AU652214B2 - Strand displacement amplification "Sealing Clerk - sealing is to be refunded" - Google Patents

Description

I

.1 -1- 221

AUSTRALIA

Patents Act 1990 BECTON DICKINSON AND COMPANY

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT 909C 00 00 909 9 9 0 9 000 1 C 00 Invention Title: 09 0 0 99 00 dO C~ 09 iCC C) Ci OCtiC 00 9 9 0 09 99 0 990 9 090909 STRAND DISPLACEMENT AMPLIFICATION The following statement is a full description of this invention including the best method of performing it known to us:r

-IA-

Field of the Invention This invention relates to a method for amplifying a target nucleic acid sequence, and more particularly relates to a method for amplification by endonuclease mediated strand displacement and detection of the amplified reaction product(s). This invention further relates to a commonly assigned application for exonuclease mediated strand displacement amplification filed of even date herewith.

o o Background of the Invention S"4 Nucleic acids may be either in the form of deoxyribonucleic acids (DNA) or in the form of ribonucleic acids (RNA). DNA and RNA are high molecular weight polymers formed from many nucleotide building blocks. Each nucleotide is composed of a base (a purine or a pyrimidine), a sugar (either ribose or deoxyribose) and a molecule of phosphoric acid. DNA is "o composed of the sugar deoxyribose and the bases adenine guanine cytosine and thymine 9°°o The nucleotides are assembled into a linear chain to form the genetic code. Each sequence of three nucleotides can be "read" as the code for one amino acid through the process of o" translation. (DNA must first be converted into RNA through the process of transcription.) By varying the combination of bases in each three base sequence, different amino acids are coded for. By linking various three base sequences together, a sequence of amino acids can be made which form proteins. The entire coding unit for one protein is referred to as a gene.

There can be one or more copies of a gene in an organism. Some genes are present in hundreds or thousands of copies. Others are present only as a single copy.

Regardless of the number of copies, genes are linked together in an organism to form higher structural units referred to as chromosomes in higher organisms. In some lower organisms, genes may occur in extra chromosomal units referred 1 -2to as plasmids. Genes need not be linked directly to each other in an end-to-end fashion. Certain non-coding regions sequences of bases that do not translate into amino acids) may occur between genes or within a gene. Thus, the arrangement of nucleotides in an organism determines its genetic makeup which may be referred to as its genome. (Hence, DNA isolated from an organism is referred to as genomic DNA.) 00 DNA in most organisms is arranged in the form of a duplex oO°I wherein two strands of DNA are paired together in the familiar double helix. In this model, hydrogen bonds are formed between S A and T and between C and G on the paired strands. Thus, on one strand, the sequence ATCG will have on its complementary strand the sequence TAGC Both strands, however, contain the same genetic code only in a complementary base-paired manner. One could read, therefore, S either strand of DNA in order to determine the genetic sequence coded for.

For a further description of the organization, structure and function of nucleic acids, see Watson, Molecular Biology of the Gene, W.J. Benjamin, Inc. (3rd edit. 1977), especially ch.s 6-14.

Understanding and determining the genetic sequence of nucleic acids present in a sample is important for mdny reasons. First, a number of diseases are genetic in the sense that the nucleotide sequence for a "normal" gene is in some manner changed. Such a change could arise by the substitution of one base for another. Given that three bases code for a single amino acid, a change in one base (referred to as a point mutation) could result in a change in the amino acid which, in turn, could result in a defective protein being made in a cell. Sickle cell anemia is a classic example of such a 2

I

-3genetic defect caused by a change in a single base in a single gene. Other examples of diseases caused by single gene defects include Factor IX and Factor VIII deficiency, adenosine deaminase deficiency, purine nucleotide phosphorylase deficiency, ornithine transcarbamylase deficiency, argininsuccinate synthetase deficiency, beta-thalassemia, a antitrypsin deficiency, glucocerebrosidase deficiency, phenylalanine hydroxylase deficiency and hypoxanthine-guanine o oa phosphoribosyltransferase deficiency. Still other diseases, o ILO such as cancers, are believed to be caused by the activation, increase in copy number and/or removal of suppression of genes known to be present in the genome referred to as oncogenes.

SFxamples of oncogenes believed to be relevant to certain cancers include N-myc for neuroblastomas, retinoblastomas and small-cell lung cancers and c-abl for chronic myelogenous leukemia. For a further description of the relevance of Soncogenes to the diagnosis of cancers and for a listing of OQd specific oncogenes, see Weinberg, Sci. Amer., Nov. 1983, Slamon et al., Science, 224:256 (1984), U.S. Pat. No. 4,699,877 and c 4,918,162.

Second, in addition to changes in the sequence of nucleic S acids, there are genetic changes that occur on a structural level. Such changes include insertions, deletions and S* 5 translocations along a chromosome and include increased or decreased numbers of chromosomes. In the former instance, such changes can result from events referred to as crossing-over where strands of DNA from one chromosome exchange various lengths of DNA with another chromosome. Thus, for example, in 3 "normal" individual, the gene for protein might reside on chromosome 1; after a crossing-over event, that gene could now have been translocated to chromosome 4 (with or without an equal exchange of DNA from chromosome 4 to chromosome 1) and the cell may not produce X.

