IL103039A - Process for amplifying, detecting and/or cloning of specific nucleic acid sequences using a thermostable enzyme - Google Patents

Description

Dira n>^ u>ny a>n>u vna>vy >"y A process for amplifying, detecting and/or cloning of specific nucleic acid sequences using a thermostable enzyme F. HOFFMANN-LA ROCHE AG.

C: 85205 - 1 - 103039/2 A PROCESS FOR AMPLIFYING, DETECTING AND/OR CLONING OF SPECIFIC NUCLEIC ACID SEQUENCES USING A THERMOSTABLE ENZYME The present application is a divisional application, divided out from co-pending, co-owned Application No. IL 83605 and constitutes a selection invention from Israel Patents Nos. 78284 and 78281.

The present invention relates to a process for amplifying, detecting and/or cloning of at least one specific nucleic acid sequence contained in a nucleic acid or a mixture of nucleic acids using a thermostable enzyme.

The invention according to the above noted IL 83605 concerns a purified thermostable Thermus aquaticus DNA polymerase enzyme, a gene coding for the enzyme, a bacteriophage vector containing the gene, and compositions containing the enzyme, all as defined in IL 83605, the above thermostable enzyme of IL 83605 being one example of a thermostable enzyme useful for carrying out the process according to the present invention.

More particularly, the present invention relates to a process for amplifying existing nucleic acid sequences if they are present in a test sample, detecting them if present using a probe, and/or cloning them into a cloning vector. The process is useful for producing any particular nucleic acid from a given sequence of DNA or RNA in amounts which are large compared to the amount initially present so as to facilitate detection of sequences and/or cloning of sequences. A thermostable enzyme is utilized for catalyzing the reaction. The DNA or RNA may be single- or double-stranded, and may be a relatively pure species or a component or a mixture of nucleic acids. The process of the invention utilizes a reaction that is repetitive to accomplish the amplification of the desired nucleic acid sequence.

Extensive research has been conducted on the isolation of DNA polymerases from mesophilic microorganisms such as E. coli. See, for example, Bessman et al . , J. Biol. Chem. (1957) 233:171-177 and Buttin and Kornberg (1966) J. Biol. Chem. 241:5419-5427.

In contrast, relatively little investigation has been made on the isolation and purification of DNA polymerases from thermophiles, such as Thermus' aquaticus. Kaledin et al . , B ok ymiya (1980) 5:644-651 discloses a six-step isolation and purification procedure of DNA polymerase from cells of 1\_ aquat cus YT1 strain. These steps involve isolation of crude extract, DEAE-cell ul ose chromatography, fractionation on hydroxyapat te, fract onation on DEAE-eellulose, and chromatography on single-strand DNA-cell ul ose. The pools from each stage were not screened for contam nating endo-and exonucl ease( s ) . The molecular weight of the purified enzyme is reported as 62,000 daltons per monomeric unit.

A second purification scheme for a polymerase from T. aquat cus is described by A. Chi en et a . , J. Bacteriol. (1976) -2- 127:1550-1557. In this process, the crude extract is applied to a DEAE-Sephadex column. The dialyzed pooled fractions are then subjected to treatment on a phosphocellul ose column. The pooled fractions are dialyzed and bovine serum albumin (BSA) is added to prevent loss of polymerase activity. The resulting mixture is loaded on a DNA-cellulose column. The pooled material from the column is dialyzed and analyzed by gel filtration to have a molecular weight of about 63,000 daltons, and, by sucrose gradient centrifugation of about 68,000 daltons.

With respect to the above noted publications, namely, Kaledin et al., Biokhimiya (Moscow), 1980, 45, 4 644-51, and Chien et al., J. Bacteriol., 1976, 127 (3), 1550-7, it should be noted that, at most, these describe the partial isolation of truncated forms of Tag polymerase. Neither publication describes the thermostable DNA polymerase, the gene encoding it or the other related aspects thereof, according to the above noted co- owned IL 83605, nor do they describe the process of nucleic acid sequence, amplification, detection and/or cloning of the present invention, as set forth hereinbelow in detail. Furthermore, in Edgar et al., in "Abstracts of the Annual Meeting of the American Society for Microbiology", Vol.75, 1975, page 151, abstract no. K26, there is disclosed essentially the same partial isolation of a truncated form of Tag polymerases as is disclosed in the published abstract (CA 85: 155559t (1976) of the above Chien et al. publication. Additionally, in another publication by Kaledin et al., Biokhimiya (Moscow), 47, 1785-91, (1982), there is disclosed a polymerase from the bacterium Thermus ruber, and not from Thermus aquaticus as is the case according to the above noted, co-owned IL 83605. In particular, in this Kaledin et al. publication, no description is made of the thermostable DNA polymerase, the gene encoding it, or the other related aspects according to IL 83605, nor is any description made of the nucleic acid sequence amplification, detection, and/or cloning process according to the present invention, as set forth hereinbelow in detail.

The use of a thermostable enzyme to amplify existing nucleic acid sequences in amounts that are large compared to the amount initially present has been suggested in European Pat. Pub. No. 200,362 published December 10, 1986. Primers, nucleotide triphosphates, and a polymerase are used in the process, which involves denaturati on, synthesis of template strands and hybridization. The extension product of each primer becomes a template for the production of the desired nucleic acid sequence. The application discloses that if the polymerase employed is a thermostable enzyme, it need not be added after every denaturat on step, because the heat will not destroy its activity. No other advantages or details are provided on the use of a purified thermostable DNA polymerase. The amplification and detection process is also described by Saiki et al . , Science, 230:1350-1354 (1985), and by Saiki et al., Bio/Technology, 3_: 1008-1012 (1985).

Accordingly, there is a desire in the art to produce a purified, stable thermostable enzyme that may be used to improve the diagnostic amplification process described above.

Accordingly, the invention described in co-pending, co-owned IL 83605, from which the present application has been divided out, provides a purified thermostable enzyme that catalyzes combination of nucleotide triphosphates to form a nucleic acid strand complementary to a nucleic acid template strand. The above purified enzyme is DNA polymerase of the Thermus aquaticus type, which may bejsolated from the_ microorganism Thermus aquaticus. or from a recombinant microorganism transformed with a vector that codes for the expression of Thermus aquaticus DNA polymerase. The above enzyme has a molecular weight of at least 86,000 daltons when compared with a phosphorylaze B standard assigned a molecular weight of 92,500 daltons as described in IL 83605. - 3 - This purified enzyme may be used in a temperature-cycling amplification process according to the present invention.

The gene encoding the enzyme from DNA polymerase from Thermus aquaticus has also been identified and provides yet another means to retrieve the thermostable enzyme of the present invention, and represents another feature of the invention according to above noted IL 83605.

Finally, the invention according to the above noted IL 83605, also encompasses a stable enzyme composition comprising a purified, thermostable enzyme as described above in a buffer containing one or more non-ionic polymeric detergents.

The above noted purified enzyme, as well as the enzymes produced by recombinant DNA techniques, according to IL 83605, provide much more specificity than the Klenovv fragment, which is not thermostable. In addition, the above purified enzyme and the recombinantly produced enzymes exhibit the appropriate activity expected when dTTP or other nucleotide triphosphates are not present in the incubation mixture with the .' . DNA template. Also, the above enzymes according to IL 83605 have a broader pH profile than that of the thermostable enzyme from Thermus aquaticus described in the literature, with more than 50% of the activity at ph 7 as at pH 8. In addition, the thermostable enzyme of IL 83605 can be stored, in a buffer with non— ionic detergents so that it is stable, not losing activity over a- period of time.

In the following there is described the various aspects and features of the present invention as well as subject matter relating to the present invention, for example a detailed description concerning the thermostable polymerase enzyme of co-owned IL 83605, from which the present application has been divided out. It is to be understood, however, w that the fol l owi ng may contai n subject matter not wi thi n the scope of the cl a ims , and whi ch i s reta i ned herei n , for the sake of compl eteness of the descri pti on . - 3a - The present invention resides in a process for amplifying one or more specific nucleic acid sequences present in a nucleic acid or mixture thereof using primers and a thermostable enzyme. The extension product of one primer when hybridized to the other becomes a template for the production of the desired specific nucleic acid sequence, and vice versa, and the process is repeated as often as is necessary to produce the desired amount of the sequence- The method 103039/2 - 4 - herein improves the specificity of the amplification reaction, resulting in a very distinct signal of amplified nucleic acid. In addition, the method herein eliminates the need for transferring reagents from one vessel to another after each ampli ication cycle. Such transferring is not required because the thermostable enzyme will withstand the high temperatures required to denature the nucleic acid strands and therefore does not need replacement. The temperature cycling may, in addition, be automated for further reduction in manpower and steps required to effectuate the amplification reaction.

More specifically, the present invention provides a process for amplifying at least. one specific nucleic acid sequence contained in - a nucleic acid or a mixture of nucleic acids, wherein the nucleic acid is ■ double-stranded, or..· consists- of two separated complementary strands of equal or unequal length, which process comp ises : (a) contacting the nucleic acid with four different nucleoside triphosphates and two oligonucleotide primers, for each different specific sequence being amplified, wherein each primer is selected to be sufficiently complementary to different strands of each specific sequence to hybridize therewith, such that the extension product synthesized from one primer, when separated from its complement, can serve as a template for synthesis of the extension product of the other primer, if the nucleic acid is double-stranded, heating the mixture for an effective time and at an effective temperature to denature each nucleic acid and cooling the mixture to a temperature which promotes hybridization of each primer to its complementary strands or, if the nucleic acid consists of two separated complementary strands, treating the mixture .at a temperature which promotes hybridization of each primer to its complementary strand; - 4a - ( b) contacting each strand, at the same time as or after step (a), with a thermostable enzyme which catalyzes the template-dependent combination of the nucleoside triphosphates to form primer extension products complementary to each strand of nucleic acid, said thermostable enzyme being a Thermus aquaticus DNA polymerase that migrates on a denaturing polyacrylamide gel faster than phosphorylase B and more slowly than does bovine serum albumin and has an estimated molecular weight of at least 86,000 daltons when compared with a phosphorylase B standard assigned a molecular weight of 92,500 daltons; (c) maintaining the mixture from step (b) at an effective temperature for an effective time to promote the activity of the enzyme, and to synthesize, for each different sequence being amplified, an extension product of each primer which is complementary to each strand, but not so high as to separate each extension product from its complementary strand; (d) heating the mixture from step (c) for an effective time and at an effective temperature to separate the primer extension products from the strands on which they were synthesized to produce single-stranded molecules, but not so high as to denature irreversibly the enzyme; (e) cooling the mixture from step (d) to an effective temperature to promote hybridization of each primer to each of the single-stranded molecules produced in step (d); and 103039/2 - 5 - maintaining the mixture from step (e) at an effective temperature for an effective time to promote the activity of the enzyme and to synthesize, for each different sequence being amplified, an extension product of each primer which is complementary to each strand produced in step (d), but no so high as to separate each extension product from its complementary strand, wherein steps (e) and (f) are conducted simultaneously or sequentially.

The above process may be carried out wherein one specific nucleic acid acid sequence is amplified and two primers are employed.

Further, the above process may be repeated until the desired level of sequence amplification is obtained, for example steps (d), (e) and (f) can be repeated at least five times.

In one embodiment of the above process, after steps (d)-(f) are repeated at least once, a second set of two primers is added, wherein the primers added are sufficiently complementary'to different strands at internal sequences of the amplified sequence to hybridize therewith, such that the extension product synthesized from one primer, when separated from its complement, can serve as a template for synthesis of the extension product of the other primer.

The preferred thermostable enzyme herein is a polymerase extracted from Thermus aquatiais (Taq polymerase). Most preferably, if the enzyme is Taq polymerase, in step (a) the nucleic acid strands are contacted with a buffer comprising about l -2mM of a magnesium salt, 150-200 μΜ each of the nucleotides, and 1 μΜ of each primer, steps (a), (e) and (f) are carried out at about 45-58°C, and step (d) is carried out at about 90-100°C.

Further, the above noted primers used in the process may be oligodeoxyribonucleotides. and the nucleic acid may be cDNA.

In another embodiment, the nucleic acid(s) are double-stranded and step (a) is accomplished by (i) heating each nucleic acid in the presence of four different nucleotide triphosphates and one oligonucleot de primer for each different specific sequence being amplified, for an effective time and at an effective temperature to denature each nucleic acid, wherein each primer is selected to be substantially complementary to different strands of each specific sequence, such that the extension product synthesized from one primer, -6-when it is separated from its complement, can serve as a template for synthesis of the extension product of the other primer; and (ii) cooling the denatured nucleic acids to a temperature which promotes hybridization of each primer to its complementary nucleic acid strand.

In another embodiment the above process may be carried out wherein the heating and cooling steps (d)-(f) are automated by a machine which controls temperature levels, transitions from one temperature to another, and the timing of temperature levels.

The above process may be carried out wherein each primer is present in a molar ration of at least 1000:1 primerxomplementary strand. Further, the above process may also be carried out wherein at least one primer contains at least one nucleotide which is not complementary to the specific sequence to be amplified, such a non-complementary nucleotide being, for example, one encoding a promoter.

Other embodiments of the above process are ones wherein steps (e) and (f) are carried out sequentially, steps (c) and (f) take place at 40°-80°C, step (d) takes place at 90°-105°C, and step (e) takes place at 35°-65°C; or wherein steps (e) and (f) are carried out sequentially, steps (c) and (f) take place at 50°-75°C, step (d) takes place at 90°-100°C, and step (e) takes place at 37°-60°C, or wherein steps (e) and (f) are carried out simultaneously at about 45°-70°C.

In other embodiments the invention relates to a process for detecting the presence or absence of at least one specific nucleic acid sequence in a sample containing a nucleic acid or mixture of nucleic acids, or distinguishing between two different sequences in said sample, wherein the sample is suspected of containing said sequence or sequences, and wherein the nucleic acid(s) are double-stranded, or each consist of two separated complementary strands of equal or unequal length, which process comprises steps (a) to (f) mentioned above, resulting in amplification in quantity of the specific nucleic acid sequence(s), if present; and a step of determining if amplification has occurred.

The above determining step may be carried out by adding to the product of step (f) a labeled oligonucleotide probe, for each sequence being detected, capable of hybridizing to said sequence or to a mutation thereof; and determining whether said hybridization has occurred.

In yet another embodiment, the invention relates to a process for detecting the presence of absence of at least one nucleotide variation in sequence in one or more nucleic acids contained in a sample, wherein the nucleic acid is double-stranded or it consists of two separated complementary strands of equal or unequal length, which process comprises steps (a)- (f) mentioned above, wherein steps (d), (e) and (f) may be repeated a sufficient number of times, as noted above, to result in detectable amplification of the nucleic acid containing the sequence, if present; and then affixing the product of step (f) to a membrane; treating the membrane under hybridization conditions with a labeled sequence-specific oligonucleotide probe capable of hybridizing with the amplified nucleic acid sequence only if a sequence of the probe is complementary to a region of the amplified sequence; and detecting whether probe has hybridized to an amplified sequence in the nucleic acid sample.

If the sample comprises cells, preferably they are heated before step (a) to expose the nucleic acids therein to the reagents. This step avoids extraction of the nucleic acids prior to reagent addition.

In another embodiment of this process, the primer(s) and/or nucleotide triphosphates are labeled so that the resulting amplified sequence is labeled. The labeled primer(s) and/or nucleotide triphosphate(s) can be present in the reaction mixture initially or added during a later cycle. The sequence-specific oligonucleot de (unlabeled) is affixed to a membrane and treated under hybridization conditions with the labeled amplificat on product so that hybridization will occur only if the membrane-bound sequence is present in the amplification product.

In yet another embodiment, the invention herein relates to a process for cloning into a cloning vector one or more specific nucleic acid sequences contained in a nucleic acid or a mixture of nucleic acids, which nucleic acid(s) is(are) double-stranded or consist(s) of two separated complementary strands, and which nucleic acid(s) are amplified in quantity before cloning, which process comprises steps (a)-(f) mentioned above, with steps (d), (e) and (f) being repeated a sufficient number of times to result in detectable amplification of the nucleic acid(s) containing the sequence(s), and wherein the sequence being amplified and cloned or each primer contains a restriction site; followed by the steps of adding to the product of step (f) a restriction enzyme for each of said restriction sites to obtain cleaved products in a restriction digest; and ligating the cleaved product(s) containing the specific sequence(s) to be cloned into one or more cloning vectors. The one or more cloning vectors may contain a promoter and/or a selectable marker.

In one more embodiment, the invention herein relates to a process for cloning into a cloning vector one or more specific nucleic acid sequences contained in a nucleic acid or mixture of nucleic acids, which nucleic acid(s), is (are) double-stranded or consist of two -8- separated complementary strands of equal or unequal length which nucleic acid(s) are amplified in quantity before cloning, which process comprises steps (a)-(f) mentioned above, with steps (d), (e) and (f) being repeated a sufficient number of times to result in effective amplification of the nucleic acid(s) containing the sequence(s) for blunt-end ligation into one or more cloning vectors; and followed by the step of ligating the amplified specific sequence(s) to be cloned as obtained from step (f) into one or more of said cloning vectors in the presence of a ligase, said amplified sequence(s) and vector(s) being present in sufficient amounts to effect the ligation.

The above blunt-end cloning procedure may be carried out wherein one nucleic acid sequence is cloned into one vector and two primers are employed.

It should be noted that various other embodiments of the above amplification, detection and cloning processes are included in the present invention, the particulars of which are set forth in the following detailed description. For example, in the above detection process, the amplified products may be first cut with a restriction enzyme and electrophoresed so that the subsequent determination step is accomplished by Southern blot analysis. Further, the nucleic acid sequence to be detected in the above process can be one known to cause a genetic, infectious or cancerous disease, examples of such genetic diseases being sickle cell anemia or hemoglobin C disease. Furthermore, in the above cloning process, the amplified products to be cloned can be those that encode a protein and the process can be extended to also include the expression of the protein encoded by the cloned sequence, examples of this being the cloning of specific nucleic acid sequences contained within the β-globin gene or the N-RAS oncogene, following the amplification of these sequences.

For carrying out the above process of the invention it is also possible to prepare and employ a kit being a composition of matter useful in amplifying at least one specific nucleic acid sequence contained in a nucleic acid or a mixture of nucleic acids, comprising four different nucleotide triphosphates and one oligonucleotide primer for each different specific sequence being amplified, wherein each primer is selected to be substantially complementary to different strands of each specific sequence, such that the extension product synthesized from one primer, when it is separated from its complement, can serve as a template for synthesis of the extension product of the other primer.

Finally, the present invention also includes the direct products of the above process of the invention, for example, a product being a sample of one or more nucleic acids comprising multiple strands of a specific nucleic acid sequence contained in the nucleic acid(s). The sample may comprise about 10-100 of the strands, about 100-1000 of the strands, or over about 1000 of the strands; or a product in the form of an amplified nucleic acid sequence from a nucleic acid or mixture of nucleic acids comprising multiple copies of the sequence.

Fi gu re 1 i s a restri cti on si te map of pl asmi d pFC83 that contai ns the -4.5 kb H ndi 11 T. aquati cus DNA i nsert subcl oned i nto pl asmi d BS 13+ . -9- Figure 2 is a restriction site map of plasmid pFC85 that contains the ~2.8 kb Hi ndlll to Asp718 T. aquaticus DNA insert subcloned into plasmid BSM13+.

As used herein, "cell", "cell line", and "cell culture" can be used interchangeably and all such designations include progeny. Thus, the words "transfor ants" or "transformed cells" includes the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same functionality as screened for in the originally transformed cell are included.

The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for procaryotes, for example, include a promoter, optionally an operator sequence, a ribosome binding site, and possibly, other as yet poorly understood sequences. Eucaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

The term "expression system" refers to DNA sequences containing a desired coding sequence and control sequences in operable linkage, so that hosts transformed with these sequences are capable of producing the encoded proteins. In order to effect transformation, the expression system may be included on a vector; however, the relevant DNA may then also be integrated into the host chromosome.

The term "gene'^as used herein refers to a DNA sequence that encodes a recoverable .. bioactive polypeptide or precursor. The polypeptide can be encoded by a full-length gene sequence or any portion of the coding sequence so long as the enzymatic activity is retained.

