The polymerase chain reaction (PCR) is a method for increasing by many orders of magnitude the concentration of a specific nucleic acid sequence in a test sample. The PCR process is disclosed in U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,965,188, each of which is incorporated herein by reference.
In PCR, a test sample believed to contain one or more targeted nucleic acid sequences is combined in a total volume of usually about 20 to 200 .mu.l with the following reagents: an aqueous buffer, pH 8-9 at room temperature, usually also containing approximately 0.05 M KCl; all four common nucleoside triphosphates (e.g., for DNA polymerase, the four common dNTPs: dATP, dTTP, dCTP, and dGTP) at concentrations of approximately 10.sup.-5 M to 10.sup.-3 M; a magnesium compound, usually MgCl.sub.2, usually at a concentration of about 1 to 5 mM; a polynucleotide polymerase, preferably a thermostable DNA polymerase, most preferably the DNA polymerase I from Thermus aquaticus (Taq polymerase and the Stoffel fragment of Taq polymerase are the subject of U.S. Pat. No. 4,889,818, incorporated herein by reference; the latter enzyme lacks the 5'.fwdarw.3' exonuclease activity of native Taq polymerase), usually at a concentration of 10.sup.-10 to 10.sup.-8 M; and single-stranded oligonucleotide primers, usually 15 to 30 nucleotides long and usually composed of deoxyribonucleotides, containing base sequences which are Watson-Crick complementary to sequences on both strands of the target nucleic acid sequence(s). Each primer usually is present at a concentration of 10.sup.-7 to 10.sup.-5 M; primers are synthesized by solid-phase methods well known in the art of nucleic acid chemistry.
In the simplest form, PCR requires two primers for each target sequence. These primers, when annealed to the opposing target strands, have their 3' ends directed toward one another's hybridization sites and separated by about 100 to 1,000 nucleotides (occasionally up to about 10,000 nucleotides). The polymerase catalyzes magnesium-dependent, template-directed extension of each primer from the 3' end of the primer, incorporating nucleoside monophosphates into the growing nucleic acid and releasing pyrophosphate.
This extension reaction continues until the polymerase reaches the 5' end of the template strand to which the extended primer was annealed, at which point the polymerase is free to bind to another primer-template duplex and catalyze extension of that primer molecule; the extension reaction also stops if the reaction mixture is heated to temperatures sufficient to separate the template from the extended primer before the enzyme has reached the 5' end of the template. After the enzyme has worked long enough to transform a large fraction of the primer-template duplexes into double-stranded nucleic acid, the latter can be denatured at high temperature, usually 90.degree. to 100.degree. C., to create two single-stranded polynucleotides, which, after cooling to a temperature where they can be annealed to new primer molecules, serve as templates for another round of enzyme-catalyzed primer extension. Because both DNA strands serve as template, each round of nucleic acid replication approximately doubles the concentration of the specific nucleic acid sequence defined at its ends by the two primer sequences. Therefore, the total concentration increase in the target nucleic acid sequence in a PCR amplification is by a factor of approximately 2.sup.n, where n is the number of completed thermal cycles between a high temperature where double-stranded DNA is denatured and a lower temperature or set of temperatures (40.degree. to 75.degree. C.) where primer-template annealing and primer extension occur.
Although one can move PCR reaction tubes manually back and forth between thermostated baths in the two temperature ranges, PCR most commonly is performed in an automated temperature-controlled machine, known as a "thermal cycler," in which a microprocessor is programmed to change the temperature of a heat-exchange block or bath containing reaction tubes back and forth among several specified temperatures for a specified number of cycles, holding at each temperature for a specified time, usually on the order of one-half to two minutes. Such a thermal cycler is commercially available from Perkin Elmer Cetus Instruments and described in the European Patent Publication No. 236,069 and U.S. patent application Ser. No. 670,545, filed Mar. 14, 1991, which is a continuation-in-part of Ser. No. 620,606, filed Nov. 29, 1990, both of which are incorporated herein by reference. The total cycle time is usually less than 10 minutes, and the total number of cycles is usually less than 40, so that a single, multi-cycle amplification, amplifying the targeted nucleic acid sequence 10.sup.5 to 10.sup.10 times, normally takes less than seven hours and often less than four hours.
The practical benefits of PCR nucleic acid amplification have been rapidly appreciated in the fields of genetics, molecular biology, cellular biology, clinical chemistry, forensic science, and analytical biochemistry, as described in the following review volumes and articles: Erlich (ed.), 1989, PCR Technology, Stockton Press (New York); Erlich et al. (eds.), 1989, Polymerase Chain Reaction, Cold Spring Harbor Press (Cold Spring Harbor, N.Y.); Innis et al., 1990, PCR Protocols, Academic Press (New York); and White et al, 1989, Trends in Genetics 5/6:185-189. PCR can replace a large fraction of molecular cloning and mutagenesis operations commonly performed in bacteria, having advantages of speed, simplicity, lower cost, and sometime increased safety. Furthermore, PCR permits the rapid and highly sensitive qualitative and even quantitative analysis of nucleic acid sequences, often with greatly increased safety because so much PCR product is made that nonisotopic detection modes suffice.