0/

C'

rl: nr* -4- In the instance of increased or decreased chromosome number (referred to as aneuploidy), instead of a "normal" individual having the correct number of copies of each chromosome two of each in humans [other than the X and Y chromosomes]), a different number occurs. In humans, for example, Down's syndrome is the result of having three copies of chromosome 21 instead of the normal two copies. Other aneuploid conditions result from trisomies involving chromosomes 13 and 18.

Q>«

1k oa Q 9 0 o Third, infectious diseases can be caused by parasites, microorganisms and viruses all of which have their own nucleic acids. The presence of these organisms in a sample of biological material often is determined by a number of traditional methods culture). Because each organism has its own genome, however, if there are genes or sequences of Snucleic acids that are specific to a single species (to several S related species, to a genus or to a higher level of S relationship), the genome will provide a "fingerprint" for that organism (or species, etc.). Examples of viruses to which this okO invention is applicable include HIV, HPV, EBV, HSV, Hepatitis B and C and CMV. Examples of microorganisms to which this invention is applicable include bacteria and more particularly include H. influenzae, mycoplasma, legionella, mycobacteria, chlamydia, candida, gonocci, shigella and salmonella.

In each example set forth above, by identifying one or more sequences that are specific for a disease or organism, one can isolate nucleic acids from a sample and determine if that sequence is present. A number of methods have been developed in an attempt to do this.

While it is critical that one or more sequences specific for a disease or organism be identified, it is not important to the practice of this invention what the target sequences are or ii C I I I L how they are identified. The most straightforward means to detect the presence of a target sequence in a sample of nucleic acids is to synthesize a probe sequence complementary to the target nucleic acid. (Instrumentation, such as the Applied BioSystems 380B, are presently used to synthesize nucleic acid sequences for this purpose.) The synthesized probe sequence then can be applied to a sample containing nucleic acids and, if the target sequence if present, the probe will bind to it to o o form a reaction product. In the absence of a target sequence to and barring non-specific binding, no reaction product will be formed. If the synthesized probe is tagged with a detectable label, the reaction product can be detected by measuring the amount of label present. Southern blotting is one example where this method is used.

A difficulty with this approach, however, is that it is not readily applicable to those instances where the number of o" copies of the target sequence present in a sample is low less than 10 In such instances, it is difficult to g" O distinguish signal from noise true binding between probe and target sequences from non-specific binding between probe and non-target sequences). One way around this problem is to increase the signal. Accordingly, a number of methods have been described to amplify the target sequences present in a sample.

One of the best known amplification methods is the polymerase chain reaction (referred to as PCR) which is described in detail in U.S. Pat. No.s 4,683,195, 4,683,202 and 4,800,159. Briefly, in PCR, two primer sequences are prepared which are complementary to regions on opposite complementary strands of the target sequence. An excess of deoxynucleosidetriphosphates are added to a reaction mixture along with a DNA polymerase Tag polymerase). If the -6target sequence is present in a sample, the primers will bind to the target and the polymerase will cause the primers to be extended along the target sequence by adding on nucleotides.

By raising and lowering the temperature of the reaction mixture, the extended primers will dissociate from the target to form reaction products, excess primers will bind to the target and to the reaction products, and the process is °1 f repeated.

ootO Another method for amplification is described in EPA No.

320,308, published June 14, 1989, which is the ligase chain reaction (referred to as LCR). In LCR, two complementary probe pairs are prepared, and in the presence of the target sequence, each pair will bind to opposite complementary strands of the target such that they abut. In the presence of a ligase, the two probe pairs will link to form a single unit. By temperature cycling, as in PCR, bound ligated units dissociate aa from the target and then serve as "target sequences" for ligation of excess probe pairs. U.S. Pat. No. 4,883,750 describes a method similar to LCR for binding probe pairs to a target sequence but does not describe an amplification step.

o° A still further amplification method is described in PCT a.a. Appl. No. PCT/US87/00880, published October 22, 1987, and is 5 referred to as the Qbeta Replicase method. In this method, a replicative sequence of RNA which has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase. The polymerase will copy the replicative sequence which then can be detected.

3o Still other amplification methods are described in GB Appl.

No. 2 202 328, published September 21, 1988, and in PCT Appl.

No. PCT/US89/01025, published October 5, 1989. In the former application, "modified" primers are used in a PCR like, -7template and enzyme dependent synthesis. The primers may be modified by labelling with a capture moiety biotin) and/or a detector moiety enzyme). In the latter application, an excess of labelled probes are added to a sample. In the presence of the target sequence, the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe.

Sog" Cleavage of the labell d probe signals the presence of the target sequence.

4 0 SFor all of the above-described methods, a variety of detection methods may be employed none of which is critical to the amplification method employed. One method is detect reaction products having a specific size via electrophoresis.

Another method is to radiolabel the primer sequence with 32, for example, and then to detect the radioactivity emitted by o the reaction products alone or in combination with 0 electrophoresis A further method is to chemically modify the primer by adding a receptor having a ligand 0'O hiotin-avidin), and enzyme alkaline phosphatase), a fluorescent dye phycobiliprotein) or a combination.

Another method is to develop a detection primer which will bind 0 4 0 4 to the reaction product and be extended in the presence of )A*4 j polymerase. The detection primer can be radiolabelled or chemically modified as described above. Many of these methods may be adapted to solid phase as well as solution systems. A number of these methods, as well as others, are described in U.S. Pat. No.s 4,358,535, 4,705,886, 4,743,535, 4,777,129, 4,767,699, and 4,767,700.

Each of the above-referenced amplification methods has one or more limitations. In most of the amplification methods, a key limitation is the requirement for temperature cycling to cause the Leaction products to dissociate from the target.

-8- This places a limitation on both the devices used to perform the method as well as on the choice of enzymes necessary to form the reaction products. Other limitations of these methods include production of RNA intermediates sensitive to endogenous nuclease degradation and difficulty in production of associated enzymes. Alternative methods to such existing amplification methods are desirable.

0 o 1 o Summary of the Invention ooI This invention provides for a method of amplification of a target nucleic acid sequence (and its complementary strand) in S a sample by endonuclease mediated strand displacement. The Smethod involves the steps of 1) isolating nucleic acids suspected of containing the target sequence from a sample, 2) generating single stranded fragments of target sequences, 3) adding a mixture comprising a nucleic acid polymerase, (b) deoxynucleosidetriphosphates including at least one substituted deoxynucleosidetriphosphate and at least one primer which is complementary to a region at the 3' end of a target fragment S and further wherein each primer has a sequence at the 5' end which is a recognition sequence for a restriction endonuclease, and 4) allowing the mixture to react for a time sufficient to generate reaction products. Where the fragments comprise double stranded nucleic acids, the method further comprises denaturing the nucleic acid fragments to form single stranded C target sequences. Where the nucleic acids comprise RNA, it is preferable to use reverse transcriptase to convert RNA to DNA.

The invention further relates to methods for the separation 3 and/or detection of reaction products generated by the above-described method. Methods for separation comprise magnetic separation, membrane capture and capture on solid supports. In each method, a capture moiety may be uound to a magnetic bead, membrane or solid support. The beads, membrane i% -9or solid support then can be assayed for the presence or absence of reaction products. An example of a capture moiety includes a nucleic sequence complementary to the reaction products produced and an antibody directed against a receptor incorporated into the primer or reaction product. The separation system may or may not be coupled to a detection system.

°e Detection systems useful in the practice of this invention oo 10 comprise homogeneous systems, which do not require separation, and heterogeneous systers. In each system, one or more detectable markers are used and the reaction or emission from the detection system is monitored, preferably by automated means. Examples of homogeneous systems include fluorescence polarization, enzyme mediated immunoassays, fluorescence energy transfer, hybridization protection acridinium luminescence) and cloned enzyme donor immunoassays. Examples of heterogeneous systems include enzyme labels (such as peroxidase, alkaline phosphatase and beta-galactosidase), fluorescent labels (such as enzymatic labels and direct fluorescence labels fluorescein and rhodamine]), chemiluminescence and bioluminescence. Liposomes or other sac like particles also can be filled with dyes and other ,,it detectable markers and used in such detection systems. In 5 these systems, the detectable markers can be conjugated directly or indirectly to a capture moiety or the reaction products can be generated in the presence of a receptor which can be recognized by a ligand for the receptor.

The invention still further relates to methods of generating reaction products which, by removing the recognition sequence at the 5' end, can function as probes. In this format, the above described method and steps are used to generate reaction products. The reaction products then may be I Y treated with a restriction enzyme to cleave the recognition sequence from the reaction product. In this manner, the recognition sequence is removed and the remaining reaction product comprises a probe which can be used in other systems.

In the presence of a single stranded target fragment, a primer will bind to its complementary target strand. In the do presence of polymerase, nucleotides and substituted nucleotides So-,o will be added to the 3' end of the primer along the remaining length of the target and nucleotides and substituted nucleotides will be added to the 3' end of the target along the primer sequence. The resulting double stranded product will have one sequence containing substituted nucleotides coupled to the 3' end of the target strand while the primer strand will have an unmodified sequence coupled 5' to an extended sequence complementary to the target sequence.

The endonuclease then cleaves the recognition sequence on the primer strand but does not cleave the complementary sequence on the target strand because its sequence contains the substituted nucleotides. The polymerase extends the 3' end at the nick and simultaneously displaces the downstream strand to the nick generating a reaction product complementary to the target strand.

The method also can function with two primers wherein one primer will bind to one strand of a target sequence and the other primer will bind to the complementary strand of the target sequence. When this embodiment is used, it will be apparent that each reaction product can function as a "target" for the other primer. In this manner, amplification proceeds logarithmically.

|i i -11- Brief Description of the Drawings FIG. 1 comprises a flow chart of the steps in an example of the method claimed in this invention for a single stranded DNA fragment.

FIG. 2 comprises a flow chart of the steps in an example of the method claimed in this invention for double stranded 0 4"" o genomic DNA.

a n o J Detailed Description ,0 In this invention, the sample may be isolated from any material suspected of containing the target nucleic acid sequence. For animals, preferably, mammals, and more preferably humans, the sources of such materials may comprise blood, bone marrow, lymph, hard tissues liver, spleen, kidney, lung, ovary, etc.), sputum, feces and urine. Other o< a sources of material may be derived from plants, soil and other materials suspected of containing biological organisms.