"Operably linked" refers to juxtaposition such that the normal function of the components can be performed. Thus, a coding sequence "operably linked" to control sequences refers to a configuration wherein the coding sequences can be expressed under the control of the sequences. -10- "Non-ionic polymeric detergents" refers to surface-active agents that have no ionic charge and that are characterized, for purposes of this invention, by their ability to stabilize the enzyme herein at a pH range of from about 3.5 to about 9.5, preferably from 4 to 8.5.

The term "oligonucleotide" as used herein is defined as a molecule comprised of two or more deoxyribonucleotides or ribonucleotides, preferably more than three. Its exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. The oligonucleotide may be derived synthetically or by cloning.

The term "primer" as used herein refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, i.e., in the presence of four different nucleotide triphosphates and thermostable enzyme in an appropriate buffer ("buffer" includes pH, ionic strength, cofactors, etc.) and at a suitable temperature. For Taq polymerase the buffer herein preferably contains 1.5-2 mM of a magnesium salt, preferably MgCl2, 150-200 μ of each nucleotide, and 1 y of each primer, along with preferably 50 mM C1 , 10 mM Tris buffer, pH 8-8.4, and 100 ug/ml gelatin.

The primer is preferably single-stranded for maximum efficiency in amplification, but may alternatively be double-stranded. If double-stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxy ribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the thermostable enzyme. The exact lengths of the primers will depend on many factors, including temperature, source of primer and use of the method. For example, depending on the complexity of the target sequence, the oligonucleotide primer -11- typically contains 15-25 nucleotides, although it may contain more or fewer nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with template.

The primers herein are selected to be "substantially" complementary to the different strands of each specific sequence to be amplified. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementa y nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternat ely, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to be amplified to hybridize therewith and thereby form a template for synthesis of the extension product of the other primer. However, for detection purposes, particulary using labeled sequence-specific probes, the primers typically have exact complementarity to obtain the best results.

As used herein, the terms "restriction endonucleases" and "restriction enzymes" refer to bacterial enzymes each of which cut double-stranded DNA at or near a specific nucleotide sequence.

As used herein, the term "DNA polymorphism" refers to the condition in which two or more different nucleotide sequences can exist at a particular site in DNA.

As used herein, the term "nucleotide variation n sequence" refers to any single or multiple nucleotide substitut ons, deletions or insertions. These nucleotide variations may be mutant or polymorphic allele variations. Therefore, the process herein can detect single nucleotide changes in nucleic acids such as occur in s-globin genetic diseases caused by single-base mutations, additions or deletions (some 6 -thalassemias, sickle cell anemia, hemoglobin C disease, etc.), as well as multiple-base variations such as are -12- involved with α-thalassemia or some ρ -thalassemias. In addition, the process herein can detect polymorphisms, which are not necessarily associated with a disease, but are merely a condition in which two or more different nucleotide sequences (whether having substituted, deleted or inserted nucleotide base pairs) can exist at a particular site in the nucleic acid in the population, as with HLA regions of the human genome and random polymorphisms such as mitochondrial DNA. The polymorphic sequence-specific oligonucleotide probes described in detail hereinafter may be used to detect genetic markers linked to a disease such as insulin-dependent diabetis mellitus or in forensic applications. If the nucleic acid is double-stranded, the nucleotide variation in sequence becomes a base pair variation in sequence.

The term "sequence-specific oligonucleotides" refers to oligonucleotides which will hybridize to specific sequences whether or not contained on alleles, which sequences span the base pair variation being detected and are specific for the sequence variation being detected. Depending on the sequences being analyzed, one or more sequence-specific nucleotides may be employed for each sequence, as described further hereinbelow.

As used herein, the the term "restriction fragment length polymorphism" ("RFLP") refers to the differences among individuals in the lengths of restriction fragments formed by digestion with a particular restriction endonuclease.

As used herein, the term "thermostable enzyme" refers to an enzyme which is stable to heat and is heat resistant and catalyzes (facilitates) combination of the nucleotides in the proper manner to form the primer extension products that are complementary to each nucleic acid strand. Generally, the synthesis will be initiated at the 3' end of each primer and will proceed in the 5' direction along the template strand, until synthesis terminates, producing molecules of different lengths. There may be a thermostable enzyme, however, which initiates synthesis at the 5' end and proceeds in the other direction, using the same process as described above.

- - The thermostable enzyme herein must satisfy a single criterion to be effective for the amplification reaction, i.e., the enzyme must not become irreversibly denatured (inactivated) when subjected to the elevated temperatures for the time necessary to effect denaturation of double-stranded nucleic acids. Irreversible denaturation for purposes herein refers to permanent and complete loss of enzymatic activity. The heating conditions necessary for denaturation will depend, e.g., on the buffer salt concentration and the length and nucleotide composition of the nucleic acids being denatured, but typically range from about 90 to about 105 °C for a time depending mainly on the temperature and the nucleic acid length, typically about 0.5 to four minutes. Higher temperatures may be tolerated as the buffer salt concentration and/or GC composition of the nucleic acid is increased. Preferably, the enzyme will not become irreversibly denatured at about 90-100°C.

The thermostable enzyme herein preferably has an optimum temperature at which it functions that is higher than about 40°C, which is the temperature below which hybridization of primer to template is promoted, although, depending on (1) magnesium and salt concentrations and (2) composition and length of primer, hybridization can occur at higher temperature (e.g., 45-70°C). The higher the temperature optimum for the enzyme, the greater the specificity and/or selectivity of the primer-directed extension process. However, enzymes that are active below 40°C, e.g., at 37°C, are also within the scope of this invention provided they are heat-stable. Preferably, the optimum temperature ranges from about 50 to 90°C, more preferably 60-80°C.

The thermostable enzyme herein may be obtained from any source and may be a native or recombinant protein. Examples of enzymes that have been reported in the literature as being resistant to heat include heat-stable polymerases, such as, e.g., polymerases extracted from the thermophilic bacteria Thermus flavus, Thermus ruber, Thermus thermophilus, Bacillus stea rothe rmoph i 1 us (which has a somewhat lower temperature optimum than the others listed), Thermus aquaticus, Thermus lacteus, Thermus rubens, and Methanothermus fervidus. -14- The preferred thermostable enzyme herein is a DNA polymerase isolated from Thermus aquaticus. Various strains thereof are available from the American Type Culture Collection, Rockv lle, Maryland, and is described by T.D. Brock, J. Bact. (1969) 98:289-297, and by T. Oshima, Arch. Microbiol. (1978) 117: 189-196. One of these preferred strains is strain YT-1.

For recover ng the native prote n the cells are grown us ng any suitable technique. One such technique is described by Kaledin et al . , Biokhimiya (1980), supra. Briefly, the cells are grown on a medium, in one liter, of nitrilotr acetic acid (100 mg), tryptone (3 g), yeast extract (3 g), succinic acid (5 g), sodium sulfite (50 mg), riboflavin (1 mg), 2HP04 (522 mg), MgS04(480 mg), CaCl2 (222 mg), NaCl (20 mg), and trace elements. The pH of the medium is adjusted to 8.0 ± 0.2 with KOH. The yield is increased if cultivated with vigorous aeration up to 20 g/liter of cells at a temperature of 70°C. Cells in the late logarithmic stage (determined by absorbance at 550 nm) are collected by cent fugation, washed with a buffer and stored frozen at -20°C.

In another method for growing the cells, described in Chien et al ., J. Bacteriol . (1976), supra, a defined mineral salts medium containing 0.3% glutamic acid supplemented with 0.1 mg/1 biotin, 0.1 mg/1 thiamine, and 0.05 mg/1 nicotinic acid is employed. The salts include nitril otri acetic acid, CaS04, MgS04, NaCl , KN03, NaN03, ZnS04, H3BO3, CuS04, NaMo04, CoCl2, F-eC13, MnS04, and Na2HP04. The pH of the medium is adjusted to 8.0 with NaOH.

In the Chien et al . technique, the cells are grown initially at 75°C in a water bath shaker. On reaching a certain density, 1 liter of these cells is transferred to 16-liter carboys which are placed in hot-air incubators. Sterile air is bubbled through the cultures and the temperature maintained at 75°C. The cells are all owed to grow for 20 hours before being collected by centrifuge.

After cell growth, the isolation and purification of the enzyme take place in six stages, each of which is carried out at a temperature below room temperature, preferably about 4°C. -15- In the first stage or step, the cells, if frozen, are thawed, disintegrated by ultrasound, suspended in a buffer at about pH 7.5, and centrifuged.

In the second stage, the supernatant is collected and then fractionated by adding a salt such as dry ammonium sulfate. The appropriate fraction (typically 45-75% of saturation) is collected, dissolved in a 0.2 potassium phosphate buffer preferably at pH 6.5, and dialyzed against the same buffer.

The third step removes nucleic acids and some protein. The fraction from the second stage is applied to a DEAE-cellulose column equilibrated with the same buffer as used above. Then-the column is washed with the same buffer and the flow-through protein-containing fractions, determined by absorbance at 280 nm, are collected and dialyzed against a 10 mM potassium phosphate buffer, preferably with the same ingredients as the first buffer, but at a pH of 7.5.

In the fourth step, the fraction so collected is applied to a hydroxy apatite column equilibrated with the buffer used for dialysis in the third step. The column is then washed and the enzyme eluted with a linear gradient of a buffer such as 0.01 M to 0.5 M potassium phosphate buffer at pH 7.5 containing 10 mM 2-mercaptoethanol and 5% glycerine. The pooled fractions containing thermostable enzyme (e.g., DNA polymerase) activity are dialyzed against the same buffer used for dialysis in the third step.

In the fifth stage, the dialyzed fraction is applied to a DEAE-cellulose column, equilibrated with the buffer used for dialysis in the third step. The column is then washed and the enzyme eluted with a linear gradient of a buffer such as 0.01 to 0.6 M KC1 in the buffer used for dialysis in the third step. Fractions with thermostable enzyme activity are then tested for contaminating deoxyribonucleases (endo- and exonucleases) using any suitable procedure. For example, the endonuclease activity may be determined eiectrophoretical ly from the change in molecular weight of phage λ DNA or superco led plasmid DNA after incubation with an excess of DNA polymerase. Similarly, exonuclease activity may be determined -16- electrophoretically from the change in molecular weight of DNA after treatment with a restriction enzyme that cleaves at several sites.

The fractions determined to have no deoxyribonuclease activity are pooled and dialyzed against the same buffer used in the third step.

In the sixth step, the pooled fractions are placed on a phosphocellulose column with a set bed volume. The column is washed and the enzyme eluted with a linear gradient of a buffer such as 0.01 to 0.4 M KC1 in a potassium phosphate buffer at pH 7.5. The pooled fractions having thermostable polymerase activity and no deoxyribonuclease activity are dialyzed against a buffer at pH 8.0.

The molecular weight of the dialyzed product may be determined by any technique, for example, by SDS PAGE using protein molecular weight markers. The molecular weight of one" of the preferred enzymes herein, the DNA polymerase purified from Thermus aquaticus, is determined by the above method to be about 86,000-90,000 daltons.

The thermostable enzyme of this invention may also be produced by recombinant DNA techniques, as the gene encoding this enzyme has been cloned from Thermus aquaticus genomic DNA. The complete coding sequence for the Thermus aquaticus (Taq) polymerase can be derived from bacteriophage CH35:Taq#4-2 on an approximately 3.5 kilobase (kb) Bgl II-Asp718 (partial) restriction fragment contained within an ~18 kb genomic DNA insert fragment. This bacteriophage was deposited with the American Type Culture Collection (ATCC) on May 29, 1987 and has accession no. 40,336., Alternatively, the gene can be constructed by li gating an ~750 base pair (bp) Bgl II-HindIII restriction fragment isolated from plasmid pFC83 (ATCC 67,422 deposited May 29, 1987) to an ~2.8 kb HindIII-Asp718 restriction fragment isolated from plasmid pFC85 (ATCC 67,421 deposited May 29, 1987). The pFC83 restriction fragment comprises the ami no-terminus of the Taq polymerase gene while the restriction fragment from pFC85 comprises the carboxyl -terminus. Thus, ligation of these two fragments into a correspondingly digested vector with appropriate - - control sequences will result in the translation of a full-length Taq polymerase.

It has also been found that the entire coding sequence of the Taq polymerase gene is not required to recover a biologically active gene product with the desired enzymatic activity. Ami no-terminal deletions wherein approximately one-third of the coding sequence is absent has resulted in producing a gene product that is quite active in polymerase assays.

In addition to the N- terminal deletions, individual am no acid residues in the peptide chain comprising Taq polymerase may be modified by oxidation, reduction, or other derivatization, and the protein may be cleaved to obtain fragments that retain activity. Such alterations that do not destroy activity do not remove the DNA sequence encoding such protein from the definition of gene.

Thus, modifications to the primary structure itself by deletion, addition, or alteration of the amino acids incorporated into the sequence during translation can be made without destroying the activity of the protein. Such substitutions or other alterations result in proteins having an amino acid sequence encoded by DNA falling within the contemplated scope of the present invention.

Polyclonal antiserum from rabbits immunized with the purified 86,000-90,000 dalton polymerase of this invention was used to probe a Thermus aquaticus partial genomic expression library to obtain the appropriate coding sequence as described below. The cloned genomic sequence can be expressed as a fusion polypeptide, expressed directly using its. own control sequences, or expressed by constructions using control sequences appropriate to the particular host used for expression of the enzyme.

Of course, the availability of DNA encoding these sequences provides the opportunity to modify the codon sequence so as to generate mutein forms also having DNA polymerase activity.

Thus, these tools can provide the complete coding sequence for Taq DNA polymerase from which expression vectors applicable to a -18- vari ety of host systems can be constructed and the coding sequence expressed. It is also evident from the foregoing that portions of the Taq polymerase-encodi ng sequence are useful as probes to retrieve other thermostable polymerase-encod ng sequences in a variety of species. Accordingly, portions of the genomic DNA encoding at least six amino acids can be replicated in E. coli and the denatured forms used as probes or oligodeoxy ribonucleotide probes can be synthesized which encode at least six amino acids and used to retrieve additional DNAs encoding a thermostable polymerase. Because there may not be a precisely exact match between the nucleotide sequence in the Thermus aquaticus form and that in the corresponding portion of other species, oligomers containing approximately 18 nucleotides (encoding the six amino acid stretch) are probably necessary to obtain hybridization under conditions of sufficient stringency to eliminate false positives. The sequences encoding six amino acids would supply information sufficient for such probes.

Suitable Hosts, Control Systems and Methods In general terms, the production of a recombinant form of Taq polymerase typically involves the following: First, a DNA is obtained that encodes the mature (used here to include all muteins) enzyme or a fusion of the Taq polymerase to an additional sequence that does not destroy its activity or to an additional sequence cleavable under controlled conditions (such as treatment with peptidase) to give an active protein. If the sequence is uninterrupted by introns it is suitable for expression in any host. This sequence should be in an excisable and recoverable form.

The excised or recovered coding sequence is then preferably placed in operable linkage with suitable control sequences in a replicable expression vector. The vector is used to transform a suitable host and the transformed host cultured under favorable conditions to effect the production of the recombinant Taq polymerase. Optionally the Taq polymerase is isolated from the medium or from the cells; recovery and purification of the protein may not be necessary in some instances, where some impurities may be tolerated. -19- Each of the foregoing steps can be done in a variety of ways. For example, the desired coding sequences may be obtained from genomic fragments and used directly in appropriate hosts. The constructions for expression vectors operable in a variety of hosts are made using appropriate repl icons and control sequences, as set forth below. Suitable restriction sites can, if not normally available, be added to the ends of the coding sequence so as to provide an excisable gene to insert into these vectors.

The control sequences, expression vectors, and transformation methods are dependent on the type of host cell used to express the gene. Generally, procaryotic, yeast, insect or mammalian cells are presently useful as hosts. Procaryotic hosts are in general the most efficient and convenient for ,the production of recombinant proteins and therefore preferred for the expression of Taq polymerase.

In the particular case of Taq polymerase, evidence indicates that considerable deletion at the N-terminus of the protein may occur under both recombinant and native conditions, and that the activity of the protein is still retained. It appears that the native proteins isolated may be the result of proteolytic degradation, and not translation of a truncated gene. The mutein produced from the truncated gene of plasmid pFC85 is, however, fully active in assays for DNA polymerase, as is that produced from DNA encoding the full-length sequence. Since it is clear that certain N-terminal shortened forms are active, the gene constructs used or expression of the polymerase may also include the corresponding shortened forms of the codi ng sequence.

Control Sequences and Corresponding Hosts Procaryotes most frequently are represented by various strains of col . However, other microbial strains may also be used, such as bacilli, for example, Bacillus subtilis, various species of Pseudomonas, or other bacterial strains. In such procaryotic systems, plasmid vectors that contain replication sites and control sequences derived from a species compatible with the host are used.

For example, Ej_ col i is typically transformed using derivatives of pBR322, a plasmid derived from an E. coli species by Bolivar, et al . , Gene (1977) 2; 95. pBR322 contains genes for ampicillin and tetracycline resistance, and thus provides additional markers that can be either retained or destroyed in constructing the desired vector. Commonly used procaryotic control sequences, which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, include such commonly used promoters as the s-lactamase (penicillinase) and lactose (lac) promoter systems (Chang, et al . , Nature (1977) 198:1056), the tryptophan (trp) promoter system (Goeddel, et al . , Nucleic Acids Res. (1980) 8^:4057) and the lambda-derived P|_ promoter (Shimatake, et al . , Nature (1981). 292:128) and N-gene ribosome binding site, which has been made useful as a portable control cassette, which comprises a first DNA sequence that is the P^_ promoter operably linked to a second DNA sequence corresponding to N^ upstream of a third DNA sequence having at least one restriction site that permits cleavage within six bp 3' of the ^g^ sequence. Also useful is the phosphatase A (phoA) system described by Chang, et al . in European Patent Publication No. 196,864 published October 8, 1986, assigned to the same assignee. However, any available promoter system compatible with procaryotes can be used.

In addition to bacteria, eucaryotic microbes, such as yeast, may also be used as hosts. Laboratory strains of Saccharomyces cerevisiae, Baker's yeast, are most used, although a number of other strains are commonly available. While vectors employing the 2 micron origin of replication are illustrated (Broach, J. R. , Meth. Enz. (1983) 101:307), other plasmid vectors suitable for yeast expression are known (see, for example, Stinchcomb, et al . , Nature . (1979) 282:39, Tschempe, et al., Gene (1980) JL0:157 and Clarke, L., et al., Meth. Enz. (1983) 101:300) . Control sequences for yeast vectors include promoters for the synthesis of glycolytic enzymes (Hess, et al . , J . Adv. Enzyme Reg. (1968) 7_:149; Holland, et al . , Biotechnology (1978) 17:4900). -21- Additional promoters known in the art include the promoter for 3-phosphoglycerate kinase (Hitzeman, et al., J. Biol. Chem. (1980) 255:2073) , and those for other glycolytic enzymes, such as glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphof ructokinase, glucose-6-phosphate isomerase, 3- phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Other promoters that have the additional advantage of transcription controlled by growth conditions are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and enzymes responsible for maltose and galactose ultilization (Holland, supra).

It is also believed that terminator sequences are desirable at the 3' end of the coding sequences. Such terminators are found in the 3* untranslated region following the coding sequences in yeast-derived genes. Many of the vectors illustrated contain control sequences derived from the enolase gene containing plasmid peno46 (Holland, M. J., et al., J. Biol. Chem. (1981) 256:1385) or the LEU2 gene obtained from YEpl3 (Broach, J., et al., Gene (1978) _8 : 121 ) ; however, any vector containing a yeast-compatible promoter, origin of replication, and other control sequences is suitable.