Despite rapid and broad adoption of PCR by a range of biological and chemical disciplines, PCR has sometimes suffered from the occurrence of side reactions which interfere with amplification of the specific target sequence or sequences. Many amplifications yield non specific side products differing in size and sequence from the sequence targeted by the primers used. Sometimes nonspecificity is caused by mis-priming, where primers have been annealed to non-target sequences, also present in the nucleic acid of the test sample similar to the target sequence. Although the genomic DNA commonly contained in PCR test samples has customarily been thought to be completely double-stranded, procedures used to prepare DNA for amplification appear to render that DNA, to a significant extent, single-stranded. Single-stranded DNA is especially susceptible to mis-priming if mixed with a complete set of PCR reagents at ambient or sub-ambient temperatures. Many PCR reagents also yield primer dimers or oligomers, double-stranded side products containing the sequences of several molecules joined end-to-end, the yield of which correlates negatively with the yield of amplified target sequence.
Recently several methodological modifications have improved PCR specificity and sensitivity significantly. In Hot Start.TM. PCR, complete mixing of PCR reagents and test sample is delayed until reactants have been heated to a temperature in the 50.degree. C.-80.degree. C. range, sufficient to minimize mis-priming and primer dimerization; thermal cycling is started immediately after mixing at elevated temperature. In manual Hot Start.TM. PCR, the operator heats the reaction tube, containing test sample and a subset of PCR reagents, to the elevated incubation temperature, opens each tube separately to add a small volume of liquid containing the missing reagent(s), and closes each tube before moving on to the next one. See Frohman et al., 1988, Proc. Natl. Acad. Sci. USA 85:8998-9002; Ward et al., 1989, Nature 341:544-546; Newton et al., 1989, Nucl. Acids Res. 17:2503-2516; and Faloona et al., Abstract 1019, 6th International Conference on AIDS, June 20-24, 1990, San Francisco, Calif. More recently, Hot Start.TM. PCR was rendered more convenient and precise by (1) replacement of the conventional mineral oil vapor barrier by a layer of wax melting in the 50.degree. C. to 80.degree. C. range, (2) assembly of reaction tubes such that before thermal cycling, PCR reactants are grouped into subsets separated by a solid wax layer, and (3) convective mixture of all reactants during the first heating step of thermal cycling after the solid wax melts into a lighter-than-water oil. Such wax-mediated, Hot Start.TM. PCR is the subject of U.S. patent application Ser. No. 481,501, filed Feb. 19, 1990, now abandoned in favor of continuation application U.S. Ser. No. 07/890,300, filed May 27, 1992, incorporated herein by reference.
Alternatively, nonspecific amplified nucleic acid resulting from primer dimerization and mis-priming while completely mixed PCR reactants stand at room temperature before thermal cycling can be destroyed by an enzymatic restriction process described in PCT U.S. patent application Ser. No. 91/05210 filed Jul. 23, 1991, which published as PCT Patent Publication No. WO 92/01814 on Feb. 6, 1992, which is a continuation-in-part of U.S. Ser. No. 609,157, filed Nov. 2, 1990, now abandoned which is a continuation-in-part of U.S. Ser. No. 557,517, filed Jul. 24, 1990, now abandoned each of which is incorporated herein by reference. To perform such restriction, one of the conventional four dNTPs is replaced by a structural analogue which is incorporated into all amplified nucleic acid by the PCR polymerase. Also included in the reaction mixture is an enzyme which digests nucleic acid at (and only at) positions which contain the structural analogue; this enzyme must be active only at temperatures below about 50.degree. C., so that it does not damage amplified nucleic acid during thermal cycling at higher temperatures. Preferably the restriction enzyme is permanently inactivated during thermal cycling, so that it cannot damage amplified nucleic acid if the latter is stored for any significant period of time at room temperature after amplification and before analysis. The most practical restriction enzymes are glycosidases which cleave from the polynucleotide phosphodiester backbone the unconventional nucleic acid base introduced by the dNTP analogue. The resulting abasic sites experience cleavage of the polynucleotide phosphodiester backbone upon heating. This restriction process has been integrated practically with PCR by replacing dTTP with dUTP and by incorporating in the reaction mixture the enzyme uracil-N-glycosidase.
A chemical variant of the Hot Start.TM. process incorporates into the PCR reagent mixture a single-stranded DNA binding protein (SSB) at a concentration sufficient to bind a significant fraction of the single-stranded DNA present before thermal cycling is started. This ssDNA comprises minimally the primers, the concentrations of which are well known by the operator, and may also include slight or considerable amounts of the test sample DNA, depending on whether the latter has been prepared in a way which might denature it. During thermal cycling, the binding of the SSB to primers and single-stranded template strands formed by PCR product denaturation must be weak enough not to interfere with primer-template annealing and enzymatic primer extension. Before thermal cycling, while reactants stand together at room temperature, SSB binding to the primers and any single-stranded regions of test sample DNA must be strong enough to block mis-priming and primer dimerization. Two heavily studied SSBs (Chase and Williams, 1986, Ann. Rev. Biochem. 55:103-136) are commercially available and have been used with PCR: gene 32 protein from the bacteriophage T4 and the 19 kilodalton SSB from E. coli (19 kda is the subunit size; the normal active species is a tetramer). SSB is the major active ingredient of Perfect Match.TM. polymerase enhancer, a mixture of E. coli SSB and bovine serum albumin sold by Stratagene (San Diego, Calif.) for the purpose of increasing PCR specificity and yield. Bacteriophage gene 32 protein has been included in PCR mixtures to improve amplification of long targets (Schwarz et al., 1990, Nucl. Acids Res. 18:1079) and to relieve polymerase inhibition by blood in the test sample (Panaccio and Lew, 1991, Nucl. