that a neutral polymer li~e polyethylene glycol can increase the rate of DNA-R~A
hybridizatioll more than dextran sulfate under optimum conditions. Although polyethylene glycol (PEG) has been used before in the hybridization medium (e.g., ~enz and Xurz, Nucleic Acids ~es., 12, 3435-3444 (1984)), no advantage was predicted over the use o~ dextran sulfate.
Applicants have observed, to their surprise, that in the presence of 10% polyethylene glycol hybridization can be virtually finished in 15 minutes. Although Amasino has shown PEG is better than dextran sulfate as an accelerator, his labeled probe was single stranded ~NA.
In the case of the present invention, the labeled material is whole gènomic sample DNA where ~oth complementary strands are present in solution. When a polymer accelerates the rate of hybridization it should also help the reannea].i.ng in solution, and hence should reduce the efficiency of hybridization, instead of increasing it.
D. Cell Lysi.s The present invention also provides a method for the efficient lysis of whole cells such that their DN~ is released and made available for photochemical labeling. While eukaryotic cells derived from nlulticell~lar animals are easily lysed under relatively 7 ~ ~L

mild conditions, single cell eukaryot~s and prokaryotes, especially Gram positive prokaryotes, are more difficult to lyse due to the complicated chemical nature and extent o~ cross-lin~ing of their cell ~alls. Methods do exist for effici~ntly lysin~ these refracto~y organisms, either by chemical--enzymatic or physica] means, hut these rnethods are ofterl complicated, time-consumlng and inappropriate for preserving the inte~rity of DNA.

SUMMARY OF THE INVENTION

The present invention provides a rapid and convenient method for detection of one or more micro-organisms or nucleic acid sequences from a prokaryotic source or an eukaryotic source in a nucleic acid-containing test sample and also a method for a simultaneous assay for the presence of more than one nucleic acid sequence.
The invention also involves direct labeling of the nucleic acid in the test sample and the use of whole chromosomal nucleic acid as the probe and/or as the test sample.
Also the invention relates to the use of oligonucleotides as immobilized probes.
The invention further relates to the use of cloning vectors and their derivatives for use as immobilized probes.
The invention also concerns the use of polyethylene glycol as a hybridization accelerator.
Still further the invention is directed to preparing a cell lysate by contacting a cell with alkali.
These advantages are realized in accordance with the present invention for a method of detecting (i) one or more microorganisms or (ii) nucleic acid sequences from a prokaryotic source or ~31~79~

an eukaryotic source in a nueleic aeid-eontaining test sample.
~he method involves the following:
(a) labeling the nueleie aeids, e.g., the nueleic acids of the organisms or eells or cell debris, in the test sample, (b) contacting, under hybriclization conditions, the labeled hybridizable sample nucleie acid and one or more immobilized hybridizable (e.g., single-stranded) nucleie acid (e.g., DNA) probes eomprising of (i) one or more ~nown microorganisms or (ii) nueleie aeid sequences from eukaryotie or prokaryotic sources, to form hybridized labeled nucleic aeids and (c) assaying for the hybridized nucleie aeids by detecting the label.
The hybridizable nucleic acid can be whole genomie nueleie aeid or a fragment thereof, e.g., an oligonueleotide.
In step (a), the nueleie aeids ean be labeled direetly in the test sample. In step (a), in sueh direet labeling, eells of the test sample ean be lysed and then a labeling reagent ean be added, whereby to label the nueleie aeid.
The method further comprises denaturing the labeled nucleie aeids from step (a) to form labeled denatured nucleic acids.
Aeeording to the invention, a labeled nueleie aeid test sample is eontaeted simultaneously with several different types of nucleie aeid, e.g., DNA, probes for hybridization. The nueleie aeid test sample is labeled and hybridized with several unlabeled immobilized probes.
The positions o~ the probes are fixed, and the labeled 13~7~

probe detected after hybridization will i.ndicate that the test sample carries a nucleic acid sequence complementary to the corresponding probe.
Nucleic acid probes for scveral microbiological systems or for different alleles of one or more genes can be immobilized separately on a solid support, for example, nitrocellulGse paper. The test sample nucleic acids are labeled and remain in solution. The solid material containing the immobilized probe is brought in contact with the labeled test nucleic acid solution under hybridization conditions. The solid material is washed free of unhybridized nucleic acid and the label is assayed. The presence of the label with one or more of the probes indicates that the test sample contains nucleic acids substantially complementary to those probes and hence originate, for example, from an infection by particular microbiological systems.
Labeling can be accomplished in a whole living cell or a cell lysate, and can be non-isotopic. The nucleic acid can be used for hybridization without further purification.
The presen~ invention also concerns specific lysis conditions to release nucleic acids from both gram positive and gram negative bacteria.
The present invention also relates to a hybridization medium which accelerates the process of hybridization. A hybridization accelerator according to the present invention is polyethylene glycol.
The present invention further concerns a kit for detecting (i) one or more microorganisms or (ii) nucleic acid sequences from a prokaryotic source or an eukaryotic source in a nucleic acid-containing test sample comprising i 131~79~

(a) a support solid containing hybridiz~ble, e.g., single-stranded, nucleic acid, e.g., DNA or an oligonucleotide, of (i) said one or more known microorganisms or (ii) said sequences from eukaryotic or prokaryotic sources immobilized thereon, e.g., a strip containing dots or spots of known microorganisms or eukaryotes or prokaryotes, (b) a reagent for labeling the nucleic acid of the test sample, (c) a reagent for releasing and denaturing nucleic acld, e.g., DNA, in the test sample, and (d) hybridi~ation reagents.
For chemil~minescence detection of the hybridized nucleic acid, the kit would further comprise a reagent for chemiluminescent detection.
In the above described kit, the reagent for labeling is given her~inbelow in a discussion on labels.
Reagents for releasin~ and denaturing DNA
include sodium hydroxide and lysing agents such as detergents and lysozymes.
Typical hybridization reagents includes a mixture of sodium chloride, sodium citrate, SDS (sodium dodecyl sulfate), bovine serum albumin, nonfat milk or dextran sulfate and optionally formamide.
The present invention further concerns a method of hybridization comprising contacting two complementary single stranded nucleic acids under hybric1ization conditions, at least one of which i~ immobilized wherein polyèthylene glycol is added.

BRIEF DESCRIPTION OF TI~E FIGURES
... . . .. _ . _ Fig. 1 is an autoradiograph of results of immobilization of an oligonucleotide sequence specific for hemoglobin mutation.

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Fig. 2 is a photograph of results of hybridization with labeled genomic DNA for non radioac-tive detection.

DETAILE~ DESCRIPTION OF TI~E INVENTION

.
The test sample in the present invention includes body fluids, e.g., urine, blood, semen, cerebrospinal fluid, pus, amniotic Fluid, tears, or semisolid or fluid discharge, e.cJ., sputum, saliva, lung aspirate, vaginal or urethal discharge, stool or solid tissue samples, such as a biopsy or chorionic villispecimens. Test samples also include samples collected with swabs ~rom the skin, genitalia, or throat.
All these test samples can be used for hybridization diagnosis after labeling the nucleie acids of the sample. The labeling can be accomplished without any processing of the sample. As for example, a urine sample (suspected of bacterial infections) can be labeled without centrifuga~ion, fil~ration or dialysis. The cells in the samples can be lysed without any separation step. It is surprising that a nucleic acid labeling reaction takes place in such a eomplex mixture as a clinical sample.
The nucleic aeid is preferably labeled by means of photochemistry, employing a photoreactive DNA-binding ~uroeoumarin or a phenanthridine eompound to link the nueleic aeid to a label whieh can be "read" or assayed in eonventional manner, ineluding fluoreseenee detection.
The end p~oduct is thus a labeled nueleie acid comprising (a) a nucleic acid component, (b) an intercalator or other DNA-binding ligand photoehemieally linked to the nucleie acid componen~, and (e) a label ehemieally linked to (b).
The photoch~mical method provides more ~avoLa~le reaction conditions than the usual chemical ~3~7~

couplin~ method for biochemically sensitive substances.
The intercalator and label can first be coupled and then photoreacted with the nucleic acid, or the n~cleic acid can first be photoreacted with the intercalator and then coupled to the label.
A general scheme for coupling a nucleic acid, exemplified by double-stranded DNA, -to apply a label, is as rollows:

Label Photoreactive Intercalator Labeled Double-Stranded DNA + Photoreactive Photoreactive Intercalator Intercalator ~- DNA h~-~