It is also, of course, possible to express genes encoding polypeptides in eucaryotic host cell cultures derived from multicellular organisms. See, for example, Tissue Culture, Academic Press, Cruz and Patterson, editors (1973). Useful host cell lines include murine myelomas N51, VERO and HeLa cells, and Chinese hamster ovary (CHO) cells. Expression vectors for such cells ordinarily include promoters and control sequences compatible with mammalian cells such as, for example, the commonly used early and late promoters from Simian Virus 40 (SV 40) (Fiers, et al., Nature (1978) 273:113), or other viral promoters such as those derived from polyoma, Adenovirus 2, bovine papiloma virus, or avian sarcoma viruses, or immunoglobulin promoters and heat shock promoters. A system for expressing DNA in mammalian systems using the BPV as a vector is disclosed in U.S. Patent 4,419,446. A modification of this system is described in U.S. Patent 4,601,978. General aspects of mammalian cell -22- host system transformations have been described by Axel, U.S. Patent No. 4,399,216. It now appears, also, that "enhancer" regions are important in optimizing expression; these are, generally, sequences found upstream of the promoter region. Origins of replication may be obtained, if needed, from viral sources. However, integration into the chromosome is a common mechanism for DNA replication in eucaryotes.

Plant cells are also now available as hosts, and control sequences compatible with plant cells such as the nopal ine synthase promoter and polyadenylation signal sequences (Depicker, A., et al . , J. MoT. Appl. Gen. (1982) J^: 561 ) are available.

Recently, in addition, expression systems employing insect cells utilizing the control systems provided by baculovirus vectors have been described (Miller, D. W., et al . , in Genetic Engineering (1986) Setlow, J. K. et al . , eds., Plenum Publishing, Vol. 8, pp. 277- 297). These systems are also successful in producing Taq polymerase.

Transformations Depending on the host cell used, transformation is done using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride, as described by Cohen, S. N., Proc. Natl. Acad. Sci . (USA) (1972) 69:2110 is used for procaryotes or other cells that contain substantial cell wall barriers. Infection with Agrobacterium tumefaciens (Shaw, C. H., et al., Gene (1983) j?3_:315) is used for certain plant cells. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Vi ol ogy (1978) j>2:546 is preferred. Transformations into yeast are carried out according to the method of Van Solingen, P., et al., J. Bact. (1977) 130:946 and Hsiao, C. L., et al . , Proc. Natl. Acad. Sci. (USA) (1979) 76:3829.

Construction of a xgtll Expression Library The strategy for isolating DNA encoding desired proteins, such as the Taq polymerase encoding DNA, using the bacteriophage -23- vector lambda gtll, is as follows. A library can be constructed of EcoRI -flanked Alul fragments, generated by complete digestion of Thermus aquaticus DNA, inserted at the EcoRI site in the lambda gtll phage (Young and Davis, Proc. Natl. Acad. Sci USA (1983) 80:1194- 1198). Because the unique EcoRI site in this bacteriophage is located in the carboxyl -terminus of the β-galactosidase gene, inserted DNA (in the appropriate frame and orientation) is expressed as protein fused with β-galactosidase under the control of the lactose operon p romte r/ope ra to r .

Genomic expression libraries are then screened using the antibody plaque hybridization procedure. A modification of this procedure, referred to as "epitope selection," uses antiserum against the fusion protein sequence encoded by the phage, to confirm the identification of hybridized plaques. Thus, this library of recombinant phages could be screened with antibodies that recognize the 86,000-90,000 dalton Taq polymerase in order to identify phage that carry DNA segments encoding the antigenic determinants of this protein.

Approximately 2 x 10^ recombinant phage are screened using total rabbit Taq polymerase antiserum. In this primary screen, positive signals are detected and one or more of these plaques are purified from candidate plaques which failed to react with preimmune serum and reacted with immune serum and analyzed in some detail. To examine the fusion proteins produced by the recombinant phage, lysogens of the phage in the host Y1089 are produced. Upon induction of the lysogens and gel electrophoresis of the resulting proteins, each lysogen may be observed to produce a new protein, not found in the other lysogens, or duplicate sequences may result. Phage containing positive signals are picked; in this case, one positive plaque was picked for further identification and replated at lower densities to purify recombinants and the purified clones were analyzed by size class via digestion with EcoRI restriction enzyme. Probes can then be made of the isolated DNA insert sequences and labeled appropriately and these probes can be used in conventional colony or plaque hybridization assays described in Maniatis et al . , Molecular Cloning: A Laboratory Manual (1982). -24- The labeled probe was used to probe a second genomic library constructed in a Charon 35 bacteriophage (Wilhelmine, A. M. et al . , Gene (1983) 26:171-179). This library was made from Sau3A partial digestions of genomic Thermus aquaticus DNA and size fractionated fragments (15-20 kb) were cloned into the BamHI site of the Charon 35 phage. The probe was used to isolate phage containing DNA encoding the Taq polymerase. One of the resulting phage, designated CH35:Taq#4-2, was found to contain the entire gene sequence. Partial sequences encoding portions of the gene were also isolated.

Vector Construction Construction of suitable vectors containing the desired coding and control sequences employs standard ligation and restriction techniques that are well understood in the -art. Isolated plasmids, DNA sequences, or synthesized oligonucleotides are cleaved, tailored, and reli gated in the form desired.

Site-specific DNA cleavage is performed by treating with the suitable restriction enzyme (or enzymes) under conditions that are generally understood in the art, and the particulars of which are specified by the manufacturer of these commercially available restriction enzymes. See, e.g., New England Biolabs, Product Catalog. In general, about 1 yg of plasmid or DNA sequence is cleaved by one unit of enzyme in about 20 yl of buffer solution; in the examples herein, typically an excess of restriction enzyme is used to ensure complete digestion of the DNA substrate. Incubation times of about one hour to two hours at about 37°C are workable, although variations can be tolerated. After each incubation, protein is removed by extraction with phenol /chl oroform, and may be followed by ether extraction, and the nucleic acid recovered from aqueous fractions by precipitation with ethanol . If desired, size separation of the cleaved fragments may be performed by polyacryl amide gel or agarose gel electrophoresis using standard techniques. A general description of size separations is found in Methods in Enzymology (1980) 65:499-560. -25- Restricti on-cleaved fragments may be blunt-ended by treating with the large fragment of E. coli DNA polymerase I (Klenow) in the presence of the four deoxynucleotide triphosphates (dNTPs) using incubation times of about 15 to 25 minutes at 20 to 25 °C in 50 mM Tris pH 7.6, 50 mM NaCl, 10 mM MgCl2, 10 mM DTT and 50-100 y dNTPs. The Klenow fragment fills in at 5' sticky ends, but chews back protruding 3' single strands, even though the four dNTPs are present. If desi ed, selective repair can be performed by supplying only one of the, or selected, dNTPs within the limitations dictated by the nature of the sticky ends. After treatment with Klenow, the mixture is extracted with phenol /chloroform and ethanol . precipitated. Treatment under appropriate conditions with SI nuclease results in hydrolysis of any single-stranded portion.

Synthetic oligonucleotides may be prepared using the tri ester method of Matteucci , et al . , (J. Am. Chem. Soc. (1981) 103:3185-3191 ) or using automated synthesis methods. Kinasing of single strands prior to annealing or for labeling is achieved using an excess, e.g., approximately 10 units of polynucleotide kinase to 1 nM substrate in the presence of 50 mM Tris, pH 7.6, 10 mM MgC^, 5 mM dithiothreitol , 1-2 mM ATP. If kinasing is for labeling of probe, the ATP will contain high specific activity γ-^Ρ.

Ligations are performed in 15-30 yl volumes under the following standard conditions and temperatures: 20 mM Tris-Cl pH 7.5, 10 mM MgCl2, 10 mM DTT, 33 yg/ml BSA, 10 mM-50 mM NaCl , and either 40 yM ATP, 0.01-0.02 (Weiss) units T4 DNA ligase at 0°C (for "sticky end" ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14°C (for "blunt end" ligation). Intermolecular "sticky end" ligations are usually performed at 33-100 yg/ml total DNA concentrations (5-100 nM total end concentration). Intermolecular blunt end ligations (usually employing a 10-30 fold molar excess of linkers) are performed at 1 yM total ends concentration.

In vector construction employing "vector fragments", the vector fragment is commonly treated with bacterial alkaline phosphatase (BAP) in order to remove the 5' phosphate and prevent -26- religation of the vector. BAP digestions are conducted at pH 8 in approximately 150 mM Tris, in the presence of Na and Mg using about 1 unit of BAP per mg of vector at 60°C for about one hour. In order to recover the nucleic acid fragments, the preparation is extracted with phenol /chloroform and ethanol precipitated. Alternatively, religation can be prevented in vectors that have been double digested by additional restriction enzyme digestion of the unwanted fragments.

Modification of DNA Sequences For portions of vectors deri ed from cDNA or genomic DNA that require sequence modifications, site-specific primer-directed mutagenesis is used. This technique is now standard in the art, and is conducted using a primer synthetic oligonucleotide complementary to a single-stranded phage DNA to be mutagenized except for limited mismatching, representing the desi ed mutation. Briefly, the synthetic oligonucleotide is used as a primer to direct synthesis of a strand complementary to the phage, and the resulting double-stranded DNA is transformed into a phage-supporti ng host bacterium. Cultures of the transformed bacteria are plated in top agar, permitting plaque formation from single cells that harbor the phage.

Theoretically, 50% of the new plaques will contain the phage having, as a single strand, the mutated form; 50% will have the original sequence. The plaques are transferred to nitrocellulose filters and the "lifts" hybridized with kinased synthetic primer at a temperature that permits hybridization of an exact match, but at which the mismatches with the original strand are sufficient to prevent hybridization. Plaques that hybridize with the probe are then picked and cultured, and the DNA is recovered.

Verification of Construction In the constructions set forth below, correct ligations for plasmid construction are confirmed by first transforming col i strain MM294, or other suitable host, with the ligation mixture. Successful transformants are selected by ampicillin, tetracycline or -27- other antibiotic resistance or using other markers, depending on the mode of plasmid construction, as is understood in the art. Plasmids from the transformants are then prepared according to the method of Clewell, D.B., et al., Proc. Natl. Acad. Sc . (USA) (1969) J52_:1159, optionally following chloramphenicol amplification (Clewell, D.B., J . Bacteriol . (1972) 210:667). The isolated DNA is analyzed by restriction and/or sequenced by the dideoxy method of Sanger, F., et al., Proc. Natl. Acad. Sci. (USA) (1977) 74:5463 as further described by Messing, et al . , Nucleic Acids Res. (1981) j 309, or by the method of Maxam, et al . , Methods in Enzymology (1980) 65:499.

Host Exemplified Host strains used in cloning and expression herein are as follows: For cloning and sequencing, and for expression of constructions under control of most bacterial promoters, col i strain MM294 obtained from E^ coli Genetic Stock Center GCSC #6135, was used as the host. For expression under control of the |_NRBS promoter, E. coli strain K12 MCIOOO lambda lysogen, 7 53CI857 SusPgQ, ATCC 39531 may be used. Used herein is E^_ coli DG116, which was deposited with ATCC (ATCC 53606) on April 7, 1987.

For M13 phage recombinants, E. coli strains susceptible to phage infection, such as E . col i K12 strain DG98, are employed. The DG98 strain has been deposited with ATCC July 13, 1984 and has accession number 39768.

Mammalian expression can be accomplished in COS-7 C0S-A2, CV-1, and murine cells, and insect cell based expression in Spodoptera f rug peida) .

Stabilization of Enzyme Activity For long-term stability, the enzyme herein must be stored in a buffer that contains one or more non-ionic polymeric detergents. Such detergents are generally those that have a molecular weight in the range of approximately 100 to 250,000, preferably about 4,000 to -28- 200,000 daltons and stabilize the enzyme at a pH of from about 3.5 to about 9.5 preferably from about 4 to 8.5. Examples of such detergents include those specified on pages 295-298 of cCutcheon's Emulsifiers & Detergents. North American edition (1983) published by the McCutcheon Division of MC Publishing Co., 175 Rock Road, Glen Rock, NJ (USA), the entire disclosure of which is incorporated herein by reference. Preferably, the detergents are selected from the group comprising ethoxylated fatty alcohol ethers and lauryl ethers, ethoxylated alkyl phenols, octylphenoxy polyethoxy ethanol compounds, modified oxyethylated and/or oxypropyl ated straight-cha n alcohols, polyethylene glycol monooleate compounds, polysorbate compounds, and phenolic fatty alcohol ethers. More particularly preferred are Tween 20, from ICI Americas Inc., Wilmington, DE, which is a polyoxyethylated (20) sorbitan monolaurate, and Iconol™ NP-40, from BASF Wyandotte Corp. Parsippany, NJ , which is an ethoxylated alkyl phenol (nonyl).

The thermostable enzyme of this invention may be used for any purpose in which such enzyme is necessary or des rable. In a particularly preferred embodiment, the enzyme herein is employed in the amplification protocol set forth below.

Amplification Protocol The amplification protocol using the enzyme of the present invention may be the process for amplifying existing nucleic acid sequences that is disclosed and claimed in European Pat. Pub. No. 200,362, supra. Preferably, however, the enzyme herein is used in the amplificat on process described below.

In general, the amplification process involves a chain reaction for producing, in exponential quantities relative to the number of reaction steps involved, at least one specific nucleic acid sequence given (a) that the ends of the required sequence are known in sufficient detail that oligonucleot des can be synthesized which will hybridize to them, and (b) that a small amount of the sequence is available to initiate the chain reaction. The product of the chain reaction will be a discrete nucleic acid duplex with termini correspond ng to the ends of the specific primers employed.

Any nucleic acid sequence, in purified or nonpurified form, can be utilized as the starting nucleic acid(s), provided it contains or is suspected to contain the specific nucleic acid sequence desired. Thus, the process may employ, for example, DNA or RNA, including messenger RNA, which DNA or RNA may be s ngle-stranded or double-stranded. In addition, a DNA-RNA hybrid which contains one strand of each may be utilized. A mixture of any of these nucleic acids may also be employed, or the nucleic acids produced from a previous amplification reaction herein using the same or different primers may be so utilized. The specific nucleic acid sequence to be amplified may be only a fraction of a larger molecule or can be present initially as a discrete molecule, so that the specific sequence constitutes the entire nucleic acid.

It is not necessary that the sequence to be amplified be present initially in a pure form; it may be a minor fraction of a complex mixture, such as a portion of the β-globin gene contained in whole human DNA (as exemplified in Saiki et al . , Science, 230, 1530-1534 (1985)) or a portion of a nucleic acid sequence due to a particular microorganism which organism might constitute only a very minor fraction of a particular biological sample. The starting nucleic acid sequence may contain more than one desired specific nucleic acid sequence which may be the same or different. Therefore, the amplification process is useful not only for producing large amounts of one specific nucleic acid sequence, but also for amplifying simultaneously more than one different specific nucleic acid sequence located on the same or different nucleic acid molecules.

The nucleic acid(s) may be obtained from any source, for example, from plasmids such as pBR322, from cloned DNA or RNA, or from natural DNA or RNA from any source, including bacteria, yeast, viruses, organelles, and higher organisms such as plants or animals. DNA or RNA may be extracted from blood, tissue material such as chorionic villi, or amniotic cells by a variety of techniques such as that described by Maniatis et al . , supra, p. 280-281. -30- If probes are used which are specific to a sequence being amplified and thereafter detected, the cells may be directly used without extraction of the nucleic acid if they are suspended in hypotonic buffer and heated to about 90-100°C, until cell lysis and dispersion of intracellular components occur, generally 1 to 15 minutes. After the heating step the amplification reagents may be added directly to the lysed cells.

Any specific nucleic acid sequence can be produced by the amplification process. It is only necessary that a sufficient number of bases at both ends of the sequence be known in sufficient detail so that two oligonucleotide primers can be prepared which will hybridize to different strands of the desired sequence and at relative positions along the sequence such that an extension product synthesized from one primer, when it is separated from its template (complement), can serve as a template for extension of the other primer into a nucleic acid sequence of defined length. The greater the knowledge about the bases at both ends of the sequence, the greater can be the specificity of the primers for the target nucleic acid sequence, and thus the greater the efficiency of the process. it will be understood that the word "primer" as used hereinafter may refer to more than one primer, particularly in the case where there is some ambiguity in the information regarding the terminal sequence(s) of the fragment to be amplified. For instance, in the case where a nucleic acid sequence is inferred from protein sequence information, a collection of primers containing sequences representing all possible codon variations based on degeneracy of the genetic code will be used for each strand. One primer from this collection will be homologous with the end of the desired sequence to be ampl fied.

The oligonucleotide primers may be prepared using any suitable method, such as, for example, the phosphotriester and phosphodi ester methods described above, or automated embodiments thereof. In one such automated embodiment, di ethyl phosphoramidites are used as starting materials and may be synthesized as described by Beaucage et al . , Tetrahedron Letters (1981), 22:1859-1862. One method for synthesizing oligonucleotides on a modified solid support is described in U.S. Patent No. 4,458,066. It is also possible to use a primer which has been isolated from a biological source (such as a restriction endonuclease digest).

The specific nucleic acid sequence is produced by using the nucleic acid containing that sequence as a template. The first step involves contacting each nucleic acid strand with four different nucleotide triphosphates and one oligonucleotide primer for each different nucleic acid sequence being amplified or detected. If the nucleic acids to be amplified or detected are DNA, then the nucleotide triphosphates are dATP, dCTP, dGTP and TTP.

The nucleic acid strands are used as a template for the synthesis of additional nucleic acid strands. This synthesis can be performed using any suitable method. Generally it occurs in a buffered aqueous solution, preferably at a pH of 7-9, most preferably about 8. Preferably, a molar excess (for cloned nucleic acid, usually about 1000:1 primer:template, and for genomic nucleic acid, usually about 10^:1 primer: tempi ate) of the two oligonucleotide primers is added to the buffer containing the separated template strands. It is understood, however, that the amount of complementary strand may not be known if the process herein is used for diagnostic applications, so that the amount of primer relative to the amount of complementary strand cannot be determined with certainty. As a practical matter, however, the amount of primer added will- generally be in molar excess over the amount of complementary strand (template) when the sequence to be amplified is contained in a mixture of complicated long-chain nucleic acid strands. A large molar excess is preferred to improve the efficiency of the process.

Preferably the concentration of nucleotide triphosphates is 150-200 y each in the buffer for amplification and MgCl2 is present in the buffer in an amount of 1.5-2 mM to increase the efficiency and specificity of the reaction. -32- The resulting solution is then treated according to whether the nucleic acids being amplified or detected are double or single- stranded. If the nucleic acids are single-stranded, then no denaturation step need by employed, and the reaction mixture is held at a temperature which promotes hybridization of the primer to its complementary target (template) sequence. Such temperature is generally from about 35°C to 65°C or more, preferably about 37-60°C for an effective time, generally one-half to five minutes, preferably one-three minutes. Preferably, 45-58°C is used for Taq polymerase and >15-mer primers to increase the specificity of primer hybridization. Shorter primers need lower temperatures.

The complement to the original single-stranded nucleic acid may be synthesized by adding one or " two oligonucleotide primers thereto. If an appropriate single primer is added, a primer extension product is synthesized in the presence of the primer, the thermostable enzyme and the nucleotide triphosphates. The product will be partially complementary to the single-stranded nucleic acid and will hybridize with the nucleic acid strand to form a duplex of strands of unequal length which may then be separated into single strands as described above to produce two single separated complementary strands. Alternatively, two appropriate primers may be added to the single-stranded nucleic acid and the reaction carried out.

If the nucleic acid contains two strands, it is necessary to separate the strands of the nucleic acid before it can be used as the template. This strand separation can be accomplished by any suitable denaturing method including physical, chemical or enzymatic means. One preferred physical method of separating the strands of the nucleic acid involves heating the nucleic acid until it is completely (>99¾) denatured. Typical heat denaturation involves temperatures ranging from about 90 to 105°C for times generally ranging from about 0.5 to 5 minutes. Preferably the effective denaturing temperature is 90-100°C for 0.5 to 3 minutes. Strand separation may also be induced by an enzyme from the class of enzymes known as heli cases or the enzyme RecA, which has helicase activity and in the presence of riboATP is known to denature DNA. The reaction conditions suitable for separating the strands of nucleic acids with helicases are described by Kuhn Hof fmann-Berl i ng, CSH-Quantitat ve Biology, 43:63 (1978), and techniques for using RecA are reviewed in C. Radding, Ann. Rev. Genetics, _l_L:405-37 (1982). The denaturation produces two separated complementary strands of equal or unequal length.