S' o& The isolation of nucleic acids from these materials can be done any number of ways. Such methods include the use of detergent lysates, sonicatibn, vortexing with glass beads and a l French press. In some instances, it may be advantageous to 1 4; purify the nucleic acids isolated where endogenous nucleases ari present). In those instances, purification of the nucleic acids may be accomplished by phenol extraction, chromatography, ion exchange, gel electrophoresis or density dependent centrifugation.

Once the nucleic acids are isolated, it will be assumed for purposes of illustration only that the genomic nucleic acid is DNA and is double stranded. In such instances, it is preferred to cleave the nucleic acids in the sample into fragments of between approximately 50-500bp. This may be done by a V- I -12restriction enzyme such as HhaI, Fok I or Dpn I. The selection of the enzyme and the length of the sequence should be such so that the target sequence sought will be contained in its entirety within the fragment generated or at least a sufficient portion of the target sequence will be present in the fragment to provide sufficient binding of the primer sequence. Other methods for generating fragments include PCR and sonication.

oo: The primers used in this method generally have a length of o 25 100 nucleotides. Primers of approximately 35 nucleotides are preferred. This sequence should be substantially homologous to a sequence on the target such that under high t stringency conditions binding will occur. The primer also should contain a sequence (toward the 5' end) that will be recognized by the endonuclease to be used in later steps. The recognition sequences generally, although not necessarily, are palindromic. The sequence selected also may be such that the 00 0 oo restriction enzyme used to cleave the fragments in the previous O0 step is the same as the endonuclease to be used in later steps.

o o Once target nucleic acid fragments are generated, they are denatured to render them single stranded so as to permit binding of the primers to the target strands. Raising the temperature of the reaction to approximately 95 0 C is a *:ar preferred method for denaturing the nucleic acids. Other methods include raising pH; however, this will require lowering the pH in order to allow the primers to bind to the target.

Either before or after the nucleic acids are denatured, a mixture comprising an excess of all four deoxynucleosidetriphosphates, wherein at least one of which is substituted, a polymerase and an endonuclease are added. (If high temperature is used to denature the nucleic acids, unless thermophilic enzym-us are used, it is preferrable to add the -13enzymes after denaturation.) The substituted deoxynucleosidetriphosphate should be modified such that it will inhibit cleavage in the strand containing the substituted deoxynucleotides but will not inhibit cleavage on the other strand. Examples of such substituted deoxynucleosidetriphosphates include 2'deoxyadenosine 2'-deoxyuridine 5'-triphosphate and 7-deaza-2'-deoxyguanosine .S It should be appreciated that the substitution of the deoxynucleosidetriphosphates may be accomplished after incorporation into a strand. For example, a methylase, such as M. Taq I, could be used to add methyl groups to the synthesized I strand. The methyl groups when added to the nucleosides are thus substituted and will function in similar manner to the S thio substituted nucleosides.

0 00 0 o It further should be appreciated that if all the 0 nucleosides are substituted, then the polymerase need not lack the exonuclease activity. The presence of the substituents throughout the synthesized strand will function to prevent such activity without inactivating the system.

o 900 0 As described for the selection of the recognition sequence incorporated in the primer, the selection of the endonuclease used in this method should be such that it will cleave a strand at or 3' (or to the recognition sequence. The endonuclease further should be selected so as not to cleave the 3o complementary recognition sequence that will be generated in the target strand by the presence of the polymerase, and further should be selected so as to dissociate from the recognition sequence at a reasonable rate. It need not be thermophilic. Endonucleases, such as HinC II which recognizes j 1 r -14the sequence 5'GTPyPuAC3', Ava I which recognizes the sequence 5CPyCGPuGPuG3', Fnu4H I which recognizes the sequence 5'GCNGC3' and Nci I which recognizes the sequence 5'CC(C,G)GG3' are particularly useful. All of these endonucleases are available from New England Biolabs.

t "7 ii 0n 0o 0O 0 00 Polymerases useful in this method include those that will initiate polymerization at a nick site and will polymerize the target commencing at a nick from the 5' end to the 3' end. The ,l0 polymerase should also displace the polymerized strand So downstream from the nick, and, importantly, should also lack any exonuclease activity. Polymerases, such as the Sklenow fragment of DNA polymerase I and the exonuclease deficient klenow fragment of DNA polymerase I (United States Biochemical) and a similar fragment from the Bst polymerase (BioRad) are useful. It should be appreciated that a o. :polymerase ordinarily having such exonuclease activity can be Sdeemed to "lack" such activity if that activity is blocked by Sthe addition of a blocking agent.

An additional feature of this method is that it need not be run at varying temperatures. Many amplification methods require temperature cycling in order to dissociate the target from the synthesized strand. In this method, a single temperature may be employed after denaturation has occurred.

The temperature of the reaction should be high enough to set a level of stringency that minimizes non-specific binding but low enough to minimize the time within which binding of the primer to the target strand takes place. 37 0 C has been found to be a 3xo preferred temperature.