h~ Chemically - Functionalized DNA
~ + Label Labeled DNA

Where the hybridizable portion of the nucleic acid is in a double stranded form, such portion is then denatured to yield a hybridizable single stranded portion. Alternatively, where the labeled DNA comprises the hybridizable portion already in single stranded form, such denaturation can be avoided if desired.
Alternatively, double stranded DNA can be labeled by the approach of the presellt invention after hybridization has occurred using a llybridization format which generates double stranded VNA only in the presence of the sequence to be detected.
To produce specific and efficient photochemical products, i.t is desirable that the nucleic acid component 131~7~4 arld the photoreac~ive intercalator compound be allowed to react in the dark in a specific manner.
For coupling to DNA, arninomethyl psoralen, aminomethyl angelicin and amlno alkyl ethidium or methidium azides are particularly useful compounds. They bind to dou~le~stranded DNA and only the comple.~ yields photoadduct. In the case where labeled double-stranded DNA must be denatured in order to yield a hybridizable single stranded region, conditions are employed so that simultaneous interaction of two strands of DNA with a single photoadduct is prevented. It is necessary that the frequency of modification alon~ a hybridizable single stranded portion of the nucleic acid not be so great as to substantially prevent hybridization, and accordingly there preferably will be not more than one site of modification per 25, more usually 50, and preferably 100, nucleotide bases. Angelicin derivatives are superior to psoralen compounds for monoadduct formation. If a single-stranded DNA nucleic acid is covalently attached to some extra dou~le-stranded DNA, use of phenanthridium and psoralen compounds is desirable since these compounds interact specifically to double-stranded DNA in the dark.
The chemistry for the synthesis of the coupled reagents to modify nucleic acids for labeling, described more ~ully hereinbelow, is similar for all cases.
I`he nucleic acid component can be single or double stranded DNA or RNA or fragments thereof such as are produced by restriction enzymes or even relatively short oligomers.
The ~NA-binding ligands of the present invention used to link the nucleic acid component -to the label can be any suitable photoreactive form of known DNA-binding ligands. Particularly preferred DNA-binding ligands are intercalator compounds such as the ~uroco~rnarins, e.9., ange~licin ~isor!soralen) or psoralen 131~794 or derivd~ es thereof which pl~o~ochemically will react with nucleic ~cids, e ~., 4 -aminomethyl-4,5 -dimethyl angelicin, 4 -an~inometllyl-trioxsalen (4 aminomethyl-4,5 ,8-trimethyl-psoralen), 3-carboxy-5- or -8-arnino-or-hydroxy-psoralen, as well as mono- or bis-azido aminoalkyl methidium or etllidium compounds.
Particularly useful photoreactive forms of intercaiating agents are the azidointercalators. Their reac~ive nitrenes are readily generated at long wavelength ultraviolet or visible light and the nitrenes of arylazides prefer insertion reactions over their rearrangement products (see W}lite et al, Methods in Enzymol., 46, 649 (1977)). Representative intercalatlng agents include azidoacridine, ethidiurn monoazide, ethidium diazide, ethi.dium dimer azide (Mitchell et al, JACS, 10~, 4265 (198?) ), i-azido-7-chloroquinoline, and 2-azidofluorene. ~ specific nucleic acid binding azido compound has been described by l:orster et al, Nuclelc Acid Res., 13, (1935), 745. The structure of such compound is as follows: ~

~IN ~H

_ C~ ( C l i ~ ) 3 - Il`' - ( C 112 ) 3 N 11 C ( 2 q ts /

Other useful photoreactable intercalators are the furocoumarins whicll ~orm (2+2) cycloadducts with pyrimidine residues. ~lkylating agents can also be used such as his-chloroetllylamines and epoxides or aziridines, e.g., aflatoY~ins, polycyclic hydrocarbon epoxides, mitomycin and norphillir~
NonlirnitincJ eY~amples oE intercalator compounds l:,r usc i~ c tj-ese~ invel~ioll include acridine dyes, ' 13147~

phenanthridines, phenazlnes, furocoumarins, phenothiazines and quinolines.
The label which is linked to the nucleic acid component according to the present invention can be any chemical group or residue having a detectable physical or chemical property, i.e., labeling can be conducted by chemical reaction or physical adsorption. The label will bear a functional chemical group to enable it to be chemically linked to the intercalator compound. Such labeling materials have been well developed in the field of immunoassays and in general most any label useful in such methods can be applied to the present invention.
Particularly useful are enzymatically active groups, such as enzymes (see Clin. Chem., (1976), 22, 1243), enzyme substrates (see British Pat. Spec. 1,548,791), coenzymes (see U.S. Patent Nos . 4,230,797 and 4,23~,565) and enzyme inhibitors (see U.S. Patent No. 4,13~,792; fluorescers (see Clin. Chem., (1979), 25/ 353), and chromophores including phycobiliproteins; luminescers such as chemiluminescers and bioluminescers (see Clin. Chem., (1979), 25, 512, and ibid, 1531); specifically bindable ligands, i.e., protein binding ligallds; and res.idues com~rising radioisotopes such as 3H, 35S, 32p, 125I, and 14C. Such labels are detected on the basis of their own physical properties (e.g., fluorescers, chromophores and radioisotopes) or their reactive or binding properties (e.g., enzymes, substrates, coenzymes and inhibitors).
For example, a cofactor-labeled nucleic acid can be detected by adding the enzyme for which the label is a cofactor and a substrate for the enzyme. A hapten or ligand (e.g., biotin) labeled n~cleic acid can be detected by adding an antibody or an antibody pi~ment to the hapten or a protein (e.g., avidin) wllich ~inds the ligand, tagged with a detectable molecule. An antigen can alsc~ be used as a ].abel. Such detec~able n-olecule ~ ~3~79~

can be some molecule with a measurable physical property ~e.g, fluorescence or absorbance~ or a participant in an enzyme reaction (e.g., see above list). For example, one can use an enzyme which acts upon a substrate to generate a product with measurable physical property.
Examples o~ the latter include, but are not limi-ted to, beta-galactosidase, alkaline phosphatase, papain and peroxidase. For ln situ hybridization studies, ideally the final product is water insoluble. Other labels, e.g., dyes, will be evident to one having ordinary skill in the art.
The label will be linked to the intercalator compound, e.g., acridine dyes, phenanthridines, phenazines, furGcoumarins, phenothiazines and ~uinolines, by direct chemical linkage such as involving covalent bonds, or by indirect linka~e such as by the incorporation of the label in a microcapsule or liposome which in turn is linked to the intercalator compound.
~ethods by which the label is linked to the intercalator compounds are essentially known in the art and any convenient method can be used to perform the present invention.
Advantageously, the intercalator compound is first combined with label chemically and thereafter combined with the nucleic acid component. For example, since biotin carries a carboxyl group, it can be combined ~ith a furocoumarin by way of amide or ester formation withou~ interfering with the photochemical reactivity of the ~urocoumarin or the biological activity of the biotin, e.g., ~31~79~

( i ) N ~ 2 ) 4 - C - O -o 8iot in-l`l-hydroxysuccinirnicle or ~ RNH 2 (ii) o ~N ~S O

H ~J--I_(CH2 ) 4 - C - (~--U2 3iotin-p-nitrophenyl ester N ~ ( C~ 2 ) 4 - C - NHR
or carbodiin ide B iot in ~ ROH > Biot in CO OR
By way o~ examp le, CH2Nilz ~

O~o ~ NH

(CH~ \ 4C00 4=~--N2 Arnt aiot in nitrophellyl es~er H

,~ ,"~7 tRNH2) CH2-NH-CO- (CH2) q b~o 1~

1 31~79~
Other aminomethylanyelicin, psoralen and phenanthridium derivatives can be similarly reacted, as can phenanthridium halides and derivatives thereof such as aminopropyl m~thidium chloride, i.~., 2 ~ - ~ NH2 Cl / C~13 ~ = C - ~H - C~ - CH~ 2 - NH2 (see Hertzberg et al, J. Amer. Chem. soc., 104, 313 (1982)).
Alternatively, a bifunctional reagent such as dithiobis succinimidyl propionate or 1,4-butanediol diglycidyl ether can be used directly to couple the photochemically reactive molecule with the label where the reactants have al~yl amino residues, again in a known manner with regard to solvents, proportions and reaction conditions. Certain bifunctional reagents, possibly glutraldehyde may not be suitable because, while they couple, they may modify nucleic acid and thus interfere with the assay. Routine precautions can be taken to prevent such difficulties.
The particular sequence used in making the labeled nucleic acid can be varied. Thus, for example, an amino-substituted psoralen can first be photochemically coupled with a nucleic acid, the product having pendant amino groups by which it can be coupled to the label, i.e., labeling can be carried out by photochemically reacting a DNA binding ligand with the nucleic acid in the test sample. Alternatively, the 13~79~

psoralen can first be coupled to a la~el such as an enzyme and then to the nucleic acid.
~ s described in pending Canadian patent application Serial N;o. 486,781, filed July 15, 1985, the present invention also encompasses a labeled nucleic acid comprising (2) a nucleic acid component, (b) a nucleic acid-bindincJ ligand photochemically linked to the nucleic acid component, (c) a label and (d) a spacer chemically linking (b) and (c).
~ dvantageously, the spacer includes a chain of up to about 40 atoms, preferably about 2 to 20 atoms, selected from the group consisting of carbon, oxygen, nitrogen and sulfur Such spacer may be the polyfunctional radical of a member selected from the gxoup consisting of peptide, hydrocarbon, polyalcohol, polyether, polyamine, polyimine and carbohydrate, e.g., -glycyl-qlycyl-glycyl-or other oligopeptide, carbonyl dipeptides, and omega-amino-alkane-carbonyl radical such as -NH
(CH~)5-CO-, a spermine or spermidine radical, an alpha, omega-alkanediamine radical such as -NH-~CH2)6-N~I or -HN-CH2-C~2-N~, or the like. Sugar, polyethylene oxide radicals, glyceryl, pentaerythritol, and like radicals can also serve as the spacers.
These spacers can be directly linked to the nucleic acid-binding ligand and/or the label or the linkages may include a divalent radical of a coupler such as dithiobis succinimidyl propionate, 1,4-butanediol diglycidyl ether, a diisocyanate, carbodiimide, glyoxal, ~lutaraldehyde, or the like.
The spacer can be incorporated at any stage of the process of making the probe.
a-b-d-c defined hereinabove. Thus, the sequence can be any of ~he ollowitlg:

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a~b+d+c, b+d+c~a, d+c+b+a, b-~d+a~-c, etc.
The conditions for the individual steps are well ~nown in chemistry.
If the label is an enzyme, for example, the product will ultimately be placed on a suitable medium and the exten~ of catalysis will be determined. Thus, if the enzyme is a phosphatase, the medium could contain nitrophenyl phospllate and one would monitor the amount of nitrophenol generated by observing the color. If the enzyme is a beta-galactosidase, the medium can contain o-nitro- phenyl-D-galacto-pyranoside which also will liberate nitrophenol.
The labeled nucleic acid of the present invention is applicable to all conventional hybridization assay formats, and in general to any format that is possible bas~d on formation of a hybridiæation product or aggregate comprising the labeled nucleic acid. In particular, the unique labeled nucleic acid of the present invention can be used in solution and solid-phase hybridization formats, including, in the latter case, formats involving immobilization of either sample or probe nucleic acids and sandwich formats.
The nucleic acid probe will comprise at least one hybridizable, e.g., single-stranded, base sequence substantially complementary to or homologous with the sequence to be detected. ~lowever, such base sequence need not be a single continuous polynucleotide segment, but can be comprised of two or more individual segments interrupted by nonhomologous sequences. These nonhomologous seque~nces can ~e linear or they can be self-complementary and form hairpin loops. In addition, ~:hc homolocJous reCJion ol the probe can be flanked at the 131~7~

3' - and 5' termini by nonhomologous sequences, such as those comprising the DNA or ~NA or a vector into which the homologous sequence had been inserted for propagation. In either instance, the probe as presented as an analytical reagent will exhibit detectable hybridization at one or mor~ points with sample nucleic acids of interest. Linear or circular hybridizable, e~g., single-stranded polynucleotides can be used as the probe element, with major or minor portions being duplexed with a complementary polynucleotide strand or strands, provided that the critical homologous segment or segments are in single-stranded form and availabl~ for hybridization with sample VNA or RNA. Useful probes include linear or circular probes wherein the homologous probe sequence is in essentially only single-stranded form (see particularly, Hu and Messing, Gene, 17:271 (1982)).
The nucleic acid probe of the present invention can be used in any conventional hybridization technique.
As improvements are made and conceptually new formats are developed, such can be readily applied to the present probes. Conventional hybridization formats which are particularly useful include those wherein the sample nucleic acids or the polynucleotide probe i5 immobilized on a solid support (solid-phase hybridization) and those wh~rein the polynucleotide species are all in solution (solution hybridization).
In solid-phase hybridization formats, one of the polynucleotide species participatill~ in hybridization is fixed in an appropriate manner in its single-stranded form to a solid support. Useful solid supports are well known in the art and include those which bind nucleic acids either coval~ntly or noncovalently. Noncovalent supports which are generally understood to involve hydro~ho~ic hondi.ng include naturally occurring and 1314L7~L

synthetic polymeric materials, such as nitrocellulose, derivatized nylon and lluorinatcd polyhydrocarbons, in a variety of forms such as filters, beads or solid sheets.
~ovalent binding supports (in the form of filters, beads or solid sheets, just to mention a few) are also useful and comprise materials having chemically reactive groups or groups, such as dichlorotria2ine, diazobenzyloxymethyl, and the like, which can be activated for binding to polynucleotides.
It is well known that noncovalent immobilization of an oligonucleotide is ineffective on a solid support, for example, on nitrocellulose paper. The present invention also describes novel methods of oligonucleotide immobilization. This is achieved by phosphorylation of an oligonucleotide by a polynucleotide ~inase or by ligation of the S'-phosphorylated oligonucleotide to produce multi-oligonucleotide molecules capable of immobilization. The conditions for ~inase and ligation reaction have been described in standard text books, e.g., Molecular Cloning, T.
Maniatis, E.F. Fri-tsch and J. Sambrook, Cold Spring Harbor Laboratory, (1982), pages 1-123.
A typical solid-phase hybridization technique begins with immobilization of sample nucleic acids onto the support in single stranded form. This initial step essentially prevents reallnealillg of complementary strands from the sample and can be used as a means for concentrating sample material on the support for enhanced detectability. The polynucleotide probe is then contacted with the support and hybriclization detected by measurement of the label as described herein. The sol.id support provides a convenient means for separating labeled probe which has hybridized to the sequence to be detected from that which has not hybridized.

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Another method of interest is the sandwich hybridization technique wherein one of two mutually exclusive frayments of the homologous sequence of the probe is immobilized and the other is labelled. The presence of the polynucleotide sequence of interest results in dual hybridlzation to the imrnobilized and labelled probe segments. See ~ethods in ~nzymology, 65:468 (1980) and Gene, 21:77-85 (1983) for further details.
For the present invention, the imrnobile phase of the hybridization system can be a series or matrix of spots of known kinds and/or dilutions of denatured DNA.
This is most simply prepared by pipetting appropriate small volumes of native DNA onto a dry nitrocellulose or nylon sheet, floating the sheet on a sodium hydroxide solution to denature the DN~, rinsing the sheet in a neutralizing solution, then baki.ng the sheet to fix the DNA. ~efore DN~:DNA hybridization, the sheet is usually treated with a solution that inhibits non-specific binding of added DNA during hybriclization.
T}lis invention involves the labelin~ of whole genomic DNA, whole nucleic acids present in cells, whole cell lysate, or unlysecl whole cells. Once the labeled material is prepared, it can be used for the detection, i.e., the presence or absence of certain specific genomic sequences by speclfic nucleic acid hybridization assays.
One method according to the invention involves the separation of celis from a human sample or the human sample directly i.s treated by mixing with a photochemically reactive nucleic acid binding intercalating ligand. The mixture is incubated depending on the type of th~ sample. If the sample is lysed cells or nucleic acids, it is incubated for a period between a few seconds to five minutes and when whole cells or par~ial].y lysed cells are used, incubation between two ~3~ ~7~

minutes to two hours is employed. After the mixing and incubation, the whole sample mixture is irradiated at a particular wavelength for the covalent interaction between the photochemically reactive DNA binding ligand and the test sample. Then this labeled material is hybridized under speciEic hybridization condi.tions with a specific probe.
After the hybridization, the unreacted unhybridized labeled test sample is removed by washing.
.~fter the washing, the hybrid is detected through the label carried by the test sample, which is specifically hybridized with a specific probe.
The present invention is surprising since in a human genomic sample the amount of a single copy gene is very low, for example, if a restriction fragment of one thousand base pairs is the region of hybridization, the frequency of such sequence in the whole human genomic sample is one in a million. This conclusion has been derived by assuming from ~he literature that a human genomic sample has 3 x 109 base pairs and 1000 base pairs will be 1/3,000,000 of that number. Automatically, in a sample of human D~A containing approximately five to ten micrograms of nucleic acids, only 5 to 10 picogram of the corresponding sequences is available and labeling the vast majority of the non-specific DNA should produce more background than the true signal. But after the reaction, i~ is surprising to observe that the results are not only specific, but also of unexpected higher sensitivity.
Without wishing to be bound by any particular theory of operability, the reason for the unexpected sensitivity may be due to the formation of a network of non-specific nucleic acid hybrids bound to the specific hybrid, thus amplifying the amount of the signal. As has been shown in a typical e~ample, a 19 nucleotide long spcciEic seyuence containing plasmld i5 imrnobilized and ~31~79~

hybridized with 5 microgram equivalent of a test sample which is labeled photochemi.cally and one detects very easily the signal resulted from such hybrid. This could not have been accomplished by any other technique because of the problems associated with tile labeling method.
The present invention relates to a novel hybridization technique where probes are immobilized and an eukaryotic nucleic acid sample is labeled and hybridized with immobilized unlabeled probe. A
surprising characteristic of the invention is the ability to detect simple or multiple copy gene defects by labeling the test sample. Since there is no requirement for an excess of labeled hybridizing sequence, the present method is more specific. In the present invention, simultaneous detection of different gene defects can be easily carried out by immobilizing specific probes.
For example, using the present invention, one can immobilize oligonucleotide probes specific for genetic defects related to hemoglobinopathines, such as sickle cell anemia and alp}-a-thalassemias on a sheet of nitrocellulose paper, label the test sample and hybridize the labeled test sample with the immobilized probes. It is surprising tl~at partially purified or unpurified nucleic acid samples (cell lysate or whole cell) can be photochemically labeled with sensitive molecules without af~ecting the specific hybridizability.
The oligonucleotide can be cloned in cloning vectors, e.g., ~1 13, PUC 19 and PBR and accordingly the vector containing the oligonucleotide acts as the probe.
The present invention is also directed to detecting eukaroytes (protists) in samples from higher organisms, such as animals or humans.
l~ukaroyte.c, include alyae, protozoa, fungi and slime molds.

13~7~

The term "algae" refers in general to chlo~ophyll-containing protists, descriptions of which are found in G.M. Smith, Cryptogamic Botany, 2nd ed. Vol.
1, Algae and Fungi, McGraw-Hill, (1955).
Eukaryotic sequences according to the present invention includes all disease sequences except for bacteria and viruses. Accordingly, genetic diseases, for example, would also be embraced by the present invention.
Non-limiting e~amples of such genetic diseases are as ~ollows:

Area Affected Diseases Metabolism Acute intermitten-t porphyria Variegate porphyria alpha1-antitrypsin deficiency Cystic fibrosis Phenylketonuria Tay-Sachs disease Mucopolysaccllaridosis I
Mucopolysaccharidosis II
Galactosaemia Homocystinuria Cystinuria Metachromic leucodystrophy Nervous System Huntington's chorea ~eurofibromatosis Myotonic dystrophy l'uberous sclerosis Neurogenic muscular atrophies Blood Sickle-cel]. anaemia Beta thalassaemia Congenital spherocytosis llaernopllilia A