If the double-stranded nucleic acid is denatured by heat, the reaction mixture is allowed to cool to a temperature which promotes hybridization of each primer present to its complementary target (template) sequence. This temperature is usually from about 35°C to 65°C or more, depending on reagents, preferably 37-60°C, maintained for an effective time, generally 0.5 to 5 minutes, and preferably 1-3 minutes. In practical terms, the temperature is simply lowered from about 95°C to as low as 37°C, preferably to about 45-58°C for Taq polymerase, and hybridization occurs at a temperature within this range.

Whether the nucleic acid is single- or double-stranded, the thermostable enzyme may be added at the denaturation step or when the temperature is being reduced to or is in the range for promoting hybridization. The reaction mixture is then heated to a temperature at which the activity of the enzyme is promoted or optimized, i.e., a temperature sufficient to increase the activity of the enzyme in facilitating synthesis of the primer extension products from the hybridized primer and template. The temperature must actually be sufficient to synthesize an extension product of each primer which is complementary to each nucleic acid template, but must not be so high as to denature each extension product from its complementary template (i.e., the temperature is generally less than about 80°C-90°C).

Depending mainly on the types of enzyme and nucleic acid(s) employed, the typical temperature effective for this synthesis reaction generally ranges from about 40 to 80°C, preferably 50-75°C. The temperature more preferably ranges from about 65-75°C when a DNA polymerase from Thermus aquaticus is employed. The period of time required for this synthesis may range from about 0.5 to 40 minutes or more, depending mainly on the temperature, the length of the nucleic -34- acid, the enzyme and the complexity of the nucleic acid mixture, preferably one to three minutes. If the nucleic acid is longer, a longer time period is generally required. The presence of dimethyl sulfoxide (DMSO) is not necessary or recommended because D SO was found to inhibit Taq polymerase enzyme activity.

The newly synthesized strand and its complementary nucleic acid strand form a double-stranded molecule which is used in the succeeding steps of the process. In the next step, the strands of the double-stranded molecule are separated by heat denaturation at a temperature effective to denature the molecule, but not so high that the thermostable enzyme is completely and irreversibly, denatured or inactivated. Depending mainly on the type of enzyme and the length of nucleic acid, this temperature generally ranges from about 90 to 105°C, more preferably 90-100°C, and the time for denaturation typically ranges from 0.5 to four minutes, depending mainly on the temperature and nucleic acid length.

After this time, the temperature is decreased to a level which promotes hybridization of the primer to its complementary single-stranded molecule (template) produced from the previous step. Such temperature is described above.

After this hybridization step, or in lieu of (or concurrently with) the hybridization step, the temperature is adjusted to a temperature that is effective to promote the activity of the thermostable enzyme to enable synthesis of a primer extension product using as template the newly synthesized strand from the previous step. The temperature again must not be so high as to separate (denature) the extension product from its template, as previously described (usually from 40 to 80°C for 0.5 to 40 minutes, preferably 50 to 70°C for one-three minutes). Hybridization may occur during this step, so that the previous step of cooling after denaturation is not required. In such a case, using simultaneous steps, the preferred temperature range is 50-70°C.

The heating and cooling steps of strand separation, hybridizat on, and extension product synthesis can be repeated as - - often as needed to produce the desired quantity of the specific nucleic acid sequence, depending on the ultimate use. The only limitation is the amount of the primers, thermostable enzyme and nucleotide triphosphates present. Preferably, the steps are repeated at least twice. For use in detection, the number of cycles will depend, e.g., on the nature of the sample. For example, fewer cycles will be requ ed if the sample being ampl fied is pure. If the sample is a complex mixture of nucleic acids, more cycles will be required to amplify the signal sufficiently for its detection. For general amplification and detection, preferably the process is repeated at least 20 times.

When labeled sequence-specific probes are employed as described below, preferably the steps are repeated at least five times. When human genomic DNA is employed with such probes, the process is repeated preferably 15-30 times to amplify the sequence sufficiently that a clearly detectable signal is produced, i.e., so that background noise does not interfere with detect on.

As will be described in further detail below, the amount of the specific nucleic acid sequence produced will accumulate in an exponential fashion.

No additional nucleotides, primers, or thermostable enzyme need be added after the initial addition, provided that the enzyme has not become denatured or inactivated irreversibly, in which case it is necessary to replenish the enzyme after each denaturing step. Addition of such materials at each step, however, will not adversely affect the reaction.

When it is desired to produce more than one specific nucleic acid sequence from the first nucleic acid or mixture of nucleic acids, the appropriate number of different oligonucleotide primers are utilized. For example, if two different specific nucleic acid sequences are to be produced, four primers are utilized. Two of the primers are specific for one of the specific nucleic acid sequences and the other two primers are specific for the second specific nucleic acid sequence. In this manner, each of the two different specific sequences can be produced exponentially by the present process. -36- After the appropriate length of time has passed to produce the desired amount of the specific nucleic acid sequence, the reaction may be halted by inactivating the enzyme in any known manner (e.g., by adding EDTA, phenol, SDS or CHCI3) or by separating the components of the reaction.

The amplification process may be conducted continuously. In one embodiment of an automated process, the reaction mixture may be temperature cycled such that the temperature is programmed to be controlled at a certain level for a certain time.

One such instrument for this purpose is an automated machine for handling the amplification reaction of this invention. This instrument utilizes a liquid handling system under computer control to make liquid transfers of enzyme stored at a controlled temperature in a f rst receptacle into a second receptacle whose temperature is controlled by the computer to conform to a certain incubation profile. The second receptacle stores the nucleic acid sequence(s) to be amplified plus the nucleotide triphosphates and primers. The computer includes a user interface through which a user can enter process parameters that control the characteristics of the various steps in the amplification sequence such as the times and temperatures of incubation, the amount of enzyme to transfer, etc.

A preferred machine that may be employed utilizes temperature cycling without a liquid handling system because the enzyme need not be transferred at every cycle. Such a machine consists of the following systems: 1. A heat-conducting container for holding a given number of tubes, preferably 500 yl tubes, which contain the reaction mixture of nucleotide triphosphates, primers, nucleic acid sequences, and enzyme. 2. A means to heat, cool, and maintain the heat-conducting container above and below room temperature, which means has an input for receiving a control signal for controlling which of the temperatures at or to which the container is heated, cooled or maintained. (These may be Peltier heat pumps available from Materials -37- Electronics Products Corporation in Trenton, NJ or a water heat exchanger. ) 3. A computer means (e.g., a microprocessor controller), coupled to the input of said means, to generate the signals that control automatically the amplificat on sequence, the temperature levels, and the temperature ramping and timing.

In another embodiment, the enzyme used for the synthesis of primer extension products can be immobilized in a column. The other reaction components can be continuously c rculated by a pump through the column and a heating coil in series. Thus, the nucleic acids produced can be repeatedly denatured without inactivating the enzyme.

The amplification protocol is demonstrated diagrammatical ly below, where double-stranded DNA containing the desired sequence [S] comprised of complementary strands [S+] and [S~] is utilized as the nucleic acid. During the first and each subsequent reaction cycle, extension of each oligonucleotide primer on the original template will produce one new ssDNA molecule product of indefinite length that terminates with only one of the primers. These products, hereafter referred to as "long products," will accumulate in a linear fashion; that is, the amount present after any number of cycles will be proportional to the number of cycles.

The long products thus produced will act as templates for one or the other of the oligonucleotide primers during subsequent cycles and will produce molecules of the desi ed sequence [S+] or [S]. These molecules will also function as templates for one or the other of the oligonucleotide primers, producing further [S+] and [S~], and thus a chain reaction can be sustained that will result in the accumulation of [S] at an exponential rate relative to the number of cycles.

By-products formed by ol gonucleotide hybridizations other than those intended are not self-catalytic (except in rare instances) and thus accumulate at a linear rate.

The specific sequence to be amplified, [S], can be depicted diagrammatically as: -38- [S+] 5' AAAAAAAAAAXXXXXXXXXXCCCCCCCCCC 3' CS"] 3' TTTTTTTTTTYYYYYYYYYYGGGGGGGGGG 5' The appropriate oligonucleotide primers would be: Primer 1: 3' GGGGGGGGGG 5' Primer 2: 5' AAAAAAAAAA 3' so that if DNA containing [S] zzzzzzzzzzzzzzzzAAAAAAAAAAXXXXXXXXXXCCCCCCCCCCzzzzzzzzzzzzzzzz zzzzzzzzzzzzzzzzTTTTTTTTTTYYYYYYYYYYGGGGGGGGGGzzzzzzzzzzzzzzzz is separated into single strands and its single strands are hybridized to Primers 1 and 2, the following extension reactions can be catalyzed by a thermostable polymerase in the presence of the four nucleotide triphosphates : 3' 5' extends ^— GGGGGGGGGG Primer 1 zzzzzzzzzzzzzzzzAAAAAAAAAAXXXXXXXXXXCCCCCCCCCCzzzzzzzzzzzzzzzz original template strand"1" original template strand" zzzzzzzzzzzzzzzzTTTTTTTTTTYYYYYYYYYYGGGGGGGGGGzzzzzzzzzzzzzzzz Primer 2 AAAAAAAAAA ^extends 5' 3' On denaturat on of the two duplexes formed, the products are: 3' 5' ....zzzzzzzzzzzzzzzzTTTTTTTTTTYYYYYYYYYYGGGGGGGGGG newly synthesized long product 1 5' 3' zzzzzzzzzzzzzzzzAAAAAAAAAAXXXXXXXXXXCCCCCCCCCCzzzzzzzzzzzzzzzz original template strand"1" 3' 5' zzzzzzzzzzzzzzzzTTTTTTTTTTYYYYYYYYYYGGGGGGGGGGzzzzzzzzzzzzzzzz original template strand" 5' 3' AAAAAAAAAAXXXXXXXXXXCCCCCCCCCCzzzzzzzzzzzzzzzz newly synthesized long product 2 If these four strands are allowed to rehybridize with Primers 1 and 2 in the next cycle, the thermostable polymerase will catalyze the following reactions: Primer 2 5' AAAAAAAAAA extends to here 3' zzzzzzzzzzzzzzzzzzTTTTTTTTTTYYYYYYYYYYGGGGGGGGGG 5' newly synthesized long product 1 extends < — GGGGGGGGGG 5' Primer 1 5 ' zzzzzzzzzzzzzzAAAAAAAAAAXXXXXXXXXXCCCCCCCCCCzzzzzzzzzzzzzz 31 original template strand Primer 2 5* AAAAAAAAAA ^extends 31 zzzzzzzzzzzzzzzzzzTTTTTTTTTTYYYYYYYYYGGGGGGGGGGzzzzzzzzzz 5 ' original template strand" extends to here<^ GGGGGGGGGG 5' Primer 1 5 ' AAAAAAAAAAXXXXXXXXXXCCCCCCCCCCzzzzzzzzzzzzzzzz ..31 newly synthesized long product 2 If the strands of the above four duplexes are separated, the following strands are found: 5' AAAAAAAAAAXXXXXXXXXXCCCCCCCCCC 3' newly synthesized [S ] 3' zzzzzzzzzzzzzzzzzzzTTTTTTTTTTYYYYYYYYYYGGGGGGGGGG 5" fi st cycle synthesized long product 1 3' zzzzzzzzzzzzzzzzzzzTTTTTTTTTTYYYYYYYYYYGGGGGGGGGG 5' newly synthesized long product 1 5 ' zzzzzzzzzzzzzzzzzzzAAAAAAAAAAXXXXXXXXXXCCCCCCCCCCzzzzzzzzz 3 ' original template strand+ 5 ' AAAAAAAAAAXXXXXXXXXXCCCCCCCCCCzzzzzzzzzzzzzzzz ...3 ' newly synthesized long product 2 31.. zzzzzzzzzzzzzzzTTTTTTTTTTYYYYYYYYYYGGGGGGGGGGzzzzzzzzzzzzzzzz ...5 ' original template strand" 3' TTTTTTTTTTYYYYYYYYYYGGGGGGGGGG 5' newly synthesized [S-] 5 ' AAAAAAAAAAXXXXXXXXXXCCCCCCCCCCzzzzzzzzzzzzzzz ...3 ' first cycle synthesized long product 2 - - It is seen that each strand which terminates with the oligonucleotide sequence of one primer and the complementary sequence of the other is the specific nucleic acid sequence [S] that is desired to be produced.

The amount of original nucleic acid remains constant in the entire process, because it is not replicated. The amount of the long products increases linearly because they are produced only from the original nucleic acid. The amount of the specific sequence increases exponentially. Thus, the specific sequence will become the predominant species. This is illustrated in the following table, which indicates the relative amounts of the species theoretically present after n cycles, assuming 100% efficiency at each cycle: •41- Number of Double Strands After 0 to n Cycles Long Specific Cycle Number Template Products Sequence [S] 0 1 1 0 2 2 1 3 3 4 5 5 26 10 10 1013 15 15 32,752 20 20 1,048,555 n (2n-n-l) When a single-stranded nucleic acid is utilized as the template, only one long product is formed per cycle.

A sequence within a given sequence can be amplified after a given number of amplifications to obtain greater specificity of the reaction by adding after at least one cycle of amplification a set of primers that are complementary to internal sequences (that are not on the ends) of the sequence to be amplified. Such primers may be added at any stage and will provide a shorter amplified fragment. Alternatively, a longer fragment can be prepared by using primers with non-complementary ends but having some overlap with the primers previously utilized in the amplification.

The method herein may be utilized to clone a particular nucleic acid sequence for insertion into a suitable expression vector. The vector may then be used to transform an appropriate host organism to produce the gene product of the sequence by standard methods of recombinant DNA technology.

The amplification process herein may yield a mixture of nucleic acids, resulting from the original template nucleic acid, the expected target amplified products, and various background non-target products. The amplified product can also be a mixture if the original template DNA contains multiple target sequences, such as in a heterozygous diploid genome or when there is a family of related genes.

The primers herein may be modified to assist the rapid and specific cloning of the mixture of DNAs produced by the amplification reaction. In one such modification, a restriction site is contained in each of the primers or in the sequence to be amplified and cloned. Preferably, the same or different restriction sites are incorporated at the 5' ends of the primers to result in restriction sites at the two ends of the amplified product. When cut with the appropriate enzymes, the amplified product can then be easily inserted into plasmid or vial vectors and cloned. This cloning allows the analysis or expression of individual amplified products, not a mixture.

If the primers have restriction sites incorporated therein, the same restriction site can be used for both primers. The use, however, of different sites allows the insertion of the product into the vector with a specific orientation and suppresses multiple insertions as well as insertions arising from amplifications based on only one of the two primers. The specific orientation is useful when cloning into single-strand sequencing vectors, when single-strand hybridization probes are used, or when the cloned product is being expressed.

One method to prepare the primers is to choose a primer sequence which differs minimally from the target sequence. Regions in which each of the primers is to be located are screened for homology to restriction sites appropriate to the desired vector. For example, the target sequence "CAGTATCCGA... " differs by only one base from one containing a BamHI site. A primer sequence is chosen to match the target exactly at its 3' end, and to contain the altered sequence and restriction site near its 5' end (for example, "CAGgATCCGA... ", where the lower case letter symbolizes a mismatch with the target -43- sequence). This minimally altered sequence will not interfere with the ability of the primer to hybridize to the original target sequence and to initiate polymerizat on. After the first amplification cycle the primer is copied, becomes the target, and matches exactly with new primers.

After the amplificat on process, the products are cleaved with the appropriate restriction enzymes, the restriction digest is optionally separated from inhibitors of ligation such as the nucleotide triphosphates and salts by, for example, passing over a desalting column, or a molecular weight chromatography column, or through a membrane, and the digestion product(s) containing the amplified sequence to be cloned is/are inserted by ligation into a cloning vector such as bacteriophage M13. The cloning vector generally has a selectable marker and may optionally also have a promoter. The gene may then be sequenced and/or expressed, if it codes for a protein, using well known techniques. The gene may also be sequenced by adding an appropriate primer during the amplification process which primer is complementary to the desired portion which is to be sequenced. The primer will form an extension product, and the extent of amplification with such extension product will provide sequence information.

Another method for preparing the primers involves taking the 3' end of the primers from the target sequence and adding the desired restriction site(s) to the 5' end of the primer. For the above example, a Hindi 11 site could be added to make the sequence "cgaagcttCAGTATCCGA... ", where lower case letters are as described above. The added bases would not contribute to the hybridization in the f st cycle of amplification, but would match in subsequent cycles. The final amplified products are then cut with restriction enzyme(s) and cloned and expressed as described above. The gene being amplified may be, for example, human beta-hemoglobin or the human HLA DQ, DR or DP-a and -β genes.

In an alternative, but less preferred and less efficient, method of cloning wherein blunt-end ligation is employed rather than -44- sticky-end ligation (using restriction enzymes), the basic amplification procedure is employed without concern for restriction enzymes in the primers or sequence(s) to be cloned. The steps must be repeated sufficiently, however, to produce enough amplified sequence(s) to effect ligation. Blunt-end ligation requires greater concentrations of sequence(s) and cloning vector(s) to be present than sticky-end ligation. In addition, the ligation must take place in the presence of a ligase, such as T4 ligase, E. col ligase and ligase. Once the amplified product is obtained, the ligation procedure is a standard procedure using conditions well known to those skilled in the art.

The cloning method which does not involve blunt end ligation controls the orientation or multiplicity of insertion of the amplified product into the cloning vector. in addition, the process herein can be used for _i_n vitro mutagenesis. The oligonucleotide primers need not be exactly complementary to the nucleic acid sequence which is being amplified. It is only necessary that they be able to hybridize to the sequence sufficiently well to be extended by the thermostable enzyme. The product of an amplification reaction wherein the primers employed are not exactly complementary to the original template will contain the sequence of the primer rather than the template, thereby introducing an ill vitro mutation. In further cycles this mutation will be amplified with an undiminished efficiency because no further mispaired primings are required. The mutant thus produced may be inserted into an appropriate vector by standard molecular biological techniques and might confer mutant properties on this vector such as the potential for production of an altered protein.

The process of making an altered DNA sequence as described above could be repeated on the altered DNA using different primers to induce further sequence changes. In this way a series of mutated sequences could gradually be produced wherein each new addition to the series could differ from the last in a minor way, but from the original DNA source sequence in an increasingly major way. In this manner changes could be made ultimately which were not feasible in a single step due to the inability of a very seriously mismatched primer to function.

In addition, the primer can contain as part of its sequence a non-complementary sequence, provided that a sufficient amount of the primer contains a sequence which is complementary to the strand to be amplified. For example, a nucleotide sequence which is not complementary to the template sequence (such as, e.g., a promoter, linker, coding sequence, etc.) may be attached at the 5' end of one or both of the primers, and thereby appended to the product of the amplification process. After the extension primer is added, sufficient cycles are run to achieve the desired amount of new template contain ng the non-complementary nucleotide insert. This allows production of large quantities of the combined fragments in a relatively short period of time (e.g., two hours or less) using a simple technique.

The method herein may also be used to enable detection and/or characterization of specific nucleic acid sequences associated with infectious diseases, genetic disorders or cellular disorders such as cancer, e.g., oncogenes. Amplification is useful when the amount of nucleic acid available for analysis is very small, as, for example, in the prenatal diagnosis of sickle cell anemia using DNA obtained from fetal cells. Amplification is particularly useful if such an analysis is to be done on a small sample using non-radioactive detection techniques which may be inherently insensitive, or where radioactive techniques are being employed but where rapid detection is desi rable.

For purposes of this invention genetic diseases may include specific deletions and/or mutations in genomic DNA from any organism, such as, e.g., sickle cell anemia, o-thalassemia, s-thalassemia, and the like. Sickle cell anemia can be readily detected via oligomer restriction analysis as described in EP Patent Publication 164,054 published December 11, 1985, or via a RFLP-like analysis following amplification of the appropriate DNA sequence by the present method. -46- α-Thalassemia can be detected by the absence of a sequence, and e- thalassemia can be detected by the presence of a polymorphic restriction site closely linked to a mutation that causes the disease.

All of these genetic diseases may be detected by amplifying the appropriate sequence and analyzing it by Southern blots without using radioactive probes. In such a process, for example, a small sample of DNA from, e.g., amniotic fluid containing a very low level of the desi ed sequence is amplified, cut with a restriction enzyme, and analyzed via a Southern blotting technique. The use of non- radioactive probes is facilitated by the high level of the amplified signal.