Referring to FIG. 1, one example of this invention is set forth. In this example, the strand labelled P represents the primer and contains at the 5' end the sequence CCGGG which is

I,

C CO lit recognized by the endonuclease Nci I. The strand labelled T is the target sequence which has already been fragmented and rendered single stranded. In the method, the primer binds to the target and in the presence of polymerase, deoxynucleosidetriphosphates and athio substituted deoxycytosinetriphosphate, the primer is extended the length of the target while the target is extended through the recognition sequence. In the presence of the endonuclease Nci I, the primer strand is nicked between the C-G residues. In the 1,.0 presence of the polymerase lacking 5' to 3' exonuclease activity, the 3' end at the nick is extended, and downstream the primer strand is displaced from the target strand beginning at the nick to create a reaction product and a new strand is S synthesized. In summary fashion (not shown), the newly synthesized strand too will be nicked by the endonuclease and the polymerase then will displace this strand generating S another until either the reaction is stopped or one of the S reagents becomes limiting.

0 o LO Referring to FIG. 2, an example, is shown for a double S stranded restriction fragment target. Thus, instead of having one primer, there are two primers (P 1 and P2 one each being complementary to the two target sequences (T 1 and T2). The reaction then proceeds as described for FIG. 1. It VS is to be noted, however, that P1 will bind to the reaction product produced from T 2 while P 2 will bind to the reaction product produced from T 1 In this manner, amplification proceeds logarithmically.

The presence of the amplified target then can be detected by any number of methods. One method is to detect reaction products of a specific size by means of gel electrophoresis.

This method is particularly useful when the nucleotides used are labelled with a radio-label, such as 32 Other methods are labelled with a radio-label, such as P. Other methods IL: i -iu -i6include the use of labelling the nucleotides with a physical label, such as biotin. Biotin-containing reaction products can then be identified by means of avidin bound to a signal generating enzyme, such as peroxidase.

An additional embodiment of this invention is in the preparation of multiple copies of a single stranded nucleic acid sequence. It will be appreciated that the production of synthetic primers on a device like the Applied Biosystems 380B 'o instrument is time consuming and is not readily applicable to generation of primers having several hundred nucleotides. The method of endonuclease mediated strand displacement need not be used to amplify a signal to detect the presence of a target for diagnostic purposes. It also is readily applicable to the generation of multiple cop;is of a single stranded nucleic acid sequence for use as a primer in any system of detection.

o 00 SIn this embodiment, a single stranded nucleic acid sequence to be amplified is prepared. This can be made from a synthetic oQ sequence or genomic nucleic acids as described above. A reaction mixture similar to that describe above is prepared using a primer complementary to the nucleic acid sequence to be S. amplified. The reaction is then allowed to take place as above. The resulting reaction products will comprise the amplified copies of the nucleic acid sequence and can be used as primers.

In order to illustrate one embodiment of this invention, reference is made to the following examples.

3o Two primers were synthesized on an Applied BioSystems 380B instrument using phosphoramidite chemistry and 3'-amine-ON CPG columns (Clontech Laboratories) which incorporate a primary 1" r A.L V Y-UU U1te ytne ror pLUULH±ll iLL.YLIL. iC;_ja chromosome 1; after a crossing-over event, that gene could now have been translocated to chromosome 4 (with or without an equal exchange of DNA from chromosome 4 to chromosome 1) and the cell may not produce X.

-17amine at the 3' terminus. Nucleotides were ammonium deprotected and purified by denaturing gel electrophoresis.

The primer sequences were: SEQ ID NO: 1, and SEQ ID NO: 2.

0 0 Plasmid pBR322 (Boerhinger Mannheim) was serially diluted with 0.05 mg/ml E. coli DNA, 50 mM K acetate, 10 mM Mg acetate, l 1 mM DTT, 12.5 mM TRIS (pH 7.9) at 25°C. Twenty pl samples containing 1 pg E. coli DNA and various amounts of pBR322 were digested 3 hours at 37 0 C with 10 Units of Fok I (New England Biolabs). The Fok I digests of pBR322/E. coli DNA were IS diluted to 100 pl in the presence of 12.5 mM K acetate, 10 mM Mg acetate, 1 mM DTT, 12.5 mM TRIS (pH 7.9) at 25 0 C, 100 pg/ml BSA, 0.3 mM each of dATP, dGTP, TTP athiodCTP (Pharmacia) and 0.1 pM of each primer. One set of samples o underwent strand displacement amplification for 4 hours at O upon addition of 4 U 5'43' exonuclease deficient klenow p fragment of DNA polymerase I (United States Biochemical) and 48 U Nci I (N igland Biolabs). A second set of samples were run without the polymerase and without Nci I as unamplified I standards.

To detect the reaction products, a pBR322 specific detection probe, SEQ ID NO: 3, was prepared and was labelled with 32 P using polynucleotide kinase. Ten pl aliquots of the amplified and unamplified Fok I/pBR322/E, coli DNA samples were mixed with 2 1p of 1.8 pM 32 P labelled detection probe, 0.5 U/pl Tag DNA polymerase (United States Biochemical). Samples were heated for 2 min. at 95 0 C, 5 min.

at 50 0 C, quenched with 50% urea, and a portion was loaded onto a 10% denaturing polyacrylamide gel. The presence of amplified it s i- c

I?

1 -18reaction products was detected through extension of the 32P labelled detection probe to a length of 44 or 61 nucleotides.

Unamplified Fok I/pBR322 was indicated by extension to 41 nucleotides. Electrophoresis 32 P labelled bands were quantified by liquid scintillation counting subtracting appropriate background bands. The results are shown TABLE I.