Bo~el Polyposis coli Kidney Polycystic disease Eyes Dominant blindness Retinoblastoma Ears Dominant early childhood deafness Dominant otosclerosls Circulation Monogenic hypercholesterolaemia slood Congenital spherocytosis Teeth Dentinogenisis imperfecta Amelogenisis imperfecta Skeleton Diaphysial aclasia Thanatophoric dwarfism Osteogenes imperfecta Marfan syndrome Achondroplasia Ehlers-Danlos syndrome Osteopetrosis tarda Cleft lip/palate Skin Ichthyosis Locomotor Muscular dystrophy A nucleic aci.d probe in accordance ~ith the present invention is a sequence which can determine the sequence of a test sample. The probes are usually DNA, RNA, mixed copolymers of ribo- and deoxyribonucleic ~ci.d5, oligonucl(~o~ides cor)~aining riborlucleotides or 131~7~4 deo~yribonucleotide residues or their modified forms.
The sequence of such a probe should be complementary to the test sequence. The extent of complementary properties will determine the stability of the double helix formed after hybridiæation. The probe can also have covalently linked non-complementary nucleic acids.
They can serve as the sites of the labeling reaction.
The nucleic acid is preferably labeled by means of photochemistry, employing a photoreactive DNA-binding furocoumarin or a phenanthridine compound to link the nucleic acid to a label which can be "read" or assayed in conventional manner, including fluorescence detection.
One use of the present invention is the identification of bacterial species in biological fluids.
In one application, samples of urine from subjects having or suspected of having urinary tract infections can provide material for the preparation of labeled DNA(s) or RNAs, while a solid support strip, e.g., made of nitrocellulose or nylon, can contain individual dots or spots of known amounts of denatured purified DNA from each of the several bacteria likely to be responsible for infection.
The format of labeled unknown and unlabeled probes, which is the converse of standard schemes, allows one to identify among a number of possibilities the species of organism in a sample with only a single labeling. It also allows simultaneous determination of the presence of more than one distinguishable bacterial species in a sample (assuming no DNA in a mixture is discriminated against in the labeling procedure).
I{owever, it does not allow in a simple way, better than an estimate of the amount of DNA (and, therefore, the concentration of bacteria) in a mixed sample. For such quantitation, sample DNA is immobilized in a series of 131~7~

dilution spots along with spots of standard DNA, and probe DN~s are labeled.
A urinary tract infection is almost always due to monoclonal growth of one of the following half dozen kinds of microorganism: Escherichia coli (60-90~ of UTI), Proteus spp. (5-20% of UTI), Klebsiella spp (3-10 of UTI), Staphylococcus spp. (4-20~ of ~TI), Streptococcus spp. (2-5% of UTI). Pseudomonas and some other gram negative rods together account for a low percentage of UTI. ~ common contaminant of urine samples that is a marker o~ improper sample collection is Lactobacillus.
The concelltration of bacteria in a urine sample that defines an infection is about 105 per milliliter.
The format for an unlabeleci probe hybridization system applicable to urinar~ tract infections is to have a matrix of DN~s from the above list of species, denatured and immobilized on a support such as nitrocellulose, and in a range of amounts appropriate for concentra~ions of bacterial DNAs that can be expected in samples of labelled unknown.
Standard hybridization with biotinylated whole genome DNA probes takes place in 5-10 ml, at a probe concentration of about 0.1 /ug/ml; hybridization oE probe to a spot containing about 10 ng denatured DNA is readily detectable. There is about 5 fg of DNA per bacterial cell, so that for a sample to contain 1 Jug of labeled DNA, it is necessary to collect 2 x 108 bacteria. If an infection produces urine having approximately 105 bacteria/ml, then bacterial DNA to be labeled from a sample is concentrated froM 2000 ml. If more than 10 ng unlabeled probe DNA is immobilized in a dot, for example, 100 nq or 1 /ug, or if the hybridization volume is reduced, then the volume of urine required for the - ~3~79~

preparation of labeled unknown is approximately a few tenths of a ml.
A strip o~ dots containing immobili~ed, denatured, unlabelled probe DNAs could have the following conLiguration:

1 ~Ig 10 ng 100 pg Escherichia o o o Proteus Klebsiella o o o Staphylococcus o o o Streptococcus o o o Pseudomonas o o o Lactobacillus o o o This procedure involves the labeling of DNA or RNA in a crude cell lysate. Ideally, preparation of labeled sample DNA or RNA will accommodate the following points:
(1) bacteria will be concentrated from a fluid sample by centrifu~ation or filtra-tion;
(2) bacteria will be lysed under conditions sufficient to release nucleic acids from the most refractory of the organisms of interest;
(3) the labeling protocol will not re~uire purification of labeled nucleic acids from unincorporated precursors, nor the puri'ication of nucleic acids prior to labeling;

(4) the labelinq protocol will be sufficiently specific for DNA and/or RNA that proteins, lipids and polysaccharides in the preparation ~Jill not interfere with hybridization nor read-out.
In the present invention, there is provided a method for efficiently and rapidly lysing whole cells, including Gram posi~ive bac~eria. The rnethod involves ~L31~7~

contacting cells, e.g., whole cells, with an alkali, e.g., sodium or potassium hydroxide solu~ion in a concentration of 0.1 to 1.6 Normal.
The important fea-tures of the present lysis protocol are its relative simplicity and speed. It employs a common chemical that requires no special storage conditions and it lyses even Gram positive organisms with high efficiency, while preserving the proper~ies of the DNA that are important for subsequent steps in the photochemical labeling process.
For the present invention, the i.mmobile phase of the hybridization system can he a series or matrix of spots of known kinds and/or dilutions of denatured DNA.
This is most simply prepared by pipet-ting appropriate small volumes of native DNA or oligonucleotides onto a dry nitrocellulose or nylon sheet, floating the sheet on a sodium hydroxide solution to denature the DNA, rinsing the sheet in a neutralizing solution, then baking the sheet to fix the DNA. Before DNA:DNA hybridization, the sheet is usually treated with a solution that inhibits non-speci~ic binding of added DNA during hybridization.
The invention will be further described in the following non-limiting examples wherein parts are by weight unless otherwise expressed.

Example 1: Preparation of ~abeling Compound The preparation of the labeling compound required 1-amino-17-N-(Biotinylamido)-3,6,9,12,15 pentaoxaheptadecane. This compound was prepared in the following four steps:
(a) 3,6,9,12,15 pentaoxaheptadecane 1,17-diol ditosylate was synthesized.
(~) 1,17-~.ipthalimido deriva~ive of 3,6,9,12, l5 pentaoxaheptadecane was prepared.

~ 3~7~

(c) 1,17-diamino derivative of 3,6,9,12,15 pentaoxaheptadecane was prepared.
(d) 1-amino, 17-biotinylamido derivative of 3,6,g,12,15 pentaoxahep~adecane was prepared.
xample l(a): Preparation of 3,6,9,12,15-Pentaoxahepta-decane-1,17-diol Ditosylate To a stirred solution containing 50 g of hexaetllylene glycol (0.177 mol) and 64 ml of triethylamine (39.36 g, 0.389 mol) in 400 ml of CH2C12 at 0C was added dropwise a solution containing 73.91 g of p-toluenesulfonyl chloride (0.389 mol) in 400 ml of CH2C12 over a 2.5 hour period. The reaction mixture was then stirred for one hour at 0C and then heated to ambient temperature for 44 hours. The mixture was then filtered and the filtrate was concentrated in vacuo. The resulting heterogeneous residue was suspended in 500 ml o~ ethyl acetate and filtered. The filtrate was then concentrated in vacuo to a yellow oil which was triturated eight times with 250 ml portions of warm hexane to remove unreacted p-toluenesulfonyl chloride.
The resulting oil was then concentrated under high vacuum to yield 108.12 g of a yellow oil (quantltative yield).
nalysis: Calculated for C26H38O11S2 Calc.: C, 52.87; H, 6.48.
found: C, 52.56; H, 6.39.

PMR: (60 MHz, CDC13) ~ : 2.45 (s, 6H); 3.4-3.8 (m~ 20H);
~.2 (m, 4H); 7.8 (AB quartet, J=8Hz, 8H).

IR: (neat) cm 1 2870, 1610, 1360, 1185, 1105, 1020, 930, 830, 785, 670.

13~7~

Example 1 (b): Preparation of 1,17 Diphthalimido-3,6,9,12,15-pentaoxaheptadecarle A stirred suspension eontaining 108 g of 3,6,9,12,15-pentaoxaheptadecane- 1,17-cliol ditosylate (0.183 mol), 74-57 g of potassium phthalimide (0.403 mol), and 700 ml of dimethylaeetarnide was heated at 160-170 C for 2 hours and was then coo].ed to room temperature. The precipitate was filtered and washed with water and acetone to yield 53.05 g of product as a white powder which was dried at 55C (0.1 mm) . mp 124-126C.
A second crop of product was obtained from the dimethylacetamid~ filtrate by cvaporatioJI in vacuo and the resultiny precipitate with was successively washed ethyl acetate, water, and acetolle. The resulting white powder was dried at 55C (0.1 mm) to yield an additional 9.7 g of product. mp 124.5-126.5C. The combined yield of product was 62:82 g (68~ yield) .
nalysis: (For first crop) Calculated for C28~332N29 1/2~320 Calc.: C, 61.19; H, 6.05; N, 5.09.
found: C, 61.08~ ll. 6.15; N, 5.05.

(For second erop) Calculated for C28H32N2O9 Calc.: C, 62.21; H, 5.97; N, 5.18.
found: C, 61.78; 13, 6.15; N, 5.13.

PMR: (60 MHz, dmso-d6) ~: 3.5 (s, 81-1); 3.6 (s, 8H); 3.8 (bt, J=3Hz, 8H): 8.1 (s, 811) .

IR: (KBr) cm : 2890, 1785, 1730, 1400, 1100, 735.

~L31 ~79~

xample l(c): Preparation of 1,17-Diamino-3,6,9,12,15-Pentaoxaheptadecane A solution containing 60 g of 1,17-diphthalimido-3,6,9~12,15-pentaoxaheptadecarle (0.118 mol), 14.8 g of hydra~i.ne hydrate (0.29G mol), and 500 ml of ethanol were hc-ated with mechanical stirring in a 100C oil bath for three hours. The mixture was then cooled and filtered. The resultant filter cake was washed four times with 300 ml portions of ethanol. The combined filtrates were concentrated to yield 32.35 g of a yellow apaque qlassy oil. The evaporative distillation at 150-200C (0.01 mm) gave 22.82 g of a light yellow oil (69% yield). lit. b.p. 175-177C (0.07 mm).