In another embodiment, a small sample of DNA may be amplified to a convenient level and then a further cycle of extension reactions performed wherein nucleotide derivatives which are readily detectable (such as ^P-labeled or biotin-labeled nucleotide triphosphates) are incorporated directly into the final DNA product, which may be analyzed by restriction and electrophoretic separation or any other appropriate method.

In a further embodiment, the nucleic acid may be exposed to a particular restriction endonuclease prior to amplification. Since a sequence which has been cut cannot be amplified, the appearance of an amplified fragment, despite prior restriction of the DNA sample, implies the absence of a site for the endonuclease within the amplified sequence. The presence or absence of an amplified sequence can be detected by an appropriate method.

A practical application of this technique can be illustrated by its use in facilitating the detection of sickle cell anemia via the oligomer restriction technique described herein and in EP 164,054, supra, and Saiki et al., Bio/Technology, 3, PP 1008-1012 (1985). Sickle cell anemia is a hemoglobin disease which is caused by a single base pair change in the sixth codon of the e-globin gene.

The method of this invention may also be used to detect directly single-base pair variations in nucleic acid sequence (such as genomic DNA) using sequence-specific oligonucleotides.- In this -47-method, the sequence variation, whether resulting from cancer, an infectious disease, or a genetic disease, e.g., a genetic lesion, is directly detected, eliminating the need for restriction digestion, electrophoresis, and gel manipulations otherwise required. The use of sequence-spec fic oligonucleot des in a dot blot format after amplification, as described herein, provides for improved specificity and sensitivity of the probe; an interpretable signal can be obtained with a 0.04 yg sample in six hours. Also, if the amount of sample spotted on a membrane is increased to 0.1-0.5 ug, non-isotopically labeled oligonucleotides may be utilized rather than the radioactive probes used in previous methods. Furthermore, the process described hereinbelow is applicable to use of sequence-specific oligonucleotides less than 19-mers in size, thus allowing use of more discriminatory sequence-specific ol igonucleotides.

Regarding genetic diseases, while RFLP requires a polymorphic restriction site to be associated with the disease, sequence-specific oligonucleotides directly detect the genetic lesion and are generally more useful for the analysis of such diseases as hemoglobin C disease, α-1-antitrypsin and β-thalassemia, which result from single-base mutations. In addition, the oligonucleotides can be used to distinguish between genetic variants which represent different alleles (e.g., HLA typing), indicating the feasibility of a sequence-specific ol igonucleoti de-based HLA typing kit that includes a thermostable enzyme.

In one embodiment of the invention herein wherein a nucleotide variation in sequence is to be detected, the sample, amplified as described above using one primer for each strand of each nucleic acid suspected of containing the nucleotide variation, is spotted directly on a series of membranes and each membrane is hybridized with a different labeled sequence-specific oligonucleotide probe. One procedure for spotting the sample on a membrane is descibed by Kafotos et al., Nucleic Acids Research, 7_:1541~1552 (1979). -48- Briefly, the DNA sample affixed to the membrane may be pretreated with a prehyb dization solution containing sodium dodecyl sulfate, Ficoll, serum albumin and various salts prior to the probe being added. Then, a labeled oligonucleotide probe which is specific to each sequence variation to be detected is added to a hybridization solution similar to the prehybridizat on solution. The hybridization solution is applied to the membrane and the membrane is subjected to hybridizat on conditions that will depend on the probe type and length, the type and concentration of ingredients, etc. Generally, hybridization is carried out at about 25 to 75°C, preferably 35 to 65°C, for 0.25-50 hours, preferably less than three hours. The greater the stringency of conditions, the greater the required complementa ity for hybridization between the probe and sample. If the background level is high, stringency may be increased accordingly. The stringent conditions can also be incorporated in the wash.

After the hybridization, the sample is washed of unhybridized probe using any suitable means such as by washing one or more times with varying concentrations of standard saline phosphate EDTA (SSPE) (180 mM NaCl , 10 mM NaHP04 and 1 M EDTA, pH 7.4) solutions at 25-75°C for about 10 minutes to one hour, depending on the temperature. The label is then detected by using any appropriate detection technique.

The sequence-specific oligonucleot de employed herein is an oligonucleot de which is generally prepared and 'selected as described above for preparing and selecting the primers. As described above, the sequence-specific oligonucleot de must encompass the region of the sequence which spans the nucleotide variation being detected and must be specific for the nucleotide variation being detected. For example, if it is desired to detect whether a sample contains the mutation for sickle cell anemia, one oligonucleot de will be prepared which contains the nucleotide sequence site characteristic of the normal $-globin gene and one ol gonucleotide will be prepared which contains the nucleotide sequence characteristic of the sickle cell allele. Each oligonucleot de would be hybridized to duplicates of the same sample to determine whether the sample contains the mutation. -49- The polymorphic areas of HLA class II genes are localized to specific regions of the first exon and are flanked by conserved sequences, so that oligonucleotide primers complementary to opposite strands of the conserved 5' and 3' ends of the first exon can be prepared.

The number of oligonucleotides employed for detection of the polymorphic areas of the HLA class II genes will vary depending on the type of gene, which has regions of base pair variation which may be clustered or spread apart. If the regions are clustered, as in the case with HLA-DQ-o, then one oligonucleotide is employed for each allele. If the regions are spread apart, as in the case with HLA-DQ-β and HLA-DR-β, then more than one probe, each encompassing an allelic variant, will be used for each allele.. In the case of HLA-DQ-β and HLA-DR-g, three probes are employed for the three regions of the locus where allelic variations may occur. For detection of insulin-dependent diabetes mellitus (IDDM) four probes for the HLA-DRg second exon are employed.

Haplotypes can be inferred from segregation analysis in families or, in some cases, by direct analysis of the individual DNA sample. Specific allelic combinations (haplotypes) of sequence-specific oligonucleotide reactivities can be identified in heterozygous cells by using restriction enzyme digestion of the genomic DNA prior to amplification- For example, if in DQB one finds three highly variable subregions A, B, and C within a single amplified region, and if there are six different sequences at each region (Al-6, Bl-6, Cl-6, then an individual could be typed in the DQe locus by sequence-specific oligonucleotide probe analysis as containing Al, A2; B2, B3; CI, C4, with the possible haplotype combinations of Al, B2, CI; Al, B2, C4; A2, B2, CI; A2, B2, C4; Al, B3, CI; Al, B3, C4; Al, B2, CI; and Al, B2, C4.

If the genomic DNA is digested with a polymorphic restriction enzyme prior to amplification, and if the enzyme cuts both alleles between the primers, there is no reactivity with the sequence- - - specific probes due to lack of amplification, and the result is uninformative. If the enzyme cuts neither allele, the probe results with digested and undigested genomic DNA are the same and the result is uninformative. If the enzyme cuts only one allele, however, then one can infer both haplotypes by comparing the probe reactivity patterns on digested and undigested DNA.

The haplotypes can be deduced by comparing sequence-specific oligonucleotide reactivities with uncut genomic DNA and genomic DNA cut with one or several enzymes known to be polymorphic and to recognize sites between the primers.

The length of the sequence-specific oligonucleotide will depend on many factors, including the particular target molecule being detected, the source of oligonucleotide, and the nucleotide composition. For purposes herein, the sequence-specific oligonucleotide typically contains 15-25 nucleotides, although it may contain more or fewer nucleotides. While oligonucleotides which are at least 19 mers in length may enhance specificity and/or sensitivity, probes which are less than 19 mers, e.g., 16-mers, may show more sequence-specific discrimination presumably because a single mismatch is more destabilizing. Because amplification increases specificity so that a longer length is less critical, and hybridization and washing temperatures can be lower for the same salt concentration, it is preferred to use oligonucleotides which are less than 19 mers in length.

Where the sample is first placed on the membrane and then detected with the oligonucleotide, the oligonucleotide must be labeled with a suitable label moiety, which may be detected by spectroscopic, photochemical, biochemical, immunochemical or chemical means. Immunochemical means include antibodies which are capable of forming a complex with the oligonucleotide under suitable conditions, and biochemical means include polypeptides or lectins capable of forming a complex with the oligonucleotide under the appropriate conditions. Examples include fluorescent dyes, electron-dense reagents, enzymes capable of depositing insoluble reaction products or being detected chromogenically , such as horseradish peroxidase, alkaline phosphatase, -51- a radioactive label such as , or biotin. If biotin is employed, a spacer arm may be utilized to attach it to the oligonucleotide. Preferably, the label moiety is horseradish peroxidase.

Alternatively, in one "reverse" dot blot format, at least one of the primers and/or at least one of the four nucleotide triphosphates is labeled with a detectable label, so that the resulting amplified sequence is labeled. These labeled moieties may be present initially in the reaction mixture or added during a later cycle of the amplification to introduce the label to the amplification product. Then an unlabeled sequence-specific oligonucleot de capable of hybridizing with the amplified nucleic acid sequence, if the sequence variation(s) (whether normal or mutant) is/are present, is spotted on (affixed to) the membrane under prehybridization conditions as described above. The amplified sample is then added to the pretreated membrane under hybridization conditions as described above. Finally, detection means are used to determine if an amplified sequence in the nucleic acid sample has hybridized to the oligonucleotide affixed to the membrane. Hybridization will occur only if the membrane-bound sequence containing the variation is present in the amplification product, i.e., only if a sequence of the probe is complementary to a region of the amplified sequence.

In another version of the "reverse" dot blot format, the amplificat on is carried out without employing a label as with the "forward" dot blot format described above, and a labeled sequence-specific oligonucleotide probe capable of hybridizing with the amplified nucleic acid sequence containing the variation, if present, is spotted on (affixed to) the membrane under prehybridization conditions as described above. The amplified sample is then added to the pretreated membrane under hybridization conditions as described above. Then the labeled oligonucleotide or a fragment thereof is released from the membrane in such a way that a detection means can be used to determine if an amplified sequence in the sample hybridized to the labeled ol gonucleotide. The release may take place, for example, by adding a restriction enzyme to the membrane which recognizes a restriction site in the probe. This procedure, known as oligomer -52- restriction, is described more fully in EP Patent Publication 164,054 published December 11, 1985.

In both the forward and reverse dot blot methods, the genetic diseases which may be detected include specific deletions, insertions and/or substitutions in any base pair mutation or polymorphism in nucleic acids, for example, genomic DNA, from any organism. Examples of diseases in which base pair variation is known include sickle cell anemia, hemoglobin C disease, o-thalassemia, e-thalassemia, and the like. Other diseases that may be detected include cancerous diseases such as those involving the RAS oncogenes, e.g., the n-RAS oncogene, and infectious diseases.

A dot blot process may also be used for HLA typing in the areas of tissue transplantation, disease susceptibility, and paternity determination. The HLA class II genes, consisting of the a and ø genes from the HLA-DR, HLA-DQ and HLA-DP regions, are highly polymorphic; their genetic complexity at the DNA level is significantly greater than the polymorphism currently defined by serological typing. In addition, the process herein may be employed to detect four DNA sequences coding for HLA class II β proteins (e.g. DRB) associated with insulin-dependent diabetes mellitus (IDDM). Briefly, the four DNA sequences associated with IDDM are selected from the group consisting of: 1 ) 5 ' -GAGCTGCGTAAGTCTGAG-3 ' , 2 ) 5 ' -GAGGAGTTCCTGCGCTTC-31 , 3) 5'-CCTGTCGCCGAGTCCTGG-3', and 4 ) 5 ' -GACATCCTGGAAGACGAGAGA-31 , or the DNA strands that are complementary thereto. Sequence-specific probes may be prepared that will hybridize to one or more of these sequences.

Various infectious diseases can be diagnosed by the presence in clinical samples of specific DNA sequences characteristic of the causative microorganism. These include bacteria, such as Salmonella, Chlamydia, Neisseria; viruses, such as the hepatitis viruses, and -53- parasites, such as the Plasmodium responsible for malaria. U.S. Patent Reexamination Certificate B14,358,535 issued on May 13, 1986 to Falkow et al . describes the use of specific DNA hybridization probes for the diagnosis of infectious diseases. A relatively small number of pathogenic organisms may be present in a clinical sample from an infected patient and the DNA extracted from these may constitute only a very small fraction of the total DNA in the sample. Specific amplification of suspected sequences prior to immobilization and hybridization detection of the DNA samples could greatly improve the sensitivity and specificity of traditional procedures.

Routine clinical use of DNA probes for the diagnosis of infectious diseases would be simplified considerably if nonradioactive^ labeled probes could be employed as described in EP 63,879 to Ward. In this procedure biotin-containing DNA probes are detected by chromogenic enzymes linked to avidin or biotin-specific antibodies. This type of detection is convenient, but relatively insensitive. The combination of specific DNA amplification by the present method and the use of stably labeled probes could provide the convenience and sensitivity required to make the Falkow and Ward procedures useful in a routine clinical setting.

A specific use of the amplification technology for detecting or monitoring for the AIDS virus is described as follows. The amplification and detection process is used with primers and probes which are designed to amplify and detect, respectively, nucleic acid sequences which are substantially conserved among the nucleic acids in AIDS viruses and specific to the nucleic acids in AIDS viruses. Thus, the sequence to be detected must be sufficiently complementary to the nucleic acids in AIDS viruses to initiate polymerization, preferably at room temperature, in the presence of the enzyme and nucleot de triphosphates.

In addition, the probe may be a biotinylated probe in which the biotin is attached to a spacer arm of the formula: H I -Y-(CH2)2-0-[(CH2)x0]y-CH2CH2-N-where Y is 0, NH or N-CHO, x is a number from 1 to 4, and y is a number from 2 to 4. The spacer arm is in turn attached to a psoralen moiety of the formula: CH3 The psoralen moiety intercalates into and crossl nks a "gapped circle" probe as described by Courage-Tebbe et al . , Biochim. Biophys. Acta, 697 (1982) 1-5, wherein the single-stranded hybridization region of the gapped circle spans the region contained in the primers. The details of this biotinylation and dot blot procedure are described more fully in commonly assigned U.S. Patent No. 4,582,789 issued April 15, 1986 and U.S. Patent No. 4,617,261 issued October 14, 1986.

The amplification process can also be utilized to produce sufficient quantities of DNA from a single copy human gene such that detection by a simple non-specific DNA stain such as ethidium bromide can be employed to diagnose DNA directly.

In addition to detecting infectious diseases and pathological abnormalities in the genome of organisms, the amplification process can also be used to detect DNA polymorphisms which may not be associated with any pathological state.

In summary, the amplification process is seen to provide a process for amplifying one or more specific nucleic acid sequences using a chain reaction and a thermostable enzyme, in which reaction primer extension products are produced which can subsequently act as templates for further primer extension reactions. The process is especially useful in detecting nucleic acid sequences which are initially present in only very small amounts.

The following examples are offered by way of illustration only and are by no means intended to limit the scope of the claimed -55- invention. In these examples, all percentages are by weight if for solids and by volume if for l quids, unless otherwise noted, and all temperatures are given in degrees Celsius.

EXAMPLE I I. Synthesis of the Primers The following two oligonucleotide primers were prepared by the method described below: 5 * -ACACAACTGTGTTCACTAGC-31 ( PC03 ) 5 ' -CAACTTCATCCACGTTCACC-3 ' ( C04 ) These primers, both 20-mers, anneal to opposite strands of the genomic DNA with their 5' ends separated by a distance of 110 base pairs.

A. Automated Synthesis Procedures: The diethylphosphoramidites, synthesized according to Beaucage and Caruthers (Tetrahedron Letters (1981) 22:1859-1862) were sequentially condensed to a nucleoside derivatized controlled pore glass support. The procedure included detritylation with trichloroacetic acid in dichl oromethane, condensation using benzotriazole as activating proton donor, and capping with acetic anhydride and dimethyl am nopyri dine in tetrahydrofuran and pyridine. Cycle time was approximately 30 minutes. Yields at each step were essentially quantitative and were determined by collection and spectroscopic examination of the dimethoxytrityl alcohol released dur ng detritylation.

B. 01 godeoxy ribonucleotide Deprotection and Purification Procedures: The solid support was removed from the column and exposed to 1 ml concentrated ammonium hydroxide at room temperature for four hours in a closed tube. The support was then removed by filtration and the solution containing the partially protected ol igodeoxynucleotide was brought to 55°C for five hours. Ammonia was removed and the residue was applied to a preparative polyacryl amide gel. Electrophoresis was carried out at 30 volts/cm for 90 minutes after which the band containing the product was identified by UV shadowing of a fluorescent plate. The band was excised and eluted with 1 ml distilled water overnight at 4°C. This solution was applied -56-to a RP-HPLC column and eluted with a 7-13% gradient of acetonitrile in 1% ammonium acetate buffer at pH 6.0. The elution was monitored by UV absorbance at 260 nm and the appropriate fraction collected, quantitated by UV absorbance in a fixed volume and evaporated to dryness at room temperature in a vacuum centrifuge.

C. Characterization of 01 igodeoxy ribonucleotides: Test aliquots of the purified oligonucleotides were P labeled with polynucleotide kinase and γ-·^Ρ-ΑΤΡ. The labeled compounds were examined by autoradiography of 14-20% polyacryl amide gels after electrophoresis for 45 minutes at 50 volts/cm. This procedure verifies the molecular weight. Base composition was -determined by digestion of the -ol igodeoxy ribonucleotide to nucleosides by use of venom di esterase and bacterial alkaline phosphatase and subsequent separation and quantitation of the derived nucleosides using a reverse phase HPLC column and a 10% acetonitrile, 1% ammonium acetate mobile phase.

II. Isolation of Human Genomic DNA from Cell Line High molecular weight genomic DNA was isolated from a T cell line, Molt 4, homozygous for normal β-globin available from the Human Genetic Mutant Cell Depository, Camden, NJ as GM2219C using essentially the method of Maniatis et al . , supra, p. 280-281.

III. Purification of a Polymerase From The mus aquaticus Thermus aquaticus strain YT1, available . without restriction from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD, as ATCC No. 25,104 was grown in flasks in the following medium: Sodium Citrate 1 mM Potassium Phosphate, pH 7.9 5 mM Ammonium Chloride 10 mM Magnesium Sulfate 0.2 mM Calcium Chloride 0.1 mM Sodium Chloride 1 g/1 Yeast Extract 1 g/1 Tryptone 1 g/1 Glucose 2 g/1.

Ferrous Sulfate 0.01 mM (The pH was adjusted to 8.0 prior to autocl avi ng. ) -57- A 10-liter fermentor was inoculated from a seed flask cultured overnight in the above medium at 70°C. A total of 600 ml from the seed flask was used to inoculate 10 liters of the same medium. The pH was controlled at 8.0 with ammonium hydroxide with the dissolved oxygen at 40%, with the temperature at 70°C, and with the stirring rate at 400 rpm.

After growth of the cells, they were purified using the protocol (with slight modification) of aledin et al., supra, through the first five stages and using a different protocol for the sixth stage. All six steps were conducted at 4°C. The rate of fractionation on columns was 0.5 columns/hour and the volumes of gradients during elution were 10 column volumes. An alternative and preferred purification protocol is provided in Example VI below.

Briefly, the above culture of the T^ aquaticus cells was harvested by centrifugation after nine hours of cultivation, in late log phase, at a cell density of 1.4 g dry weight/1. Twenty grams of cells were resuspended in 80 ml of a buffer consisting of 50 mM Tris'HCl pH 7.5, 0.1 mM EOT A. Cells were lysed and the lysate was centrifuged for two hours at 35,000 rpm in a rotor at 4°C. The supernatant was collected (fraction A) and the protein fraction precipitating between 45 and 75¾ saturation of ammonium sulfate was collected, dissolved in a buffer consisting of 0.2 M potassium phosphate buffer, pH 6.5, 10 mM 2-mercaptoethanol , and 5% glycerine, and finally dialyzed against the same buffer to yield fraction B.

Fraction B was applied to a 2.2 x 30-cm column of DEAE-cellulose, equilibrated with the above described buffer. The column was then washed with the same buffer and the fractions containing protein (determined by absorbance at 280 nm) were collected. The combined protein fraction was dialyzed against a second buffer, containing 0.01 M potassium phosphate buffer, pH 7.5, 10 mM 2-mercaptoethanol , and 5% glycerine, to yield fraction C.