TABLE I nii ooQ *1~ EI II 4 i* 04 4l 4 I t* ItIi DBR322 Molecules Amplified (±50 cpm) 3x10 8 3x10 7 3x10 6 3x15 52900 18200 5690 298 37 215 24 21 0

ND

Unamplified (±50 cpm) ND not determined As can be seen from TABLE I, as the amount of pBR322 DNA in the aliquot decreases, the number of counts per minute also decreases.

In another example, a synthetic nucleic acid target was constructed having the sequence of SEQ ID NO: 4. Primers for the amplification reaction using the restriction enzyme HinC II 3o (New England BioLabs) were synthesized to provide a 3'-NH 2 cap using 3'-amine-on CPG columns. The primer sequences used were: i i: i: 1 ii i .I -19- SEQ ID NO: 5 and SEQ ID NO: 6.

A probe for the detection of the reaction products was of the sequence! SEQ ID NO: 7. All synthetic sequences were synthesized on an Applied Biosystems 380B instrument as above, and were gel purified on 10% or 15% polyacrylamide gels containing 50% urea. Excised bands were electroeluted in 1/2X TBE buffer.

<f SEQ ID NO: 4 was diluted into 0.3 pM of the primers SEQ ID NO: 5 and SEQ ID NO: 6) to provide a final stock Sconcentration of 600,000 molecules of target/pl. This mixture was boiled for 3 minutes and placed at 37 0 C. Serial 4 fold dilutions of this stock solution were then prepared in the o presence of the primers. (In the control, only amplification primers were present.) 0D Twenty pi of the diluted stock target solutions were 4 6 added to a mixture to provide a final volume of 60 p1 and a final concentration of the following components: 20 mM TRIS (pH 7.2) at 25 0 C, 0.1 pM of the primer sequences, 20 mM ammonium sulfate, 50 mM KC1, 50 U HinC II, 5 U exo-klenow "5 polymerase (UNSN Biochemical), 1 mM DTT, 5 mM MgCl 2 and 300 -A pM each of 5'dCTP, 5'dGTP, 5'dTTP and R'athio-dATP. The amplification reaction was allowed to proceed at 37 0 C for 1 or 2 hours. In one reaction set, an additional 50 U of HinC II was added after 1 hour and the reaction was allowed to proceed for an additional hour.

At the end of the reaction times, a 10 pl aliquot of each mixture was placed on ice. To this 10 pl was added 1 p1 of a 1 pM stock solution of capture probe freshly labelled with

V

i 32. This mixture was boiled for 3 minutes and cooled to 37 0 C, whereupon 1 pl of 1 U/pl of Sequenase 2.0 (U.S.

Biochemical) was added. (This enzyme will polymerize the capture probe along the full length of any reaction product when the capture probe is bound to a reaction product.) This extension reaction was allowed to proceed for 15 minutes at 37 0 C. To this mixture was added an equal volume of loading dyes in 50% urea. Samples were boiled again for 3 minutes before loading onto a 10% polyacrylamide gel containing .o urea. Samples loaded on the gel represented 2.5 pi of the t original 60 pl reaction mixture. Electrophoresis was allowed to proceed for 1 to 1.5 hours at 59 W after which the gel was S removed and placed on film overnight at -70°C. Bands were rendered visible after exposure, were excised and quantified by liquid scintillation.

oo d TABLE II Target 1 Hour 2 Hour 2 Hour with O Additional HinC II (cpm) (cpm) (cpm) 0 0 0 0 2000 ND 2 8 8000 4 12 36 30,000 37 78 129 125,000 175 196 746 500,000 824 1858 2665 Referring to TABLE II, it can be seen that the two hour reaction time is sufficient to allow this system to distinguish between 0 reaction products and 2000 reaction products.

Presently, a one hour reaction time is st;,ficient to allow this system to distinguish between 0 reaction products and 8000.

Y i i; 1 -21- In still another example, the following primer sequences were used: SEQ ID NO: 8 and SEQ ID NO: 9.

These sequences were generated as in the other examples and were used to detect a target sequence in the plasmid pBR322.

One pg of pBR322 was digested for 2 hours at 37 0 Q with 8 U of Fok I, and then was serially diluted with 0.05 mg/ml Shuman placental DNA, 50 mM KC1, 5 mM MgCl, 20 mM HQ)2SO 4 1 mM DTT and 20 mM TR S (pH 7N2 at 25 C)N Ten pl samples containing 0.5 pg human placental DNA and various amounts of pBR322 were diluted to 100 pV in the ,oo 0 presence of 50 mM KC1, 5 mM MgCl 2 20 mM (NH4) 2

SO

4 1 SmM DTT and 20 mM TRIS (pH 7.2 at 25°C) 100 mg/ml BSA, 0.1 mM each of dGTP, TTP, dCTP (Pharmacia), 0.5 mM catniodATP q.O (Pharmacia) and 0.1 pM of each probe. One set of samples underwent strand displacement amplification for 3.5 hours at :39 0 C upon addition of 5 U of exonuclease deficient S klenow fragment of DNA polymerase I and 50 U of HinC II. A Ssecond set of samples were run without polymerase and without HinC II as unamplified standards.