PMR: (60 MHz, CDC13) ~ : 1.77 (s, 4H, NH2);
2.85 (t, J=5Hz, 4H); 3.53 (t, J=5Hz, 4H); 3.67 (m, 16H).

IR: (CHC13) cm : 3640, 3360, 2860, 1640, 1585, 1460, 1350, 1250, 1100, 945, 920, 870.

Mass Spectrum: (EI) m/e = 281.2 (0.1%, M+1).
(FAB) m/e = 281.2 (100%, M+1).

12 28 2 5-1/2 H~O
Calc.: C, 49.80, I1, 10.10; N, 9.68.
~ound: C, 50.36; II, 9.58; N, 9.38.

Literature Re~erence: W. Kern, S. Iwabachi, H. Sato and V. Bohmer, Makrol _Chem., 180, 2539 (1979).

ple l(d): Preparation of_1-Amino-17-N-(Bi.otinyl-amido?-3 ! 6,9,12,15-pent:aoxaileptadecane . _ A solution contalning 7.2 y of 1~l7-dia~.no-3~9~l2~]s-p~ntaoxaheptadecane (25 rnmol) in 75 ml of DMF under an aryon atmosphere was treated with 13~ ~7~

3.41 g of N-succinimidyl biotin (10 mmol) added in portions over 1.0 hour. The resulting solution was stirred for four hours at ambient temperature. TLC
(SiO2, 70:10.1 CHCL3-CH3OH-cone. NH4 OH) visualized by dimethylaminocinnamaldehyde spray reayent showed exeellent conversion to a new product (Rf=0.18). The reaction mixture w~s divided in half and each h~lf was absorbed onto SiO2 and flash-ehromatographed on 500 g of SiO2-60 (230-400 mesh) usiny a 70:10.1 CHCl3-C~3O~I-eonc.
NH40H solvent mixture. Fractions eon~aining the product were polled and concentrated to a yield 2.42 g of a gelatinous, waxy solid. The product was precipitated as a solid from isopropanol-ether, washed with hexane, and dried at 55C (0.1 mm) to give 1.761 g of a white powder (35~ yield).
nalysis: Calculated for C22H42N4O7S.3/2 H2C:
C, 49.51; H, 8.50; N. 10.49.
found: C, 49.59; H, 8.13; N, 10.39.
MR: (90 MHz, dmso-d6)~ : 1.1-1.7 (m, 6H)i 2.05 (t, J=711z, 2H);
2.62 (t, J=4Hz, lH); 2.74 (t, J=4Hz, lH)i 3.0-3.4 (m, 14H).
3.50 (s, 141-1); 4.14 (m, lH); 4.30 (m, lH); 6.35 (d, J=4Hz, lH); 7.80 (m, lH).

CMR: (22.5 MHz, dmso-d6)~ : 25.2, 28.0, 28.2, 35.1, 40.6, 55.3, 59.2, 61.1, 69.6, 69.8, 71,2, 162.7, 172.1.

IR: (KBr) cm : 2900, 2350, 1690, 1640, 1580, 1540, 1450, 1100.

Mass ~,pectrurn (FA13) m/e: 507 . 3 (M~l, 56% ) ... . .. .. _ . ., .... , .. . __ .. ... . _ .. _._.. ... ..... _ .-- .. . .... _ _ .. _ .. _ __ _ .__., ._.__ _ . _ _ ~3~47'~

Example 2: Preparation of 4'-Biotinyl-PEG-4,5'-dimethylangelicin -A solution of 203 mg of 1-amino-17-N-~biotinyl-amido)-3,6,9.12,15-pentaoxaheptadecane (0.4 mmol) in 1 ml of DM~ under an argon atmosphere was trea~ed with 78 mg of N,~-carbonyldimidazole ~0.48 mmol). Tl-e resulting mi~ture was stirred for four hours and was then treated with 55 mg of 4'-aminomethyl-4,5'dimethylingelicin hydrochloride (0.2 mmol), 140 ~ll of diisopropylethyl-amine, and 100 ~1 of DMF`. The resulting mixture ~as stirred overnight at 50C. The mixture was then evaporated onto SiO2 in vacuo and the resultant impregnated solid flash was chromatographed on 60 g.of SiO2 (230-900 mesh) eluted with 1.5 liters of 7%
CH3-CHC13 followed by 1 liter of 10~ CH3OH-CHC13.
Fractions containing the product were pooled and concentrated to yield 72 mg of a glassy solid (47 yield).

PMR: (90 MH~, dmso-d6):~ 1.1-1.8 (m~ 6H); 2.04 (bt, J=7Hz, 2H); 2.5 (s, 6H); 2.56 (m, lH); 2.74 (bd, J=4Hz, lH); 2.8-3.4 (m, 14H); 3.40 (m~ 14H); 4 14 tm, lH); 4.25 (m, lH); 4.40 (bd, J=6Hz, 211); 6.5 (m, lH); 6.35 (s, lH); 7.02 (s, lH); 7.45 (d, J=8Hz, lH); 7.62 (d, J=8Hz, lH); 7.80 (m, lH).

C~lR: (22.5 MHz, dmso-d6) ~ : 11.9, 18.9, 25.3, 28.2 28.3, 33.4, 35.2, 55.4, 59.2, 61.0, 69.2, 69.~, 69.8, 70.0, 89.0, 107.8, 112.0, 113.1, 114.3, 120.6, 121.6, 153.6, 154.4, 155.6. 157.9, 159.5, 162.7, 172.1.

Literature Refer~nce: F. Dall'Acqua, D. Vedaldi, S.
Caffieri, ~. Guiotto, P. Rodighiero, F. Baccichetti, F.
Carlassare and F. Bordin, J. Med Chem., 24, 178 (1981).

.. ....... ~.. __ .... .. . ..... ~ .__ .____ _ 131479~

Example 3: Colorimetric or Chemiluminescent Detection of the Nuclei.c Acid Hybrids E~ample 3(a): Colorimetrlc Detection ____ Colorimetric detection of the biotinylated hybrids is carried out following the procedure and kit developed ~y Bethesda ~esearch Laboratories (B~L), Gaithersburg, Maryland 20877, U.S.A. The procedure is descrihed in detail in a manual supplied with a kit by B~L, entitled "Products for Nucleic Acid Detection", "DNA
Detection System Instructlon Manual", Catalogue No.
8239SA.
.

Example 3(b): Chemiluminescent Detection . . .
Chemiluminescent detection of the biotinylated hybrids is identical to the above method: the filters with the hybrids are saturated with BSA (bovine serum albumin) by immersing the paper in 3% BSA at 42C for 20 minutes. Excess BSA is removed by taking the paper out of the container, and blotting it between two pieces of filter paper. The paper is then incubated ln a solution containing Streptavidin (0.25 mg/ml, 3.0 ml total volume), for 20 minutes at room temperature. It i5 then washed three times with a buffer containing 0.1 M
Tris-HCl, p~l 7.5, 0.1 M NaCl, 2 mM MgCl2, 0.05% "TRITON
X-100". Next the filter is incubated with biotinylated horseradish peroxidase (0.10 mg/ml) for 15 minutes at room temperature. This is ~ollowed by three washings with 0.1 M Tris-HCl, pH 7.5, 0.1 M NaCl, 2 mM MgCl2 and 0~05% Triton X-100, and one washing with 10 mM Tris (pH
8.0) buffer. Chemiluminescent activation is conducted in two ways. (1) Spots are punched out and the discs containing the DNA are placed in a microtiter plate with wells that are painted black on the sides. After the punched paper circ].e.~ arc placed in the microtiter plate `b~

~3:L~7~

wells, 0.8 ml buffer containing 40 mM Tris and 40 mM
ammonium acetate (pH 8.1) is added to each well. Then 10 ~1 of 1:1 mixture of 39 mM Luminol (in DM~) and 30 mM
~2~ (in wa~er) is added. Light emission is recorded on a "POLAROID" instant film by exposing it direc~ly in the film holder. Alterna~ively (2), the paper is soaked in a solution containing 1:1 mixture of 0.5 mM Luminol and H202 and wrapped with a transparent "SARAN WRAP". The light e~ission is recoxded on a "POLAROID" film as above.

Example 4: General Method of Labeling the Test Sa~ple Nucleic Acids .. .. . .
Hi~h molecular weight DNA from a patient's sample is isolatecl by a method described in U.S.P.
4,395,486 (Wilson et al), _ The nucleic acid is dissolved in 10 mM borate buffer (p~l 8.0) to a final concentration of approximately 20 ~g/ml. To the nucleic acid solution "angelicin-peg-biotin" in aqueous solution is added to a final concentration of 10 ~g/ml. The mixture is then irradiated at long wavelength irradiation for about 60 minutes using a black ray UVL 56 lamp. The product is ready for hybridization without purification.
~owever, the product can be purified by dialysis or alcohol precipitation (~.S.P. 4,395,486) as is usually followed for nucleic acids.
Instead of nucleic acids, whole cell lysate can also be labeled following an identical procedure. The lysis is conducted by boiling the cells with 0.1 N sodium hydro~ide, followed by neutralization with hydrochloric acid.
When whole cells are used, the mixture of "PEG-ang-bio" and cells are incubated for at least 60 minutes prior to irradiation for effi.cient tran~port of *Trade Mark A

7 ~ ~

the ligands Many different variations of the above described methods can be adopted for labeling.