Fraction C was appl ed to a 2.6 x 21-cm column of hydroxyapat te, equilibrated with a second buffer. The column was then washed and the enzyme was eluted with a linear gradient of 0.01- -58- 0.5 potassium phosphate buffer, pH 7.5, containing 10 mM 2- mercaptoethanol and 5% glycerine. Fractions containing DNA polymerase activity (90-180 mM potassium phosphate) were combined, concentrated four-fold using an Amicon stirred cell and YM10 membrane, and dialyzed against the second buffer to yield fraction D.

Fraction D was applied to a 1.6 x 28-cm column of DEAE- cellulose, equilibrated with the second buffer. The column was washed and the polymerase was eluted with a linear gradient of 0.01-0.5 M potassium phosphate in the second buffer. The fractions were assayed for contaminating endonuclease(s) and exonuclease(s) by electrophoretically detecting the change in molecular weight of phage λ DNA or supercoiled plasmid DNA after incubation with an excess of DNA polymerase (for endonucl ease) and after treatment with a restriction enzyme that cleaves the DNA into several fragments (for exonuclease) . Only those DNA polymerase fractions (65-95 mM potassium phosphate) having minimal nuclease contamination were pooled. To the pool was added autoclaved gelatin in an amount of 250 yg/ml , and dialysis was conducted against the second buffer to yield Fraction E.

Fraction E was applied to a phosphocellulose column and eluted with a 100 ml gradient (0.01-0.4 M KC1 gradient in 20 mM potassium phosphate buffer pH 7.5). The fractions were assayed for contaminating endo/exonuclease(s) as described above as well as for polymerase activity (by the method of Kaledin et al . ) and then pooled. The pooled fractions were dialyzed against the second buffer, then concentrated by dialysis against 50% glycerine and the second buffer.

The molecular weight of the polymerase was determined by SDS PAGE. Marker proteins were phosphorylase B (92,500), bovine serum albumin (66,200), ovalbumin (45,000), carbonic anhydrase (31,000), soybean trypsin inhibitor (21,500), and lysozyme (14,400).

Preliminary data suggest that the polymerase has a molecular weight of about 86,000-90,000 daltons, not 62,000-63,000 daltons reported in the literature (e.g., by Kaledin et al.). -59- The polymerase was incubated in 50 yl of a mixture containing 25 mM Tris-HCl pH 6.4 and pH 8.0, 0.1 M KC1 , 10 mM MgCl2, 1 mM 2-mercaptoethanol , 10 nmoles each of dGTP, dATP, and TTP, and 0.5 yCi (3H) dCTP, 8 yg "activated" calf thymus DNA, and 0.5-5 units of the polymerase. "Activated" DNA is a native preparation of DNA after partial hydrolysis with DNase I until 5% of the DNA was transferred to the acid-soluble fraction. The reaction was conducted at 70°C for 30 minutes, and stopped by adding 50 yl of a saturated aqueous solution of sodium pyrophosphate containing 0.125 M EDTA-Na2» Samples were processed and activity was determined as described by Kaledin et al . , supra .

The results showed that at pH 6.4 the polymerase was more than one-half as active as at pH 8.0. In contrast, Kaledin et al . found that at pH about 7.0, the enzyme therein had 8% of the activity at pH 8.3. Therefore, the pH profile for the thermostable enzyme herein is broader than that for the Kaledin et al . enzyme.

Finally, when only one or more nucleotide triphosphates were eliminated from a DNA polymerase assay reaction mixture, very little, if any, activity was observed using the enzyme herein, and the activity was consistent with the expected value, and with an enzyme exhibiting high fidelity. In contrast, the activity observed using the Kaledin et al . (supra) enzyme is not consistent with the expected value, and suggests mis ncorporation of nucleotide triphosphate(s) .

IV. Amplification Reaction One microgram of the genomic DNA described above was diluted in an initial 100 yl aqueous reaction volume containing 25 mM Tris'HCl buffer (pH 8.0), 50 mM KC1 , 10 mM MgCl2, 5 mM dithiothreitol , 200 yg/ml gelatin, 1 yM of primer PC03, 1 yM of primer PC04, 1.5 mM dATP, 1.5 mM dCTP, 1.5 mM dGTP and 1.5 mM TTP. The sample was heated for 10 minutes at 98°C to denature the genomic DNA, then cooled to room temperature. Four microliters of the polymerase from Thermus aquaticus was added to the reaction mixture and overlaid with a 100 yl mineral oil cap. The sample was then placed in the aluminum heating -60- block of the liquid handling and heating instrument described above employing pipettes programmed to deliver liquids and a temperature- control device to effect temperature changes.

The DNA sample underwent 20 cycles of amplification in the machine, repeating the following program cycle: 1) heating from 37°C to 98°C in heating block over a period of 2.5 minutes; and 2) cooling from 98°C to 37°C over a period of three minutes to allow the primers and DNA to anneal.

After the last cycle, the sample was incubated for an additional 10 minutes at 55 °C to complete the final extension reaction.

V. Synthesis and Phosphorylat on of 01 igodeoxy ibonucleotide Probes A labeled DNA probe, designated RS24, of the following sequence was prepared: 51 -*CCCACAGGGCAGTAACGGCAGACTTCTCCTCAGGAGTCAG-3 ' ( RS24 ) where * indicates the label. This probe is 40 bases long, spans the fourth through seventeenth codons of the gene, and is complementary to the normal β-globin allele (g^). The schematic diagram of primers and probes is given below: j 110 bp ^ 6-globin ' PC03 RS24 PC04 This probe was synthesized according to the procedures described in Section I of Example I. The probe was labeled by contacting 20 pmole thereof with 4 units of T4 polynucleotide kinase and about 40 pmole γ-32Ρ-ΑΤΡ (about 7000 Ci/mmole) in a 40 yl reaction volume containing 70 mM Tris buffer (pH 7.6), 10 mM MgCl2» 1·5 mM spermine, and 10 mM d th othreitol for 60 minutes at 37 °C. The total volume was then adjusted to 100 yl with 25 mM EDTA and purified according to the procedure of Maniatis et al . , supra, p. 466-467 over a 1 ml spin dialysis column equilibrated with Tris-EDTA (TE) buffer (10 mM Tris buffer, 0.1 mM EDTA, pH 8.0). TCA precipitation of the -61- reaction product indicated that for RS24 the specific activity was 4.3 Ci/pmole and the final concentration was 0.118 pmole/ul.

VI. Dot Blot Hybridizations Four microliters of the amplified sample from Section IV and 5.6 yl of appropriate dilutions of 8-globin plasmid DNA calculated to represent amplification efficiencies of 70, 75, 80, 85, 90, 95 and 100% were diluted with 200 ul 0.4 N NaOH, 25 mM EDTA and spotted onto a nylon filter by first wetting the filter with water, placing it in an apparatus for preparing dot blots which holds the filters in place, applying the samples, and rinsing each well with 0.1 ml of 20 x SSPE (3.6 M NaCl, 200 mM NaH2P04, 20 mM EDTA), as disclosed by Reed and Mann, Nucleic Acids Research, _13« 7202-7221 (1985). The filters were then removed, rinsed in 20 x SSPE, and baked for 30 minutes at 80°C in a vacuum oven.

After baking, each filter was then contacted with 16 ml of a hybridization solution consisting of 3 x SSPE, 5 x Denhardt's solution (1 x = 0.02% polyvinylpyrrolidone, 0.02% Ficoll, 0.02% bovine serum albumin,, 0.2 mM Tris, 0.2 mM EDTA, pH 8.0), 0.5% SDS and 30% formamide, and incubated for two hours at 42°C. Then 2 pmole of probe RS24 was added to the hybridization solution and the filter was incubated for two minutes at 42 °C.

Finally, each hybridized filter was washed twice with 100 ml of 2 x SSPE and 0.1% SDS for 10 minutes at room temperature. Then the filters were treated once with 100 ml of 2 x SSPE, 0.1% SDS at 60°C for 10 minutes.

Each filter was then autoradiographed, with the signal readily apparent after two hours.

VII. Discussion of Autoradiogram The autoradiogram of the dot blots was analyzed after two hours and compared in intensity to standard serial dilution f?-globin reconstructions prepared with Jjael 11 /Mae_I -digested pBR:eA, where βΑ is the wild-type allele, as described in Saiki et al., Science, supra. -62- Analysis of the reaction product indicated that the overall amplification efficiency was about 95%, corresponding to a 630,000- fold increase in the β-globin target sequence.

EXAMPLE II I. Amplification Reaction Two 1 samples of genomic DNA extracted from the Molt 4 cell line as described in Example I were each diluted in a 100 yl reaction volume containing 50 mM KCl, 25 mM Tris-HCI buffer pH 8.0, 10 mM MgCl2. 1 uM of primer PC03, 1 μ of primer PC04, 200 ug/ml gelatin, 10% dimethyl sulfoxide (by volume), and 1.5 mM each of dATP, dCTP, dGTP and TTP. After this mixture was heated for 10 minutes at 98°C to denature the genomic DNA, the samples were' cooled to room temperature and 4 μΐ of the polymerase from Thermus aquaticus described in Example I was added to each sample. The samples were overlaid with mineral oil to prevent condensation and evaporative loss.

One of the samples was placed in the heating block of the machine described in Example 1 and subjected to 25 cycles of amplification, repeating the following program cycle: (1) heating from 37 to 93 °C over a period of 2.5 minutes; (2) cooling from 93°C to 37°C over a period of three minutes to allow the primers and DNA to anneal; and (3) maintaining at 37 °C for two minutes.

After the last cycle the sample was incubated for an additional 10 minutes at 60°C to complete the final extension reaction.

The second sample was placed in the heat-conducting container of a temperature cycling (heating and cooling) machine. In this machine, the heat-conducting container is attached to Peltier heat pumps which adjust the temperature upwards or downwards and a microprocessor controller to control automatically the amplificat on sequence, the temperature levels, the temperature ramping and the timing of the temperature. -63- The second sample was subjected to 25 cycles of ampli ication, repeating the following program cycle: (1) heating from 37 to 95°C over a period of three minutes; (2) maintaining at 95 °C for 0.5 minutes to allow denaturation to occur; (3) cooling from 95 to 37 °C over a period of one minute; and (4) mainta ning at 37 °C for one m nute.

II. Analysis Two tests were done for analysis, a dot blot and an agarose gel analysis.

For the dot blot analysis, a labeled DNA probe, designated RS18, of the following sequence was prepared. 5 ' -*CTCCTGAGGAGAAGTCTGC-3 ' ( S18 ) where * indicates the label. This probe is 19 bases long, spans the fourth through seventeenth codons of the gene, and is complementary to the normal β-globin allele (βΑ). The schematic diagram of primers and probes is given below: v 110 bp ^ β-gl obin 7 PC03" RS18 PC04 This probe was synthesized according to the procedures described in Section I of Example I. The probe was labeled by contacting 10 pmole thereof with 4 units of T4 polynucleotide kinase and about 40 pmole γ"32Ρ-ΑΤΡ (about 7000 Ci/mmole) in a 40 μ1 reaction volume containing 70 mM Tris*HCl buffer (pH 7.6), 10 mM MgCl2, 1.5 mM spermine and 10 mM dithiothrei tol for 60 minutes at 37 °C. The total volume was then adjusted to 100 yl with 25 mM EDTA and purified according to the procedure of Maniatis et al . , supra, p. 466-467 over a 1 ml spin dialysis column equilibrated with Tris-EDTA (TE) buffer - - (10 mM THS-HCI buffer, 0.1 mM EDTA, pH 8.0). TCA precipitation of the reaction product indicated that for RS18 the specific activity was 4.6 yCi/pmole and the final concentration was 0.114 pmole/gl.

Five microliters of the amplified sample from Section I and of a sample amplified as described above except using the lenow fragment of col i DNA Polymerase I instead of the thermostable enzyme were diluted with 195 ul 0-4 N NaOH, 25 mM EDTA and spotted onto two replicate nylon filters by first wetting the filters with water, placing them in an apparatus for preparing dot blots which holds the filters in place, applying the samples, and rinsing each well with 0.4 ml of 20 x SSPE (3.6 M NaCl , 200 mM NaH2P04, 20 mM EDTA), as disclosed by Reed and Mann, supra. The filters were then removed, rinsed in 20 x SSPE, and baked for 30 minutes at 80°C in a vacuum oven.

After baking, each filter was then contacted with 6 ml of a hybridization solution consisting of 5 x SSPE, 5 x Denhardt1 s solution (1 x = 0.02% polyvinylpyrrolidone, 0.02% Ficoll, 0.02% bovine serum albumin, 0.2 mM Tris, 0.2 mM EDTA, pH 8.0) and 0.5% SDS, and incubated for 60 minutes at 55°C. Then 5 gl of probe RS18 was added to the hybridization solution and the filter was incubated for 60 minutes at 55°C.

Finally, each hybridized filter was washed twice with 100 ml of 2 x SSPE and 0.1% SDS for 10 minutes at room temperature. Then the filters were treated twice more with 100 ml of 5 x SSPE, 0.1% SDS at 60 °C for 1) one minute and 2) three minutes, respectively.

Each filter was then autoradiographed, with the signal readily apparent after 90 minutes.

In the agarose gel analysis, 5 gl each amplification reaction was loaded onto 4% NuSieve/0.5% agarose gel in 1 x TBE buffer (0.089 M Tris, 0.089 M boric acid, and 2 mM EDTA) and electrophoresed for 60 minutes at 100V. After staining with ethidium bromide, DNA was visualized by UV fluorescence. -65- The results show that the machines used in Example I and this example were equally effective in amplifying the DNA, showing discrete high-intensity 110-base pair bands of similar intensity, corresponding to the desired sequence, as well as a few other discrete bands of much lower intensity. In contrast, the amplification method that involves reagent transfer after each cycle using the Klenow fragment of col i Polymerase I gave a DNA smear resulting from the non-specific amplification of many unrelated DNA sequences- It is expected that similar improvements in amplification and detection would be achieved in evaluating HLA-DQ, D and DP regions.

If in the above experiments the amplification reaction buffer contains 2 mM MgCl2 instead of 10 mM MgCl2 and 150-200 μΜ of each nucleotide rather than 1.5 mM of each, and if the lower temperature of 37°C is raised to 45-58°C during amplification, better specificity and efficiency of amplification occurs. Also, DMSO was found not necessary or preferred for amplification.

EXAMPLE III Amplification and Cloning For amplification of a 119-base pair fragment on the human β-globin gene, a total of 1 microgram each of human genomic DNA isolated from the Molt 4 cell line or from the GM2064 cell line (representing a homozygous deletion of the β- and 6-hemoglobin region and available from the Human Genetic Mutant Cell Depository, Camden, NJ) as described above was amplified in a 100 μΐ reaction volume containing 50 mM KC1 , 25 mM Tris'HCl pH 8, 10 mM MgCl2, 200 g/ml gelatin, 5 mM 2-mercaptoethanol , 1.5 mM each of dATP, dCTP, TTP, and dGTP, and 1 yM of each of the following primers: 51 -CTTCTGcagCAACTGTGTTCACTAGC-31 (GH18) 5'-CACaAgCTTCATCCACGTTCACC-3' (GH19) where lower case letters denote mismatches from wild-type sequence to create restriction enzyme sites. GH18 is a 26-base oligonucleotide complementary to the negative strand and contains an internal Pstl site. GH19 is a 29-base oligonucleotide complementary to the plus strand and contains an internal Hindi II recognition sequence. These primers were selected by first screening the regions of the gene for homology to the Pstl and Hindi 11 restriction sites. The primers were then prepared as described in Example I.

The above reaction mixtures were heated for 10 minutes at 95°C and then cooled to room temperature. A total of 4 yl of the polymerase described in Example I was added to each reaction mixture, and then each mixture was overlayed with mineral oil. The reaction mixtures were subjected to 30 cycles of ampl fication with the following program: 2.5 m n. ramp, 37 to 98°C 3 m n. ramp, 98 to 37 °C 2 min. soak, 37 °C After the last cycle, the reaction mixtures were incubated for 20 minutes at 65°C to complete the final extension. The mineral oil was extracted with chloroform and the mixtures were stored at -20°C.

A total of 10 ΐ of the amplified product was digested with 0.5 yg M13mpl0 cloning vector, which is publicly available, in a 50 yl volume containing 50 mM NaCl , 10 mM Tris'HCl, pH 7.8, 10 mM MgCl2, 20 units Pstl and 26 units Hindi 11 for 90 minutes at 37°C. The reaction was stopped by freezing at -20°C. The volume was adjusted to 110 yl with TE buffer and loaded (100 yl ) onto a 1 ml BioGel P-4 spin dialysis column. One 0.1 ml fraction was collected and ethanol precip tated.

(At this point it was discovered that there was p-globin amplif cation product in the GM2064 sample. Subsequent experiments traced the source of contamination to the primers, either GH18 or GH19. Because no other source of primers was available, the experiment was continued with the understanding that some cloned sequences would be derived from the contaminating DNA in the primers.) The ethanol pellet was resuspended in 15 yl water, then adjusted to 20 yl volume containing 50 mM Tris'HCl, pH 7.8, 10 mM MgCI 2 » °·5 mM ATP> 10 dithiothre tol , and 400 units ligase. [One unit is the amount of enzyme required to give 50¾ ligation of Hindi 11 digested xDNA in 30 minutes at 16°C in 20 μΐ at a 5' termini concentration of 0.12 mM (about 330 Vml)]. This mixture was incubated for three hours at 16°C.

Ten microliters of ligation reaction mixture containing Molt 4 DNA was transformed into E. col strain JM103 competent cells, which are publicly available. The procedure followed for preparing the transformed strain is described in Messing, J. (1981) Third Cleveland Symposium on Macromol ecu! es: ecombinant DNA, ed. A. Walton, Elsevier, Amsterdam, 143-163. A total of 651 colorless plaques (and 0 blue plaques) were obtained. Of these, 119 had a (+)- strand insert (18%) and 19 had a (-)- strand insert (3%). This is an increase of almost 20-fold over the percentage of e-globin positive plaques among the primer-positive plaques from the amplification technique using Klenow fragment of E. coli Polymerase I, where the reaction proceeded for two minutes at 25°C, after which the steps of heating to 100°C for two minutes, cooling, adding Klenow fragment, and reacting were repeated nine times. These results confirm the improved specificity of the amplification reaction employing the thermostable enzyme herein.

In a later cloning experiment with GM2064 and the contaminated primers, 43 out of 510 colorless plaques (8%) had the (+)- strand insert. This suggests that approximately one-half of the 119 clones from Molt 4 contain the contaminant sequence.

Ten of the ( + )- strand clones from Molt 4 were sequenced. Five were normal wild-type sequence and five had a single C to T mutation in the third position of the second codon of the gene (CAC to CAT). Four of the contaminant clones from GM2064 were sequenced and all four were normal.

Restriction site-modified primers may also be used to amplify and clone and partially sequence the human N-ras oncogene and to clone base pair segments of the HLA DQ-a, DQ-B and DR-e genes using the above technique. -68- Again, if the concentrations of MgC^ and nucleotides are reduced to 2 mM and 150-200 yM, respectively, and the minimum cycling temperature is increased from 37°C to 45-58°C, the specificity and efficiency of the amplification reaction can be increased.

EXAMPLE IV Gene Retrieval A. IDENTIFICATION OF A DNA SEQUENCE PROBE FOR THE TAQ POLYMERASE GENE A specific DNA sequence probe for the Taq pol gene was obtained following immunological screening of a xgtll expression library. T. aquaticus DNA was digested to completion with Alul , li gated with EcoRI 12-mer linkers (CCGGAATTCCGG, New England Biolabs), digested with EcoRI and li gated with dephosphorylated, E^coRI -digested Xgtll DNA (Promega Biotech). The li gated DNA was packaged (Gigapack Plus, Strategene) and transfected into E_. coli K-12 strain Y1090 (provided by R. Young).

The initial library of 2 x 10^ plaques was screened (Young, R.A., and R.W. Davis (1983) Science, 222:778-782) with a 1:2000 dilution of a rabbit polyclonal antiserum raised to purified Taq polymerase (see Examples I and VI). Candidate plaques were replated at limiting dilution and rescreened until homogeneous (~3 cycles). Phage were purified from candidate plaques which failed to react with preimmune serum and reacted with immune serum.