To detect the reaction products, the pBR322 detection primer having SEQ ID NO: 7 was used having been labelled with 32P. Ten pi aliquots of the amplified and unamplified Fok 3O I/pBR322/human placental DNA samples were mixed with 2 pl of 1 pM 32P labelled detection primer, and were heated 2 minutes at 95 0 C. Two U of Sequenase 2.0 were then added, and samples were incubated for 5 minutes at 37 0 C. Samples were quenched with 50% urea and loaded onto a 10% denaturing

Q

i :i -22polyacrylamide gel. The presence of amplified reaction products was detected through extension of the 32 P labelled detection primer to lengths of 58 and 79 nucleotides.

Unamplified samples were indicated by extension to nucleotides. Electrophoresis of the labelled bands was quantified by liquid scintillation counting subtracting appropriated background bands. The results are shown in TABLE

III.

t .4 pBR322 Molecules 1$ 0 C 04 0.

4 0, C C 0 0 4 10 108 10 7 106 10 10 10 3 0 TABLE III Amplified cpm)

ND

ND

ND

135408 13841 2324 380 139 139 Unamplified (±10 cpm) 1963 257

ND

ND

ND

ND

ND

ND

ND not determined The amplified sample with zero added pBR322 molecules exhibited faint amplified target specific bands (58- and 79-mer) due to inadvertent contamination with pBR322.

Comparing the unamplified samples with 10 and p r 'P322 molecules with respective samples containing 10 and 1l" .pBR322 molecules indicates an amplification factor of over 10 5 fold. Further, it has been found that by adjusting the buffer composition and deoxynucleosidetriphosphate -23concentrations one can improve amplification performance.

Inclusion of (NH4) 2 SO4, a relatively low pH and a athiodATP:dGTP ratio of 5:1 have been found to enhance amplification efficiency.

All publications and patent applications mentioned in this specification are indicative of the level of brdinary skill in 0o0 the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the o 9o oo* same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

o It will be apparent to one of ordinary skill in the art that many changes and modifications can be made in the invention without departing from the spirit or scope of the a appended claims.

0 0 oa a -24- SEQUENCE LISTING SEQ ID NO: 1dTCATTTCTTACTTTACCGGGAAAAATCACTCAGGGTCAA SEQ ID NO: 2-- 0044 4 dTCATTTCTTACTTTACCGGGACCCTGTGGAACACCTACAT 110 SEQ ID NO: 3dCCAGCGCTTCGTTAATACA SEQ ID NO: 4o dACCCTGTGGA.ACA-CCTACATCTGTATTA.ACGAAGCGCTGGCATTGACCCTGAGTGATTTT 44: TC o~a~SEQ ID NO: dGGATATTTATTGTTGACTTACCCTGTGGAACAC-NH 2 Got t SEQ ID NO: 6dGGAATAATAATATGTTGACTTGAAAAATCACTCAG-NH 2 SEQ ID NO: 7dACATCTGTATTAACGAAGCG SEQ ID NO: 8dTTGAAGTAACCGACTATTGTTGACTACCCTGTGGAACACCT-N-1 2

I

SEQ ID NO: 9-- 6.TTGAATAGTCGGTTACTTGTTGACTCAGAGAAAAATCACTCAG-NH 2 0 0004 O 04 00 0 0090 09 0 0 0 0 90 4 Ge 0* *9.0~ 0 00 00 0 00 o 0 01 o a t 000 U 00600 a I ii

Claims (2)