E~ample 5:
Alpha-thalassemia is associated with gene deletion. The detection of gene deletion by hybridization in a dot/slot blot format requires that the total amount of sample and its hybridizability are accurately known.
Since the beta-globin gene is a slngle copy gene, simultaneous hybridization of a sample with beta-globin and alpha-globin and their relative amounts will indicate the amount of alpha-globin with the sample.
The format and hybridization conditions are the same as Rubin and Kan, supra, except probes, not test VNA, is immobilized. }Iybridization conditions are also similar. The detection is done by using the BRL kit described supra following BRL's specifications.
The hybridization detection process are conducted in three steps as follows:

Step 1: Immobilization of the Probes As described in Rubin and Kan, supra, 1.5 kb PstI fragment containing alpha2 globin gene is used as a probe for alpha-thalassemia and for the beta-globin gene a 737 base pair probe produced by the digestion of pBR
beta Pst (4.4 kb) is used. The beta-globin gene probe has been described in U.S.P. 4,395,486 (column 4). For the detection of gene deletion related -to alpha-thalassemia, the amount of starting nucleic acid, hybridization efficiency and control samples are needed.
The present invention avoids these problems by simultaneous hybridization with a single copy essential gene (e.g., beta-globin gene) when simi.lar amounts of probes are immobilized side by sid~, labeled sample is hybri.dized, ~ tive strength of signal intensity is a ~3~ ~7~

measure of relative amount of gene dosage present in the sample.
The probes (0.5, 1, 3 and 5 ~ per 100 ~1) are suspended in 10 m~ tris HC1 ~pH 7) buffer, denatured with 20 ~1 3 M sodium hydroxide, at 100C, for 5 minutes, an equivalent volume of 2 M ammonium acetate, pH 5.O is added to neu~ralize the solution, immediately after neutralization the probes for beta- and alpha-globin genes are applied in parallel rows to nitroc~llulose filter paper under vacuum in a slot blot manifold, purchased from Scleicher and Schuell, ~Keene, New Hampshire, U.S.A.). The filter is then dried in vacuum at 80C for 60 minutes. It is then prehybridiz~d for 4 hours in a mixture containing 50 mM sodium phosphate (pH
7) 45 mM sodium citrate, 450 mM sodium chloride, 50%
~v/v) formamide, 0.2% each ~w/v) of polyvinyl pyrrolidine, "FICOLL 400" and bovine serum albumin and 0.2 mg/ml alkali boiled salmon sperm DNA and 0.15 mg/ml yeast RNA.

Step 2: Lab_ling of the Test Sample This was described above.

Step 3: Hybridization The nitrocellulose strip containing the immobilized probes are hybridized with the labeled test sample in plastic ba~s (e.g., "SEAL-A-MEAL~', "SEAL and SAVE'', etc.J. ~Iybridization solution is the same as prehybridization solution plus 10% dextran sulphate.
Hybridization is done at 42C for 16 hours. After hybridization detection of biotin is conducted with a kit and procedure supplied by ~ethesda Research Laboratory, Maryland, U.S.A., (catalogue No. 8239SA). Results of relative intensity of alpha- and beta- regions are used to estimate the exterlt o~ dele~ion o~ alpha-globin genes:

*Trade Mark .. ..... .... .. ........ , ..... , ~ ....... . ... ... . ... . .. .

l3~-~r~

No signal on the alpha-globin side: all 4 alpha-globin genes missinq.
Signal on the alpha-globin side is half as stronq as on the corresponding beta-side: 3 alpha-globin genes missing.
Signals on alpha and beta side equivalent: 2 alpha-globin genes missing.
Signals on alpha side is s~ronger than the corresponding ~eta side (2 alpha = 3 beta):
alpha-globin gene missing.

Example 6: Immobilization of an Oli~onucleotide Sequence Specific for Hemoq]obin Mutation It is known that an oligonucleotide cannot be easily immobilized onto nitrocellulose paper by a simple adsorption process. The present invention encompasses three different methods to incorporate an oligonucleotide sequence into a larger molecule capable of adsorption.

~ethod 1: Two oligonucleotides, one a 43mer and the other a 16-mer, have been chemically synthesized in an automated synthesizer (Applied Biosystem 3gOB) by the phosphoramidite-method and phosphorylated at the 5' end by a T4-polynucleotide kinase mediated process according to Maniatis et al, Molecular Clonlng, page 122. These oligonucleotides contain a segment of a 19 nucleotide long sequence specific ~or the detection of the mutation associated with sickle cell anemia.
43mer A & S (A = normal globin gene; S = sickle globin gene) were kinased according to Maniatis et al, Molecular Clonlng, page 122, in two separate reactions, namely, one with 3 P-ATP and one with no radioactive label. 0.4 ~g 32P-43mer and 0.6 mg cold 43mer were mixed and purified on a spun column (G-25med in TE (Tris EDI`A
buf~er~) to ~ ~inal volume of ~O Jul. I'wo dilutiolls were ~3~7~

spotted on S & S (Schleicher & Schuell) nitrocellulose and nytran (nylon) membranes at 50 and 0.5 ng.

Method 2: The phosphorylated oligonucleotide products of method 1 were further elongated by making multimers of sequences by a ligase mediated process. The principle is described as follows:

(X) . _ . . . _ 3' 5' 3' 5' ligase \l /
. .

3' 5' strand separation \ , . _ _ 3' 5' .
(X) The product being o~ a higher molecular weight than an oligonucleotide it should be immobiliza~le by adsorpt:ion on to a nitrocellulose paper.

~3~79~

Aqueous soluti.ons containing 4~g of 32P43mer and 3.7 lug 16mer linker (X) were mixed and dried under vacuum. 6 mg of cold kinased 43mer was added and the sample was heated to 55C and cGoled slowly to 0C to anneal. Liqation was carried ou-t in 20 ~l total reaction volume with 800 units of ligase (Pharmacia) at 15C for 4 hours. 1 mg (2 ~l) was purified on a spun column (G-25med in T~) to a final volume of 40 ~l. Two dilutions were spotted on nitrocellulose nylon membranes at 50 and 0.5 ng.

~1ethod 3: The same as method 2, but ligation was not conducted. Instead of liyation, cross linking was conducted with an intercalator to keep the double stranded regions intact. Hence, the cross linked molecule will have several oligonucleotide sequences covalently linked to each other.
2 lug of 32P43mer (for sequence P-50) was added to 2.9 mg of a l6mer (for sequence P-50) linker and purified on a spun column (G25med in TE) to a final volume of 40 ~l. 6 mg of kinased 43mer was added and the samples were heated to 55C and cooled slowly to 0C to anneal. 25 ~1l of intercalation compound aminomethyltrioxsalen was added and the sample was irradiated for 30 minutes on ice in 500 ~l total 10 mM
borate buffer pH 8.2 with a long wave UV lamp model (UVL-21, ~ = 366 nM).
The probes modified by all three methods were then immobilized on to nitrocellulose and nylon paper and hybridized with labelled oliyonucleotides. The results indicate that the sequence are immobilizable and hybridization fidelity remains intact.
Two dilutions of the products of rnethods 1 to 3 were spotted on nitrocellulose and nylon membranes at 50 and 0.5 ngs.

. . ~, . .:.;
.,.. ; - ~
..... :. `:.. .

1 3:~7~

Whole filters were baked for 30 minutes in 80C
vacuum oven and prehybr-dized in blotto ~5~ nonfat dry milk, 6XSSC, 20 mM Na-pyrophospllate) for 30 minutes in 50C oven.
Hybridization was carried out with primer extended l9'A ~ l9S' probes at 50C for one hour (3 strips/probe).
Filters were s-tringently washed for 15 minutes at room temperature in 6XSSC with slight agitation and 2 x 10 minutes at 57C.
Air-dried filters were place on Whatman paper and autoradlographed at -70C overnight.
The results presented in Fig. 1 surprisingly indicate specific hybridization are obtained by immobilizing oligonucleotide probes.

Example 7: H~/bri~ization with labeled gerlomic DNA for ~on ~adioactive Detection .
Human normal genomic (~X) DNA was photolabeled with "biotin-PEG-angelicin" (BPA) in 10 mM borate buffer pH 8.2 at a weight ratio of 0.3 to 1 (~PA:DNA) for 15 minutes on ice with a long wave UV lamp model UVL-21, J~= 366 nm. No puri~ication is necessary.
Target DNA oligonucleotides were directly immobilized on S & S nitrocellulose in 1 ~1 aliquots at the following concentratioll, and then baked in an \30C
vacuum oven for 30 minutes. The amounts of the different in~obilized probes are as follows:
.3-mer (A) - Kinased (method 1) 200 ng 43-mer (A) 200 ng 43-mer (S) - Kinased (method 1) 200 ng 43-mer (S) 200 ng ~11319Ass 50 ng M1319Sss 50 ng M]3737~ss 50 ng 1 3~ 7~4 sRL Co~nercially biotinylated DNA 200 pg pUC19 50 ng 43-merA: 5' GGAT3AAT4CTCCTGAGGAGAAGTCT
GCT4AATCTTAA 3' * =T for 43-mer S
16-mer (Common to both A and S) 3' - TTAGAATTCCTAAATT-5' Filters were prehybridized in blotto (5% nonfat dry milk, 6XSSC, 20 mM Na-pyrophosphate) for 30 minutes in a 45C H2O bath.
All 4 strips were ilybridized in 2 mls solution containing 2 /ug laheled XX DNA containing normal beta-ylobin gene (hybridization solu~ion was blotto with 10~ PEG) for 2 hours in 45C in a 112O bath.
A stringency wash was carried out as follows:
1 X 20' at room temperature in 6XSSC
2 X 20' at temperatures indicated in Fig. 2 witii very little agitation.
50 ml centrifuge tubes were used for elevated temperature washes. ~ecults are shown in Fig. 2.
Detection of biotin in the hybrid was carried out according to the Bethesda ~esearch Laboratory, Bethesda, Maryland, ~.S.h., manual using their kit for biotin detection. The results indicated specific hybridization.