Candidate phage were used to lysogenize E_. coli K-12 strain Y1089 (R. Young). Lysogens were screened for the production of an IPTG inducible fusion protein (larger than β-galactosidase) which reacted with the Taq polymerase antiserum. Solid phase, size-fractionated fusion proteins were used to affinity purify epitope-specific antibodies from the total polyclonal antiserum (Goldstein, L.S.B., et al. (1986) J. Cell Biol. 102:2076-2087).

The "fished", epi tope-selected antibodies were used, in turn, in a Western analysis to identify which xgtll phage candidates encoded DNA sequences uniquely specific to Taq polymerase. One xgtll - - phage candidate, designated xgt:l, specifically selected antibodies from the total rabbit polyclonal Taq polymerase antiserum which uniquely reacted with both purified Taq polymerase and crude extract fractions containing Taq polymerase. This phage, xgt:l, was used for further study.

The —115 bp EcoRI-adapted Alul fragment of Thermus aquaticus DNA was labeled (Maniatis et al . , supra) to generate a Taq polymerase- specific probe. The probe was used in Southern analyses and to screen a J_. aquaticus DNA random genomic library.

B. CONSTRUCTION AND SCREENING OF A THERMUS AQUATICUS RANDOM GENOMIC LIBRARY Lambda phage Charon 35 (Wilhelmine, A. M. et al . , supra ) was annealed and l gated via its cohesive ends, digested to completion with BamHI , and the annealed arms were purified from the "stuff er" fragments by potassium acetate density gradient ul tracentrifugation (Maniatis, et al ., supra ) . 1_. aquaticus DNA was partially digested with Sau3A and the 15-20 kb size fraction purified by sucrose density gradient ul tracentrifugation. The random genomic library was constructed by li gating the target and vector DNA fragments at a 1:1 molar ratio. The DNA was packaged and transfected into E. col -12 strains LE392 or K802. A library of >20,000 initial phage containing >99¾ recombinants was amplified on _E_. coli K-12 strain LE392.

The CH35 Taq genomic phage library was screened (Maniatis et al . , supra ) with the radiolabeled EcoRI insert of xgtll : 1. Specifically hybridizing candidate phage plaques were purified and further analyzed. One phage, designated Ch35: :4-2, released >_ four T\ aquaticus DNA fragments upon digestion with Hindi I I (~8.0, 4.5, 0.8, 0.58 kb) The four Hindi 11 T. aquaticus DNA fragments were li gated with Hindi 11 digested plasmid BSM13+ (3.2 kb, Vector Cloning Systems, San Diego) and individually cloned following transformation of E_. col i K-12 strain DG98.

The -8.0 kb Hindi 11 DNA fragment from CH35::4-2 was isolated in plasmid pFC82 (11.2 kb), while the 4.5 kb Hindi I I DNA fragment from CH35::4-2 was isolated in plasmid pFC83 (7.7 kb).

E_. coli strain DG98 harboring pFC82 was shown to contain a thermostable, high temperature DNA polymerase activity (Table 1). In addition, these cells synthesize a new ~60 kd molecular weight polypeptide which is immunologically related to Taq DNA polymerase.

The Taq polymerase coding region of the 8.0 kb Hindi 11 DNA fragment was further localized to the lac-promoter proximal 2.8 kb Hindi 11 to Asp718 portion of the 8.0 kb Hindi 11 fragment. This region was subcloned to yield plasmid pFC85 (6.0 kb). Upon induction with IPTG, E_. coli DG98 cells harboring plasmid pFC85 synthesize up to 100-fold more thermostable, Taq polymerase-rel ated activity (Table 1) than the original parent clone (pFC32/DG98) . While cells harboring pFC85 synthesize a significant amount of a thermostable DNA polymerase activity, only a portion of the Taq pol DNA sequence is translated, resulting in the accumulation of a ~60 kd Taq polymerase-rel ated polypeptide.

TABLE 1 Expression of a Thermostable DNA Polymerase Activity in E. coli^ Sample Units*/ml IPTG +IPTG BSM13/DG98- - 0.02 pFC82/DG98 2.2 2.7 pFC85/DG98 11.9 643.8 ffCells were grown to late log phase (+/- IPTG, 10 m ), harvested, sonicated, heated at 75°C for 20 minutes, centrifuged and the clarified supernatant assayed at 70°C for DNA polymerase activity. * 1 unit = 1 nM dCTP incorporated in 30 minutes.

, EXAMPLE V Expression of Tag Polymerase The thermostable gene of the present invention can be expressed in any of a variety of bacterial expression vectors including DG141 (ATCC 39588) and pPLNRB$ATG. Both of these host vectors are pBR322 derivatives that have either a sequence containing a tryptophan promoter-operator and ribosome binding site with an operably linked ATG start codon (DG141) or a sequence containing the lambda PL promoter and gene N ribosome binding site operably linked to an ATG start codon (pP|_NRB^ATG) . Either one of these host vectors may be restricted with Sac I , and blunt ended with Klenow or SI nuclease to construct a convenient restriction site for subsequent insertion of the Taq polymerase gene.

The full -length Taq polymerase gene was constructed from the DNA insert fragments subcloned into plasmids pFC83 and pFC85 as follows. Vector BSM13+ (comrnercially available from Vector Cloning Systems, San Diego, CA) was digested at the unique Hindi 11 site, repa red with Klenow and dNTPs, and li gated with T4 DNA ligase to a Bgl II octanucleotide linker, 5 ' -CAGATCTG-31 , and transformed into Ej_ col i strain DG98. Plasmids were isolated from Amp^ lacZ "1" transf ormants. One of the clones was digested with Bgl II and Asp718 restriction enzymes, and the large vector fragment purified by gel electrophores s.

Next, plasmid pFC83 was digested with Bgl II and Hindi 11 and the ~750 base pair fragment was isolated. Plasmid. pFC85 was digested with Hindi 11 and Asp718 and the ~2.8 kb fragment isolated and joined in a three-piece ligation to the ~750 base pair Bgl II-HindIII fragment from pFC83. and the Bgl II-Asp718 vector fragment of BSM13+. This ligation mixture was used to transform col i strain DG98 (ATCC 39,768 deposited July 13, 1984) from which AmpR colonies were selected and an ~6.75 kilobase plasmid (pLSGl) was isolated. Isopropyl -&-D-thiogalactoside ( IPTG)-induced DG98 cells harboring pLSGl synthesized Taq DNA polymerase indistinguishable in size from the native enzyme isolated from T^ aquaticus. Plasmid pLSGl can then be used to - - generate a single strand DNA template according to the procedure recommended by Vector Cloning Systems. 01 i gonucleoti de-di rected mutagenesis (see Zoller and Smith, Nuc. Acids Res. (1982) 10 6487-6500) can then be used to introduce an SphI restriction site as part of the ATG start codon (upstream of the internal Hindi 11 site in the coding sequence of the Taq polymerase gene). Similarly, a Bglll site can be introduced after the carboxyl- terminus of the gene (~0.7 kb upstream from the Asp718 site) to facilitate subcloning of the Taq polymerase gene into an expression vector. After the site-directed mutagenesis is performed, the gene can be isolated from the BS 13+ vector on an ~3.2 kb Sphl-BstEII restriction fragment, treated with Klenow fragment and all four dNTPs, and inserted with T4 DNA ligase (blunt-end conditions) into either one of the aforementioned expression vectors, which have been digested with Sacl , repaired with Klenow and dNTPs, and treated with calf intestine phosphatase to generate dephosphorylated blunt ends. This ligation mixture is used to transform E. coli DG116 and the resulting transf ormants are screened for production of Taq polymerase. Expression of the enzyme can be confirmed by Western immunoblot analysis and activity analysis.

A greater proportion of the Taq polymerase gene contained within the ~2.8 kb HindIII-Asp718 fragment of plasmid pFC85 can be expressed using, for example, plasmid ρΡ^Ν^β^ΑΤΰ, by operably linking the ami no- terminal Hindi 11 restriction site encoding the Taq pol gene to an ATG initiation codon. The product of this fusion upon expression will yield an ~66, 000-68, 000 dal ton truncated polymerase.

This specific construction can be made by digesting plasmid pFC85 with Hindi 11 and treating with Klenow fragment in the presence of dATP, dGTP and dCTP. The resulting fragment is treated further with SI nuclease to remove any single-stranded extensions, and the resulting DNA digested with Asp718 and treated with Klenow fragment in the presence of all four dNTPs. The recovered fragment can be li gated using T4 DNA ligase to dephosphorylated plasmid which had been digested with Sacl and treated with Klenow fragment in the - - presence of dGTP to construct an ATG blunt end. This ligation mixture can then be used to transformed E_;_ col DG116 and the transf ormants screened for production of Taq polymerase. Again, expression can be confirmed by Western immunoblot analysis and activity analysis.

EXAMPLE VI Purification The thermostable polymerase may be purified directly from a culture of Thermus aquaticus following the example disclosed below or, alternatively, from a bacterial culture containing the recombinants produced enzyme with only minor modifications necessary in the preparation of the crude extract.

After harvesting by centrifugation, 60 grams of cells were resuspended in 75 ml of a buffer consisting of 50 mM Tris-Cl pH 8, 1 mM EDTA. Cells were lysed in a French Press at 14,000-16,000 PSI after which 4 volumes (300 ml) of additional Tris-EDTA were added. Buffer A (β-me captoethanol to 5 mM and NP-40 and Tween 20 to 0.5% (v/v) each) was added and the solution was sonicated thoroughly while cooling. The resultant homogeneous suspension was diluted further with Buffer A such that the final volume was 7.5-8 times the starting cell weight; this was designated Fraction I.

The polymerase activity in Fraction I and subsequent fractions was determined in a 50 μΐ mixture containing 0.025 M TAPS-HC1 pH 9.4 (20°C), 0.002 M MgCl2, 0.05 M KC1 , 1 mM 2-mercaptoethanol , 0.2 mM each dGTP, dATP, TTP, 0.1 mM dCTP [ct-32P, .05 Ci/mM], 12.5 ug "activated" salmon sperm DNA and 0.01-0.2 units of the polymerase (diluted in 10 mM Tris-HCl, pH 8, 50 mM KC1 , 1 mg/ml autoclaved gelatin, 0.5% NP-40, 0.5% Tween 20, and 1 mM 2-mercaptoethanol). One unit corresponds to 10 nM product in 30 minutes. "Activated" DNA is a native preparation of DNA after partial hydrolysis with DNase I until 5% of the DNA was transferred to the acid-soluble fraction. The reaction was conducted at 74°C for 10 minutes and then 40 yl was transferred to 1.0 ml of 50 yg/ml carrier DNA in 2 mM EDTA at 0°C An equal volume (1.0 ml) of 20% TCA, 2% sodium pyrophosphate was added.

- - After 15-20 minutes at 0°C the samples were filtered through Whatman GF/C discs and extensively washed with cold 5% TCA-1% pyrophosphate, followed by cold 95% ethanol , dried and counted.

Fraction I was centrifuged for two hours at 35,000 rpm in a Beckman TI 45 rotor at 2°C and the collected supernatant was designated Fraction II.

The Taq polymerase activity was precipitated with Polymin P (BRL, Gaithersburg, MD) (10%, w/v, adjusted to pH 7.5 and autoclaved) after the minimum amount of Popymin P necessary to precipitate 90-95% of the activity was determined, which amount was generally found to be between 0.25% and 0.3% final volume.

An appropriate level of Polymin P was added slowly to Fraction II while st rring for 15 minutes at 0°C. This solution was centrifuged at 13,000 rpm for 20 minutes in a Beckman JA 14 rotor at 2°C. The supernatant was assayed for activity and the pellet was resuspended in 1/5 volume of 0.5X Buffer A (diluted 1:2 with H2O). This suspension was recentri fuged and the pellet resuspended in 1/4 volume of Buffer A containing 0.4 M KC1. This suspension was homogenized thoroughly and left overnight at 4°C. The homogenate was centrifuged as above and the collected supernatant designated Fraction III.

The protein fraction was collected by "precipitation" at 75% saturation of ammonium sulfate, centrifuged (at 27,000 rpm, SW27 rotor, 30 minutes) and the float ng pell cle was resuspended in 50 m Tris-Cl pH 8, 1 mM EDTA. These steps were repeated and the protein suspension was dialyzed extensively with P-cell buffer (20 mM KPO4 pH 7.5, 0.5 mM EDTA, 5 mM β-mercaptoethanol , 5% (w/v) glycerol, 0.5% (v/v) NP-40 and Tween 20) containing 80 mM C1.

The dialysate was transferred to a centrifuge bottle to which was added any recovered protein from sacks rinsed with the P-cell buffer containing 80 mM KC1. Centrifugation was performed at 20,000 x g and the time was reduced to 15 minutes. The supernatant was saved and any pellet remaining was washed, extracted with P-cell buffer and 80 mM KC1 , and recentri fuged. The supernatants were then combined to form Fraction IV.

- - Fraction IV was applied to a 2.2 x 22-cm column of phosphocellul ose, equilibrated with the P-cell buffer containing 80 mM KCl. The column was washed (2.5-3 column volumes) with the same buffer and the protein eluted using a linear gradient of 80 to 400 mM KCl in P-cell buffer. Fractions containing DNA polymerase activity (-0.18-0.20 M KCl) were pooled and concentrated 3-4 fold on an Ami con stirred cell and Y 30 membrane. The cell was rinsed with the P-cell buffer without KCl and added to the fraction concentrate (0.15 M KCl adjusted final volume) to form Fraction V.

Fraction V was applied to a 5 ml Heparin Sepharose CL-6B column (Pharmacia) equilibrated with P-cell buffer and 0.15 M KCl. The column was washed with 0.15 M KCl buffer (3-4 column volumes) and the protein eluted with a linear gradient from 0.15 to 0.65 M KCl in P-cell buffer. A 1:10 dilution into diluent without gelatin was made for SDS-PAGE analysis and a subsequent 1:20 dilution into diluent with 1 mg/ml gelatin was made for use in enzyme assays. The activity fractions (eluting at ~0.3 M KCl) were assayed on supercoiled DNA template for specific and non-specific endonucleases/topoisomerase by electrophoretically detecting the change in molecular weight of supercoiled plasmid DNA after incubation with an excess of DNA polymerase. Exonuclease contamination was detected following incubation with small linear DNA fragments. In peak fractions, an ~88 kd protein was found to be the major band. The major pool, designated Fraction VI, had the highest polymerase activity with minimal detectable endonuclease activity when this pool was assayed for 30 minutes at 55°C with ~3-5 polymerase units/600 ng DNA.

Fraction VI was dialyzed" against 10 mM KP04 pH 7.5, 5 mM ø-mercaptoethanol , 5¾ glycerol, 0.2% NP-40, and 0.2% Tween 20 (HA buffer). The dialyzed sample was applied to a 3 ml column of hydroxyapatite and the enzyme eluted with a linear gradient of 10 to 250 mM KPO4 pH 7.5, HA buffer. DNA polymerase activity began to elute at 75 mM KP04 with the peak at 100 mM KPO4. Active peak fractions were assayed at 1:100-1:300 dilution. As in the prior chromatography step, a 1:10 dilution in diluent was prepared without gelatin for SDS--PAGE analysis. Fractions with no significant endonuclease or double- - - strand exonuclease when assayed at 55°C with 5 polymerase units were pooled and designated Fraction VII.

Fraction VII was dialyzed against a solution of 25 mM sodium acetate pH 5.2, 5% glycerol, 5 mM g-mercaptoethanol , 0.1 mM EDTA, 0.1% NP-40, and 0.1% Tween 20, adjusted to pH 5 at room temperature. The dialyzed sample was applied to a 2 ml DEAE-Tr s-Acryl -M (LKB) column pre-equil ibrated and subsequently washed with the same buffer. The fraction containing polymerase activity that did not adhere to the column was pooled and adjusted to 50 mM NaCl in the same buffer to yield Fraction VIII.

Fraction VIII was applied to a 2 ml CM-Tri s-Acryl M (LKB) column equil ibrated -with the same buffer (25 mM sodium acetate, 50 mM NaCl, 5% glycerol 0.1 mM EDTA, 0.1% NP-40, and 0.1% Tween 20). The column was washed with 4-5 column volumes of the same buffer and the enzyme eluted with a linear gradient from 50 to 400 mM NaCl in sodium acetate buffer. The polymerase activity peak eluted ~0.15-0.20 M NaCl. The polymerase activity was assayed at 1:300 to 1:500 dilution with the fi st dilution 1:10 into diluent without gelatin for the SDS-PAGE analysis. An assay across the activity peak on supercoiled DNA templates for specific and non-specific endonuclease/topoisomerase using DNA polymerase assay salts (25 mM TAPS-HC1 pH 9.4, 2.0 mM MgCl2 and 50 mM KC1 ) at 74°C was performed, as well as assays for nucleases on M13 ss DNA and pBR322 fragments. Active fractions with no detectable nuclease(s) were pooled and run on a silver stained SDS-PAGE mini gel. The results show a single ~88 kd band with a specific activity of ~250,000 units/mg.

This specific activity is more than an order of magnitude higher than that claimed for the previously isolated Taq polymerase and is at least an order of magnitude higher than that for E_;_ col i polymerase I.

EXAMPLE VII The Taq polymerase purified as described above in Example VI was found to be free of any contaminating Taq endonuclease and - - exonuclease activities. In addition, the Taq polymerase is preferably stored in storage buffer containing from about 0.1 to about 0.5% volume/volume of each non-ionic polymeric detergent employed. More preferably the storage buffer consists of 50% (v/v) glycerol, 100 mM KCl, 20 mM Tris-Cl pH 8.0, 0.1 mM ethyl enedi ami netetraacetic acid (EDTA), 1 mM dithiothreitol , 0.5% v/v NP-40, 0.5% v/v Tween 20, and 200 ug/ml gelatin, and is preferably stored at -20°C.

The stored Taq polymerase was diluted in a buffer consisting of 25 mM Tris CI pH 8.0, 20 mM KCl , 1 mM 6-mercaptoethanol, 0.5% NP- 40, 0.5% Tween-20, and 500 yg/ml gelatin. A reaction buffer was then prepared containing 50 mM KCl, 10 mM Tris-Cl, pH 8.3, 1.5 mM MgCl2, 0.01% (w/v) gelatin, 200 μΜ each dNTP, 1 μ each of the primers that define a 500 base pai target sequence on a control template from bacter ophage λ, and 2.0-2.5 units Taq polymerase/assay in a final volume of 100 μΐ . Template was added to the reaction buffer, the sample placed in a 0.5 ml polypropylene tube, and the sample topped with 100 μΐ of heavy white mineral oil to prevent evaporation.

At least a 10^-fold amplification was achieved when the following conditions were employed, using 1 ng of control template (bacteriophage λ DNA) where the target sequence represented approximately 1% of the starting mass of DNA.

First the template mixture was denatured for one minute, 30 seconds at 94°C by placing the tube in a heat bath. Then the tube was placed in a heat bath at 37 °C for two minutes. Then the tube was placed in a heat bath at 72 °C for three minutes, and then in the heat bath at 94 °C for one minute. This cycle was repeated for a total of 25 cycles. At the end of the 25th cycle, the heat denaturation step at 94 °C was omitted and replaced by extending the 72 °C incubation step by an additional three minutes. Following termination of the assay, the samples were allowed to cool to room temperature and analyzed as described in previous examples.

The template may be optimally amplified with a different concentration of dNTPs and a different amount of Taq polymerase. Also, the size of the target sequence in the DNA sample will di ectly impact the minimum time required for proper extension (72°C incubation step). An optimization of the temperature cycling profile should be performed for each individual template to be amplified, to obtain maximum efficiency.

EXAMPLE VIII Taq polymerase purified as described above in Example I was formulated for storage as described in the previous example, but without the non-ionic polymeric detergents. When assayed for activity as described in that example, the enzyme storage mixture was found to be inactive. When the NP-40 and Tween 20 were added to the storage buffer, the full enzyme activity was restored, indicating that the presence of the non-ionic 'detergents is necessary to the stability of the enzyme formulation.