1. 26- THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:- 1. A method for amplifying a target nucleic acid sequence comprising the steps of: a) binding an oligonucleotide primer to a single stranded nucleic acid fragment containing the target nucleic acid sequence, the primer comprising a recognition sequence for a restriction endonuclease which nicks one strand of double stranded DNA when a substituted deoxynucleoside triphosphate is incorporated into the recognition sequence, and; b) amplifying the target nucleic acid sequence at a single temperature of about 37 0 C to 450C by i) extending the primer on the fragment in the presence of a DNA polymerase lacking exonuclease activity, deoxynucleoside triphosphates, at least one substituted deoxynucleoside triphosphate and the restriction endonuclease, thereby producing a double stranded first reaction product comprising the primer, a first newly S: synthesized strand and a hemimodified restriction S 20 endonuclease recognition sequence, ii) nicking the double stranded first reaction product with the restriction endonuclease, the restriction endonuclease dissociating from the first reaction product at a rate which allows binding of the polymerase to the nick, iii) polymerizing from the nick using the polymerase, thereby displacing the first newly synthesized strand from the first reaction product and generating a second newly synthesized strand, and iv) repeating the nicking, extending and displacing v r .j steps such that the target sequence is amplified. 30 2. The method of claim 1 comprising the additional step of detecting the presence of the reaction products produced. 3. The method of claim 1 wherein the target sequence is double-stranded and is rendered single stranded before step a). s s k L I L .L'AJ L 1 t.L da±lu UY PL 3 tIlI J tv L k-.L I &%L&L further should be selected so as to dissociate from the recognition sequence at a reasonable rate. It need not be thermophilic. Endonucleases, such as HinC II which recognizes 27 4. A method for detecting a target nucleic acid sequence in a sample of biological material comprising the steps of: a) isolating nucleic acids from the sample; b) preparing a double stranded fragment of the nucleic acids in the sample, said double stranded fragment containing the target sequence; c) denaturing the double stranded fragment to produce complementary single stranded nucleic acid fragments; d) binding oligonucleotide primers to the complementary single strands, each of the primers comprising a recognition sequence for a restriction endonuclease which nicks one strand of double stranded DNA when a substituted deoxynucleoside triphosphate is incorporated into the recognition sequence; e) amplifying the target nucleic acid sequence at a single temperature of about 37 C to 45 0 C by i) extending the primers on the single stranded fragments in 14: the presence of a DNA polymerase lacking exonuclease 20 activity, deoxynucleoside triphosphates, at least one substituted deoxynucleoside triphosphate and the S°restriction endonuclease, thereby producing a double stranded first reaction product comprising the primer, a first newly synthesized strand and a hemimodified 25 restriction endonuclease recognition sequence, ii) nicking 6 the double stranded first reaction product with the restriction endonuclease, the restriction endonuclease 'J 'dissociating from the first reaction product at a rate S.0 which allows binding of the polymerase to the nick, iii) polyrmnrizing from the nick using the polymerase, thereby displacing the first newly synthesized strand from the first reaction product and generating a second newly synthesized strand, and iv) repeating the nicking, extending and displacing steps such that the target sequence is amplified; and I t "il! C _1 28 f) detecting the amplified target sequence. The method of claim 4 wherein the polymerase is selected from the group consisting of the klenow fragment of DNA polymerase I, the exonuclease deficient klenow fragment of DNA polymerase I and the klenow fragment of Bst polymerase. 6. The method of claim 4 wherein detection of the reaction products is carried out by tagging the primer with a label. 7. A method for detecting a target nucleic acid sequence in a sample of biological material comprising the steps of: a) isolating nucleic acids from the sample; b) preparing a double stranded fragment of the nucleic acids by adding a restriction endonuclease to the sample, the double stranded fragment containing the target sequence; Sc) denaturing the double stranded fragment to produce complementary single stranded nucleic acid fragments; d) binding oligonucleotide primers to the complementary single strands, each of the primers comprising a recognition sequence of 5'-GTPyPuAC-3'; e) amplifying the target nucleic acid sequence at a single temperature of about 37 0 C to 45 C by i) extending the primers on the single stranded fragments 7 in the presence of a DNA polymerase lacking 5'-3' exonuclease activity, deoxynucleoside triphosphates, at least one substituted deoxynucleoside triphosphate and HinCII restriction endonuclease, thereby producing a double stranded first reaction product comprising the primer, a first newly synthesized strand and a hemimodified HinCII recognition sequence, ii) nicking the double stranded first reaction product with the HinCII restriction endonuclease, the HinCII dissociating from the first reaction product at a rate which allows binding of Dy any numner ot methods. One method is to detect reaction products of a specific size by means of gel electrophoresis. This method is particularly useful when the nucleotides used 32 are labelled with a radio-label, such as P3. Other methods
2. 77- 29 the polymerase to the nick, iii) polymerizing from the nick using the polymerase, thereby displacing the first newly synthesized strand from the first reaction product and generating a second newly synthesized strand, and iv) repeating the nicking, extending and displacing steps such that the target sequence is amplified; and f) detecting the amplified target sequence. 8. The method of any of claims 1 and 7 wherein the primers are specific for a viral nucleic acid sequence. 9. The method of any of claims 1 and 7 wherein the primers are specific for a bacterial nucleic acid sequence. A method for generating multiple copies of a target nucleic acid sequence comprising the steps of: a) preparing one or more single stranded nucleic acid fragments containing the target nucleic acid sequence; b) binding oligonucleotide primers to the single strands, each of the primers comprising a recognition sequence for a restriction endonuclease which nicks one strand of double stranded DNA when a substituted 20 deoxynucleoside triphosphate is incorporated into the o" recognition sequence; c) generating copies of the target nucleic acid sequence at a single temperature of about 370C to 45 0 C by i) extending the primers on the single stranded S 25 fragments in the prence of a DNA polymerase lacking newly synthesized strand from the f irst reaction productimer, a a n d eneatin a seond newly syntsied s trand and a heod ed f)restriction endonucleasemplif recognition sequence, ii) nicking the double mestranded first reaction pr1 anoduct with the 9 The method oe any of claims 1 and 7 wherein the primers are specific for a bacterial nucleic acid sequence nucleic acid sequence comprising the steps of: restriction endonuclease, the restriction endonuclease dissociating from the first reaction productleict t a rate which allows binding of the polymerase to the nick, which allows binding of the polymerase to the nick, h l l l l lll l l l l ll. l~ l i Cc 30 iii) polymerizing from the nick using the polymerase, thereby displacing the first newly synthesized strand from the first reaction product and generating a second newly synthesized strand, and iv) repeating the nicking, extending and displacing steps such that multiple copies of the target sequence are generated. DATED this 22nd day of June 1994 BECTON DICKINSON AND COMPANY Patent Attorneys for the Applicant: F.B. RICE CO. 4 0r 0 a *U S0 00 t 090 0~0 0 a o 4 0 00 a a 4 Y i 4;- STRAND DISPLACEMENT AMPLIFICATION Abstract This invention relates a nucleic acid target amplification and detection method which operates at a single temperature and makes use of a polymerase in conjunction with an endonuclease that will nick the polymerized strand such that the polymerase will displace the strand without digestion while generating a newly polymerized strand. 4 S4 34.e a 0 #1 1 I1 44 0 00 C 1S a 4A