E~ample_8: Immobilization of Whole Genomic_DNA As Probes Tens of milligram to gram amounts of DNA were prepared in the following manner ~rom bacterial cells harvested from fermentor cultures. Bac-teria were collected by centrifugation from 10 liter nutrient broth cultures grown in a Ncw Brunswic~ Scientific Microferm Fermentor. Generally, cells in concen~rated suspension were lysed by exposure to an ionic dete~gent such as SDS
(~a dodecyl sulEate), then nucleic aci~s were puri~ie~
from proteins and lipids by e~traction with phenol and/or chloroform (J. Marmur, J. Mol. siol., 3, 208-218, 1961).
R~A was removed from the nucl~ic acids preparation by treatment of the DNA solu~ion with 0.2 mg/ml ribonuclease at 37C, then DNA was precipitated from solution by the addition of two volumes of ethanol. Bacterial DNA
redissolved from the precipitate in a low salt buffer such as TE (10 mM Tris-HCl, pH 7.5, 1 n~ Na2 EDTA) was characterized with respect to purity concentration and molecular size, then approximately 1 microgram aliquots were denatured and immobilized as spots on nitrocellulose or nylon membranes for hybridization (Kafatos et al., Nuclelc_Acids, Res. 7, 15~1-1552, (1979)). Denaturation was accomplished by exposure of the DNA with approximately 0.1 N NaOH. After ~enaturation the solution was neutralized, then the Inembrane was rinsed in NaCl/Tris-HCl, p~l 7.5, and dried.

Example 9: Processing of a Test Sample for Cellular DNA
Labeling Samples of urine, for example (although the following can equally apply to suspensions of material form gonorrhea-suspect swabs, from meningitis-suspect cerebrospinal fluid, frorn contamination-suspect water samples, etc.), are centrifuged or filtered to wash and concentrate any bacteria in the sample. The bacteria are then lysed by exposure to either (i) 2 mg~ml lysozyme or lysostaphin then exposure to approximately 90C heat, (ii) 0.1 to 1.6 N NaO~I, or (iii) 1~ Na dodecyl sulfate.
After (ii) NaOH, the cell lysate solution is neutralized before labelling; after (iii) detergent lysis, DNA
l~be]lincJ is preceded by removal o~ the SDS with 0.5 M K

7~4 acetate on ice. Angelicin should be able to permeake intact cells so that DNA labeling can be accomplished before cell lysis. This in situ labeling simplifies the e~traction procedure, as alkaline or detergent lysates can be incorporated directly into a hybridi~ation solution.
Prior to hybridi~ation, the labeled sample is denatured, and it should also preferably be reduced to short single stranded lengths to facilitate specific annealing with the appropriate unlabeled probe DN~.
~.ethods of denaturation are ~nown in the art. These methods include treatment with sodium hydroxide, organic solvent, heating, acid treatment and combinations thereof. Fragmentation can be accomplished in a controlled way be heating the DNA to approximately 80C
in NaOH for a determined length of time, and this, of course, also denatures the DNA.

Example 10: Labelin~ of the Products of Example 9 (i) A test sample of about 10ml urine will contain 104 or more infectious agents. After separation by centrifugation and washing, the pretreated cell lysate (step 2) was resuspended in 0.2 ml 10 mM sodium borate buffer (pH approximately 8). To this suspenslon, 10 ~g of photolabelling reagent dissolved in ethanol (10 mg/ml), was added and mixed by shaking on a vortex mixer.
The rnixture was then irradiated at 365 nm for 30 minutes with a UVGL 25 device at its long wavelength setting.
The UVGL device is sold by UVP Inc., 5100 Walnut Grove Avenue, P.O. Box 1501, San Gabriel, CA 91778, U.S.A.
(ii) The sample was also labeled with N-(4-azido-2-nitrophenyl)-N'-(N-d-biotinyl-3-aminopropyl)-N'-methyl-1,3-propanediamine (commercially available from BRESA, G.P.O. Box 498, Adelaide, South 131479~

~ustralia 5001, Australia), following the procedure described by Forster et al (19~5), supra for DNA.
(iii) When unlysed cells were used, the cell suspension in 0.~ ml 10 mM borate was incubatcd with the photoreagent for 1 hour prior to irradiation.
.Yample 11: Hybridization of the Products of Examples 8 and 10 Prior to hybridi~ation, the membrane with spots of denatured unlabeled probe DNA was treated for up to 2 hours with a "prehybridi~a~ion" solution to block sites in the membrane itself that could bind the hybridization probe. This and the hybridization solution, which also contained denatured labelecl sample DNA, was comprised of appro~imately 0.9 M Na , 0.1~ SDS, 0.1-5~ bovine serum albumin or nonfat dry milk, and optionally formamide.
With 50~ formamide, the prehybridization and hybri.dization steps were done at approximately 42C;
without, the temperature was approximately 68C.
Prehybridized membranes can be stored for some time. DNA
hybridization was allowed to occur ror about 10 minutes or more, then unbound laheled DNA was washed from the membrane under conditions such as 0.018 M Na (0.1 x SSC), 0.1% SDS, 68C, that dissociate poorly base paired hybrids. After posthybridization washes, the membrane was rinsed in a low salt solution without detergent in anticipation of hybridization detection procedures.
~ample_12: Detection of a Nucleic Acid Hybrid with Inunu ogold Affinity isolated goat antibiotin antibody ~purchased from Zymed Laboratories, San Francisco, California, U.S.A.) was adsorbed onto colloidal ~old (~0 nm) following the method described by its supplier (Jans:er, instruction booklet, Janssen Life Sciences ~ 3~7~

Products, Piscataway, New Jersey, U.S.A.) and reacted with hybridized bio~inylated DNA after blocking as in a colorimetric method. The signals were silver enhanced using a Janssen (B2340 BEERSE, Belgium) silver enhancement kit and protocol.

Example 13: Detection of Urinary Tract Infection ln a Urine Sample Urine samples were collected from a hospital where they were analyzed by microbiological methods and the results were kept secret until the hybridization diagnosis was conducted. Then they were compared ascertain the validity of the hybridization results.
1 ml of clinical sample (urine) suspected of UTI infection was centrifuged in a Brinkman micro centrifuge for 5 minutes. Then 0.1 ml of 1.2 N sodium hydroxide was added and the suspension was heated to 100C to lyse the cells. The suspension was then diluted to 1 ml with 10 mM sodium borate buffer p~ 8 and was neutralized with hydrochlorine acid to a p~; of 7. To the solution, 50 ~ug "biotin-PEG-anyelicin" (see Example 2) is added and the mixture was irradiated with a UVL 56 long wavelength UV lamp for 15 minutes. The irradiated sample (0.1 ml) was added to 3 ml 3XSSC o~ 5~ nonfat dry milk 10~ PEG with 0.2 M sodium pyrophosphate and hybridization was conducted with probes (whole genomic DNA) immobilized onto nitrocellulose paper at 68C for 5 minutes to overnight. After hybridization detection was conducted according to Examples 3 or 12, the spots or the photoyraphs were visually interpreted for the presence of specific bacteria in the test sample. A spot of human DNA was also present in the nitrocellulose paper for the detection of leucocytes. The presence of leucocytes was further verified wi-th a common method using "LEUKOSTIX"*
~Miles Laboratories, Elkhart, Indiana, U.S.A.).

~8 *Trade Mark 13~79~

Typical res~llts (Tables 1 and 2) indicate that the hybridization diagnosis produces similar results in a shorter time then the corresponding microbiological assays. The present invention not only provides information related to species identification, but also the le~cocyte content in a clinical sample.

DIAGNOSIS OF CLINICAL URINE S~PLES
_ _ * HOSPITALAPPLICANTS' HYBRIDIZATION DETECTION
DIAGNOSIS RESULTS SYSTEM
-NEG NEG GOLD
NEG NEG GOLD
NEG NEG GOLD
NEG E.c.-M C~E~
NEG E.c.-VW GOLD
NEG E.c.-VW GOLD
____________________~___________________________________ __________ S+, C- N~ GOLD
S+, C- E.c.-S CHEMI
S+, C- E.c.-S, Kl.-M CHEMI
S+, C- NEG GOLD
S+, C- NEG GOLD
S+, C- NEG GOLD
S+, C- E.c.-VW GOLD
S+, C- NEG GOLD
S+, C- E.c.-VW GOLD
S+, C- NE~ GOI,D
S+, C- NEG - GOLD
_________ _________________________________________________________ 100,000/mL E.c.E.c.-S GOLD
100,000/mL E.c.E.c.-S Cl~
100,000/mL E.c.E.c.-W GOLD
50,000/mL E.c.E.c.-M C~E~
50,000/mL E.c. NEG GOLD
E. coli E.c.-S, Kl.-M Cl~
E. coli E.c.-VS, Kl.-S CHEMI
E. coli E.c.-S, Kl.-S Cl~
E. coli/Klebsiella mix E.c.-S, Kl.-W GOLD
E. coli/Staph mix E.c.-S, St.-M CHEMI
__ _ _____________________________________________________________ ~9 ... . . _ ..... .... ..

79 ~

TABLE 1 (Continued) DIAGNOSIS OF CLINIGU,URINE SAMPLES
*~IOSPITALAPPLIC~S'~DIZATION DETECTION
DIAGNCSIS RFAS TS SYSTEM
~lebsiella spp. E.c.-M, Kl.-W CH~
100,000/mL K. pneumoniae E.c.-W, Kl-VW GOLD
Fnterobacter spp. NEG** GOLD
100,000 Candida NE~** GOLD
100,000/mL Proteus Pr.-S, E.c.-W GOLD
_____________________________________________________ _____________ 10,000/n~ Strep NEG ~E~I
Mixture of 3 unidentified ~n(-~) NEG GOLD
___________________________________________________________________ * diagnosis conducted ~y streaking urine on an agar plate and treating the plate under condltions so that the infectious organism can grow.