EXAMPLE IX Several 1 v9 samples of human genomic DNA were subjected to 20-35 cycles of amplificat on as described in Example V, with equivalent units of either Klenow fragment or Taq polymerase, and analyzed by agarose gel electrophoresis and Southern blot. The primers used in these reactions, PC03 and PC04, di ect the synthesis of a 110-bp segment of the human beta-globin gene. The Klenow polymerase amplifications exhibited the smear of DNA typically observed with this enzyme, the apparent cause of which is the nonspecific annealing and extension of primers to unrelated genomic sequences under what were essentially non-stringent hybridizat on conditions (lx Klenow salts at 37°C). Nevertheless, by Southern blot a specific 110-bp beta-globin target fragment was detected in all lanes. A substantially different electrophoretic pattern was seen in the ampl ficat ons done with Taq polymerase where the single major band is the 110-bp target sequence. This remarkable specificity was undoubtedly due to the temperature at which the primers were extended.

Although, like Klenow fragment amplifications, the anneal ng step was performed at 37°C, the temperature of Taq-catalyzed reactions had to be raised to about 70°C before the enzyme exhibited sign ficant activity. During this transition from 37 to 70°C, poorly matched primer- tempi ate hybrids (which formed at 37°C) disassociated so that by the time the reaction reached an enzyme-activating temperature, only highly complementary substrate was available for extension. This specificity also results in a greater yield of target sequence than similar amplifications done with Klenow fragment because the nonspecific extension products effectively compete for the polymerase, thereby reducing the amount of 110-mer that can be made by the Klenow fragment.

EXAMPLE X Amplification was carried out of a sample containing 1 yg Molt 4 DNA, 50 mM KC1 , 10 mM Tris pH 8.3, 10 mM MgCl2, 0.01% gelatin, 1 uM of each of the following primers (to amplify a 150 bp region): 5'-CATGCCTCTTTGCACCATTC-3' ( S79) and 51 -TGGTAGCTGGATTGTAGCTG-31 ( RS80 ) 1.5 mM of each dNTP, and 5.0 units of Taq polymerase per 100 pi reaction volume. Three additional samples were prepared containing 2.5, 1.3, or 0.6 units of Taq polymerase. The ampl fication was carried out in the temperature cycling machine described above using the following cycle, for 30 cycles: from 70 to 98°C for 1 minute hold at 98°C for 1 minute from 98°C to 35, 45 or 55 °C for 1 minute hold at 35, 45 or 55°C for 1 minute from 35, 45 or 55°C to 70°C for 1 minute hold at 70°C for 30 seconds At 35°C annealing temperature, the 2.5 units/100 yl Taq enzyme dilution gave the best-signal -to noise ratio by agarose gel electrophoresis over all other Taq polymerase concentrations. At 45°C, the 5 units/100 yl Taq enzyme gave the best signal-to-noise ratio over the other concentrations. At 55°C, the 5 units/100 yl Taq enzyme gave the best signal-to-noise ratio over the other concentrations and over the 45°C annealing and improved yield. The Taq polymerase has more specificity and better yield at 55°C.

In a separate experiment the Molt 4. DNA was 10-fold serially diluted into the cell l ne GM2064 DNA, containing no β- or δ-globin sequences, available from the Human Genetic Mutant Cell Depository, Camden, New Jersey, at various concentrations representing varying copies per cell , and amplification was carried out on these samples as described in this example at annealing temperatures of 35°C and 55°C. At 35°C, the best that can be seen by agarose gel electrophoresis is 1 copy in 50 cells. At 55°C, the best that can be seen is 1/5,000 cells (a 100-fold improvement over the lower temperature), illustrating the importance of increased annealing temperature for Taq polymerase specificity under these conditions.

In a third experiment, DNA from a cell line 368H containing HIV-positive DNA, available from B. Poiesz, State University of New York, Syracuse, NY, was similarly diluted into the DNA from the SCI cell line (deposited with ATCC on March 19, 1985; an EBV-transf orrned ø cell line homozygous for the sickle cell allele and lacking any HIV sequences) at various concentrations representing varying copies per cell, and amplification was carried out as described in this Example at annealing temperatures of 35°C and 55°C, using the primers SK38 and SK39, which amplify a 115 bp region of the HIV sequence: 51 -ATAATCCACCTATCCCAGTAGGAGAAAT-3 ' (SK38) and 5 ' -TTTGGTCCTTGTCTTATGTCCAGAATGC-3 ' ( SK39 ) The results by agarose gel electrophoresis showed that only the undiluted 368H sample could be detected with the annealing temperature at 35°C, whereas at least a 10" dilution can be detected with the annealing temperature at 55°C, giving a 100-fold improvement n detection.

EXAMPLE XI cDNA was made from 1 pg of rabbit reticulocyte mRNA (Bethesda Research Laboratories) in a 100 μΐ reaction volume containing 150 mM KC1, 50 mM Tris'HCl (pH 8.3), 10 mM MgCl2, 5 mM DTT, 0.5 mM dATP, 0.5 mM dCTP, 0.5 mM TTP, 0.5 mM dGTP, 0.2 yg oligo(dT)12-18 (Pharmacia), 40 units RNasin (Promega Biotec), and 5 units AMV reverse transcr ptase (BRL) and incubated for 30 minutes at 42°C. The reaction was stopped by heating for 10 minutes at 95°C. Two ug RNase A was added to the sample (2 yl of a 2 mg/ml solution in water) and incubated for 10 minutes at 37 °C.

Three amplification reactions were done with the Klenow fragment using different pairs of primers. The primer pair PC03/PC04 define a 110-bp product. The primer pair RS45/oligo(dT)25-30 define an about 370-bp product, and the primer pair PC03/oligo(dT)25-30 an about 600-bp product. PC03, PC04, and RS45 are complementary to the human s-glob n gene and each has two mismatches with the rabbit gene. PC03 and PC04 are described in Example I. RS45 has the sequence: 5 ' -CAAGAAGGTGCTAGGTGCC-31.

The amplification reactions were performed with l/20th (5 yl of the cDNA described above in a 100 μΐ reaction volume containing 50 mM NaCl, 10 m Tris'HCl (pH 7.6), 10 mM MgCl2> 200 ug/ml gelatin, 10% DMS0, 1 yM PC03 or RS45, 1 UM PC04 or ol igo(dT)25-30, 1.5 mM dATP, 1.5 mM dCTP, 1.5 mM TTP and 1.5 mM dGTP. The samples were heated for five minutes at 98°C, then cooled to room temperature and overlayed with 100 μΐ mi neral oil .

The samples were subjected to 10 cycles of automated amplification using the machine described in Example I and using the follow ng program: 1) heating from 37 °C to 98°C in a heating block over 2.5 minutes (denature); 2) cooling from 98°C to 37°C over 3.0 minutes .(anneal ) ; . 3) adding 1 unit Klenow fragment; and 4) maintaining at 37°C for 20 minutes (extend).

The final volume of each sample was about 140 μΐ .

One-twentieth (7 ul) of each sample was analyzed by electrophoresis on a 2% agarose gel. After staining with ethidium bromide, discrete bands were seen in the PC03/PC04 and RS45/ol i'go(dT) samples. The sizes of the bands were consistent with the expected lengths: 110-bp for the former, about 370-bp for the latter. No evidence of amplification of an about 600-bp fragment with the PC03/oligo(dT) primer pair was observed.

The contents of the gel were Southern blotted onto a Genatran nylon membrane and hybridized with a nick-translated human p- globin probe, pBR328:betaA, described in Saiki et al . , Science, supra, using standard techniques. The resulting autorad ogram extended the conclusions reached previously - the 110 and about 370-bp fragments were β-globin specific amplification products and no significant amplification of the about 600-bp band was detected.

Three additional samples were amplified with the Taq polymerase obtained as described above using the same primer pairs described previously. Five microliter portions of cDNA'were amplified in 100 μΐ reaction volumes containing 50 mM C1 , 25 mM Tris'HCl (pH 8.0), 10 mM MgCl2, 200 ug/ml gelatin, 10% DMS0, 1 yM PC03 or RS45, 1 μΜ PC04 or oligo-(dT)25-30, 1.5 mM dATP, 1.5 mM dCTP, 1.5 mM TTP and 1.5 mM dGTP. The samples were heated for five minutes at 98°C, then cooled to room temperature. One microliter of Taq polymerase (1/8 dilution of lot 2) was added to each and overlayed with about 100 μΐ mineral oil .

The samples were subjected to 9 cycles of amplification in the Peltier device described in the previous example using the following program: 1) 1 mi n., 35 to 60°C ramp; 2) 12 mi n., 60 to 70 °C ramp (extend); 3) 1 min., 70-95°C ramp (denature); 4) 30 sec, 95°C soak; 5) 1 min., 95 to 35 °C ramp (anneal); and 6) 30 sec, 35°C soak.

After the last cycle, the samples were incubated an additional 10 minutes at 70°C to complete the final (10th cycle) extension. The final volume of each was about 100 yl .

- - As before, l/20th (10 yl ) of each sample was analyzed on a 2% agarose gel. In this gel, amplification products were present in all three samples: 110-bp for PC03/PC04, about 370-bp for RS45/oligo(dT), and about 600-bp for PC03/oligo(dT). These results were confirmed by Southern transfer and hybridization with the pBR328:betaA probe.

The production of the 600-bp product with Taq polymerase but not with the lenow fragment is significant, and suggests that Taq polymerase is capable of producing longer DNA than the Klenow fragment.

The following bacteriophage and bacterial strains were deposited with the Cetus Master Culture Collection, 1400 Fifty-Third Street, Emeryville, California, USA (C CC) and with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, USA (ATCC). These deposits were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for purposes of Patent Procedure and the Regulations thereunder (Budapest Treaty). This assures maintenance of a viable culture for 30 years from the date of deposit. The organisms will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between applicants and ATCC that assures unrestricted availability upon issuance of the pertinent U.S. patent. Availability of the deposited strains is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.

Deposit Designation CMCC No. ATCC No Deposit CH35:Taq#4-2 3125 40,336 5/29/87 E. coli DG98/pFC83 3128 67,421 5/29/87 £· co i DG98/pFC85 3127 67,422 5/29/87 In summary, the present invention is seen to provide a process for amplifying one or more specific nucleic acid sequences using a tempe ature-cycled chain reaction and a thermostable enzyme, in which reaction primer extension products are produced that can subsequently act as templates for further primer extension reactions. The process is especially useful in detecting nucleic acid sequences that are initially present in only very small amounts and in detecting nucleotide variations using sequence-specific oligonucleotides. Also, the ampl fication process herein can be used for molecular cloning.

The process herein results in increased yields of amplified product, greater specificity, and fewer steps necessary to carry out the amplification procedure, over what has been previously disclosed.

Claims (37)

- 84 - 103039/3 CLAIMS:

1. A process for amplifying at least one specific nucleic acid sequence contained in a nucleic acid or a mixture of nucleic acids, wherein the nucleic acid is double-stranded or consists of two separated complementary strands of equal or unequal length, which process comprises: (a) contacting the nucleic acid with four different nucleoside triphosphates and two oligonucleotide primers, for each different specific sequence being amplified, wherein each primer is selected to be sufficiently complementary to different strands of each specific sequence to hybridize therewith, such that the extension product synthesized from one primer, when separated from its complement, can serve as a template for synthesis of the extension product of the other primer, if the nucleic acid is double-stranded, heating the mixture for an effective time and at an effective temperature to denature each nucleic acid and cooling the mixture to a temperature which promotes hybridization of each primer to its complementary strands or, if the nucleic acid consists of two separated complementary strands, treating the mixture at a temperature which promotes hybridization of each primer to its complementary strand; (b) contacting each strand, at the same time as or after step (a), with a thermostable enzyme which catalyzes the template-dependent combination of the nucleoside triphosphates to form primer extension products complementary to each strand of nucleic acid, said thermostable enzyme being a Thermus aquaticus DNA polymerase that migrates on a denaturing polyacrylamide gel faster than phosphorylase B and more slowly than does bovine serum albumin and has an estimated molecular weight of at least 86,000 daltons when compared with a phosphorylase B standard assigned a molecular weight of 92,500 daltons; - 84a - 103039/1 (c) maintaining the mixture from step (b) at an effective temperature for an effective time to promote the activity of the enzyme, and to synthesize, for each different sequence being amplified, an extension product of each primer which is complementary to each strand, but not so high as to separate each extension product from its complementary strand; (d) heating the mixture from step (c) for an effective time and at an effective temperature to separate the primer extension products from the strands on which they were synthesized to produce single-stranded molecules, but not so high as to denature irreversibly the enzyme; (e) cooling the mixture from step (d) to an effective temperature to promote hybridization of each primer to each of the single-stranded molecules produced in step (d); and (f) maintaining the mixture from step (e) at an effective temperature for an effective time to promote the activity of the enzyme and to synthesize, for each different sequence being amplified, an extension product of each primer which is complementary to each strand produced in step (d), but no so high as to separate each extension product from its complementary strand, wherein steps (e) and (f) are conducted simultaneously or sequentially.

2. The process of claim 1, wherein one specific nucleic acid acid sequence is amplified and two primers are employed.

3. The process of claim 1 or 2, wherein steps (d), (e) and (f) are repeated at least five times.

4. The process of claim 3, wherein after steps (d)-(f) are repeated at least once, a second set of two primers is added, wherein the primers added are sufficiently complementary to different strands at internal sequences of the amplified sequence to hybridize therewith, such that the extension product synthesized from one primer, when separated from its complement, can serve as a template for synthesis of the extension product of the other primer.

5. The process according to any one of claims 1 - 4, wherein said thermostable enzyme is a polymerase from Thermus aquaticus.

6. The process of claim 5 wherein in step (a) the triphosphates and primer(s) are contained in a buffer comprising 1.5-2 mM of a magnesium salt, 150-200 μΜ each of the triphosphates, and 1 μΜ of each primer, steps (a), (e) and (f) are carried out at about 45°- 58° C, and step (d) is carried out at about 90-100° C.

7. The process according to any one of claims 1-6, wherein said primers are oligodeoxyribonucleotides.

8. The process according to any one of claims 1-7, wherein said nucleic acid is cDNA. - 86 - 103039/3

9. The process according to any one of claims 1-8, wherein the heating and cooling steps (d)-(f) are automated by a machine which controls temperature levels, transitions from one temperature to another, and the timing of temperature levels.

10. The process according to any one of claims 1-9, wherein each primer is present in a molar ratio of at least 1000:1 primerxomplementary strand.

11. The process according to any one of claims 1-10, wherein at least one primer contains at least one nucleotide which is not complementary to the specific sequence to be amplified.

12. The process of claim 11, wherein said primer that contains at lest one nucleotide which is not complementary to the specific sequence to be amplified encodes a promoter.

13. The process according to any one of claims 1-12, wherein steps (e) and (f) are carried out sequentially, steps (c) and (f) take place at 40-80°C, step (d) takes place at 90-105 °C, and step (e) takes place at 35-65°C.

14. The process of claim 13, wherein steps (e) and (f) are carried out sequentially, steps (c) and (f) take place at 50-75°C, step (d) takes place at 90-100°C, and step (e) takes place at 37-60°C.

15. The process according to any one of claims 1-14, wherein steps (e) and (f) are carried out simultaneously at about 45-70°C.

16. The process according to any one of claims 1-15, wherein said specific nucleic acid sequence is contained in a mixture of nucleic acids.

17. The process of claim 16, wherein said specific nucleic acid sequence is contained in a larger sequence.

18. A process for detecting the presence of a specific nucleic acid sequence in a sample, said process comprising: (a) amplifying said sequence by a process according to any one of claims 1-17, and (b) combining the product of step (a) with a hybridization probe for determining whether amplification has occurred and detecting whether said hybridization has occurred.

19. The process of claim 18, wherein step (b) is carried out by adding to the product of step (a) a labeled oligonucleotide probe for each sequence being detected capable of hybridizing to said sequence or to a mutation thereof; and by determing whether said hybridization has occurred.

20. The process of claim 18 or 19, wherein after step (a) and before step (b) the sample is cut with a restriction enzyme and electrophoresed, and step (b) is accomplished by Southern blot analysis.

21. The process according to any one of claims 18-20, wherein the specific nucleic acid sequence to be detected can cause a genetic, infectious or cancerous disease.

22. The process of claim 21, wherein the genetic disease is sickle cell anemia or hemoglobin C disease.

23. A process for detecting the presence or absence of at least one nucleotide variation in a sequence in one or more nucleic acids contained in a sample, said process comprising (a) amplifying said sequence by a process according to anyone of claims 1-17 and (b) affixing the product of step (a) to a membrane; (c) treating the membrane under hybridization conditions with a labeled sequence-specific oligonucleotide probe capable of hybridizing with the amplified nucleic acid sequence only if a sequence of the probe is complementary to a region of the amplified sequence; and (d) detecting whether the probe has hybridized to an amplified sequence in the nucleic acid sample.

24. A process for detecting the presence or absence of at least one nucleotide variation in sequence in one or more nucleic acids contained in a sample, said process comprising (a) amplifying said sequence by a process according to any one of claims 1- 17 wherein at least one primer and/or at least one of the four nucleoside triphosphates used for amplification is labeled with a detectable moiety; - 88 - 103039 (b) affixing to a membrane a sequence-specific oligonucleotide capable of hybridizing with the amplified nucleic acid sequence only if a sequence of the oligonucleotide is complementary to a region of the amplified sequence; (c) treating the membrane under hybridization conditions with the product of steps (a); and (d) detecting whether an amplified sequence in the nucleic acid sample has hybridized to the oligonucleotide affixed to the membrane.

25. A process for detecting the presence or absence of at least one nucleotide variation in sequence in one or more nucleic acids contained in a sample, said process comprising (a) amplifiying said sequence according to any one of claims 1-17; (b) affixing to a membrane a labeled sequence-specific oligonucleotide probe capable of hybridizing with the amplified nucleic acid sequence, only if a sequence of the probe is complementary to a region of the amplified sequence; (c) treating the membrane under hybridization conditions with the product of step (a); (d) treating the product of step (c) with a restriction enzyme which will cleave any hybrids formed if the probe and variation(s) to be detected both contain a restriction site recognized by the enzyme; and (e) detecting whether a labeled restriction fragment of the required length is present in the restriction digest to indicate that hybridization occurred.

26. A process for cloning into a cloning vector at least one specific nucleic acid sequence contained in a mixture of nucleic acids, which process comprises (a) amplifying said sequence in quantity by a process in accordance with any one of claims 1-17 wherein the sequence being amplified and cloned contains restriction sites or each primer contains a restriction site; (b) adding to the product of step (a) a restriction enzyme for each of said restriction sites to obtain cleaved products in a restriction digest; and - 89 - 103039/2 (c) ligating the cleaved product(s) of step (b) containing the specific sequence to be cloned into a suitably cleaved cloning vector, said cleaved product and said cleaved vector being present in sufficient amounts to effect the ligation.

27. The process according to claim 26, wherein the cloning vector contains a selectable marker.

28. The process of claim 26 or 27, further comprising the step of passing the restriction digest of step (b) through a desalting column or membrane before step (c).

29. The process according to any one of claims 26-28, further comprising, after step (c), sequencing the cleaved product ligated into the vector.

30. The process according to any one of claims 26-28, wherein said specific nucleic acid sequence encodes a protein and wherein said method further comprises, after step (c), a further step (d) wherein the specific nucleic acid sequence contained in said cloning vector is expressed by using a suitable host-organism.

31. The process according to any one of claims 26-28, wherein each primer contains a restriction site which is the same as in the other primer.

32. The process according to any one of claims 26-28, wherein one specific sequence is being amplified, the restriction sites are different on each primer, and the product of step (b) is ligated into one cloning vector with a specific orientation.

33. The process of claim 32, wherein the specific nucleic acid sequence amplified is contained within the β-gJobin gene or the N-RAS oncogene.

34. The process of claim 31 of 32, wherein the restriction sites are on the 5' end of the primers.

35. A process for cloning into a cloning vector at least one specific nucleic acid sequence contained in a nucleic acid or mixture of nucleic acids, which process comprises (a) amplifying said sequence by a process according to any one of claims 1-17; (b) ligating the amplified nucleic acid containing the specific sequence for blunt- end ligation into a suitably cleaved cloning vector in the presence of a ligase, said - 90 - 103039/2 amplified sequence and vector being present in sufficient amounts to effect the ligation.

36. The process of claim 35, wherein one nucleic acid sequence is cloned into one vector and two primers are employed.

37. A kit comprising a thermostable enzyme for carrying out the process according to any one of claims 1-36. For the Applicants, DR. REINHOLD COHN AND PARTNERS