** Enterobacter/Candida probes not included in the hybridization assay, t:herefore, negative results are not surprising; given the high stringency conditiolls employed in the assay, cross-hybridizati.on with species related to Enterobacter was not detected.

Abbreviations: VS=very strong; S=strong; ~I=medium;
W=weak; and ~l=very weak hybridization signals;
GOLD=detection method according to Example 12;
CHEMI=chemiluminescent detection according to Example 3(b) Applicants' hybridization results represent the result of a subjective interpretation of the intensity of the hybridization signals obtained after detection. DNAs from the orqanisms listed in column two are the only ones for which ally hybridization signal was obtained. The panel of DNAs used for hybridization included E._coli ("E.c."), Klebsiella pneumoniae ("Kl"), Proteus vul~aris ("Pr"), Pseudornonas aer ~inosa, Sta~hylococcus epidermatis ("SE"), Streptococcus faecalis and Homo sapiens.

13~79~

COMPARISON OE` AMES LEUl<OSTIX ASSAY
WITH APPLICANT ' S ASSAY
. ~
"LEUKOSTIX" APPLICANTS ' l1YBRIDIZATION DETECTION
RESULT RESULT SYSTEM
. .
3+ VS GOLD
3+ S CHEMI
3+ S CHEMI
3+ M CHEMI
3+ M CHEMI
3 + S GOLD
3+ S GOLD
3 + VS GOLD
3+ VS GOLD
3 + VS GOLD
3+ VS GOLD
__________________________________~____________________ 2+ S CHEMI
2 + S CHEMI
2+ S (::HEMI
2 + S C HEMI
2+ S GOLD
2 + S GOLD
2+ S GOLD
2 + S GOLD
2+ S GOLD
____ ________________________________________________ 1+ S GOLD
1 + VS GOLD
1 + VW GOLD
TRACE/1+ M CHEMI
TRACE VS CHEMI
TRACE W GOLD
TRACE W GOLD
TRACE VS GOLD
_____________________ _________________________________ NEG S CHEMI
NEG S CHEMI
N EG M CHEMI
NEG VW GOLD
NEG VW GOLD
NEG NEG GOLD
N E(`, NL;'G GOLD
NEG W GOLD
NEG W GOLD
NEG W GOLD
_______________________________________________________ , 51 ~3~79~

~ The hybrldization results summarized in column 2 of Table 2 represerlt subjecti~e interpretations of the in~ensity of hybridization signal obtained whe;l labeled urine samples described in Table l were hybridized with genomic human DNA.
The "LEUKOSTIX" assay is a colorimetric reagent strip assay. Color development on the reagent strip is compared to a chart provided with the assay reagent strips and ranges frorn negative (no color development) to 3+ (very strong color development).

Example 14: Lysis of Cells A 1.0 mL aliquot of cell suspension was centrifuged and the cell pellet resuspended in 100/uL of unbuffered NaOH solution. The sample was then exposed to high temperature for a short time and then diluted to the original volume usir,g 10 mM borate buffer. The pH of the solution was then adjusted to neutral with HCl.
Table ~ shows the efficiency of lysis of two different Gram positive cocci, Star~hylococcus epidermidis and Streptococcus faecalis, at varying NaOH
concentrations at either 68C or 100'C. In this Example, the absorbance of the 10 mL aliquots at 600 nrn was recorded before centrifugation. After centrifugation, the cell pellets were resuspended in varying concentrations of NaOH (100 JuL) and duplicate samples of each exposed to 68C for 10 minutes or 100C for 5 minutes. Each sarnple was then diluted to 1.0 mL and the absorbance at 600 nm again recorcded. Since the beginning and ending volumes are identical, the beginning and ending absorbance at 600 nm provi.des a direct measurement of lysis efficiency.
Whereas Gram negative organisms lysed efficiently in as low as 0.1 N NaOI~, Table ~ shows ~ 3 ~ 4 clearly that ef~icient lysis is a function of both NaOH
concentration and temperature, such tha~ higher NaOH
concentra~ions are rec~uired as the lncubation temperature decreases. ~t 100C (maximum temperature at 1 atmosphere) a concentratioll of a-t least 1.6 N NaOH was required for efficient lysis of S. epidermidis and S.
faecalis. If lower temperatures are desirable or necessary, then higher concentrations of NaOH will be re~uired to maintain lysis efficiency.

FFICIENCY OF LYSIS OF GRAM POSITrVE BAC~RIA
AT VARIO~S CONC~l~ATIONS OF NaOH AT 68C and 100C
-Streptococcus faecalis 100C/5 Minutes 68C/10 Minutes [NaOH] OD600 PREOD600 POST%LYSISOD600 P~EOD6 POST %LYSIS
0 N 0.475 0.366 23 0.512 0.357 30 0.1 .509 .261 50 .513 .238 54 0.2 .512 .194 62 .514 .259 50 0.4 .504 .175 65 .513 .150 71 0.8 .506 .113 78 .505 .147 71 1.2 .498 .082 84 .498 .150 70 1.6 .487 .061 88 .426 .099 77 Staphylocuccus epideL~idis 100C/5Minutes 68C/10 Minutes [NaOH] OD600 PREOD600 POST%LYSISOD600 PREC)D600 POST %LYSIS
0 N 0.667 0~55S 16 0.690 0.560 19 0.1 .681 .396 42 .701 .441 37 0.2 .674 .296 60 .699 .414 41 0.4 .699 .183 74 .730 .309 58 0.8 ~705 .091 87 .715 .187 74 1.2 .680 .070 90 .719 .090 88 1.6 .693 .035 95 .660 .040 94 Example 15: Direct Labelill~ of Nucleic Acids in an Infected Urine Sample A set of clinical urine test samples was collected from a hospital. 1 ml of urine was deposited in a polypropylene tuhe (1.5 ml). To l:he urine 150 1 of 8 N-sodium hydroxide solution in wa~er was added. The ~L3:L~r~ ~4 mixture was maintained in a boiling water bath for five minutes. The alkaline suspension was then transferred to another tube containing 0.3 g of solid boric acid for neutralization. The mixture was shaken well by hand for mixing and solubillzation. To this mixture, 10 microgram of the photolabelillg reagent "bio-PEG-Ang", as used in E~ample 10 (i) was added (10 microliter of 1 mg/ml in water). The mixture was then irradiated for 60 minutes using a hand held UV lamp (WL25) at a long wavelength setting. The tube was kept Oll ice during irradiation.
A nitrocellulose paper strip containing immobilized unlabeled genomic D~A probes (as used in Example 8), 700 microliter mixture containing 5% non-fat dry mil~, 10% polyethylene glycol (MW-6000), lOmM sodium pyrophosphate, all in 3 x SSC and 300 ~1 labeled test samples were placed in a seal-a-meal bag. The bag was heat sealed. Hybridi~ation was then conducted for two hours at 6~C by incubating tlle sealed bag in a water bath.
~ fter hybridization, the nitrocellulose strip was washed at 68C with 0.1 x SSC, 0.1% ~DS for 30 minutes. The unsaturated sites were then blocked by immersing the paper in 3% BSA solution in 0.1 mM 'crls, 0.1 M sodium chloride, 2 m~ magnesium chloride for 30 minutes. The hybrid was then de'cected by either immunogold, or by chemiluminescence or by the BRL method (see Examples 2, 3(a) or 3(b)). For every sample, the diagnosis was also done by bacteriological growth methods. The results were then compared for validity of the present method.
It will be appreciated that the instant specification and claims are set forth by way of illustration and not limitation, and that various modifications and changes may be made without departing from thc spiri.t an~ scope of the present i.nvention.

5~

Claims (11)

1. A method for detecting at least one polynucleotide sequence from a microorganism or eucaryotic source in a biological test sample, comprising the steps of:
(a) without prior centrifugation or filtration treating said biological test sample to lyse cells and to release nucleic acids therefrom, (b) without purification or isolation of specific nucleic acid sequences, specifically labeling released sample nucleic acids directly in the lysate produced in step (a), (c) contacting the resulting labeled nucleic acids, under hybridization conditions, with at least one immobilized oligonucleotide or nucleic acid probe hybridizable with the sequence or sequence to be detected, thereby to form hybridized labeled nucleic acids, and (d) assaying for the hybridized nucleic acids by detecting the label.

2. The method according to claim 1, wherein the biological test sample is selected from urine, blood, cerebrospinal fluid, pus, amniotic fluid, tears, sputum, saliva, lung aspirate, vaginal discharge, stool, a solid tissue sample, a skin swab sample, a throat swab sample and a genital swab sample.

3. The method according to claim 1, wherein said labeling is effected by photochemically reacting a labeled form of a nucleic acid binding ligand with the sample nucleic acids.

4. The method according to claim 1, wherein the nucleic acids released from the sample are in double-standard form and, prior to step (c), the labeled nucleic acids are denatured to provide labeled single-stranded nucleic acids.

5. The method according to claim 1, wherein the biological sample is a eucaryotic source selected from algae, protozoa, fungi, slime molds and mammalian cells.

6. The method according to claim 1, wherein the biological sample is a microorganism selected from Escherichia, Proteus, Klebsiella, Staphylococcus, Streptococcus, Pseudomonas and Lactobacillus.

7. The method according to claim 1, wherein lysis in (a) is effected by treatment with alkali.

8. The method according to claim 1, wherein the label in (b) is selected from a protein binding ligand, haptens, antigen, fluorescent compound, dye, radioactive isotope and enzyme.

9. The method according to claim 1, wherein in (c) the probe is immobilized by covalent coupling or adsorption to a solid surface.

10. The method according to claim 9, wherein the probe is immobilized in the form of at least one dot on a solid support strip.

11. The method according to claim 1, wherein in (c) the hybridization is accelerated by the addition of polyethylene glycol.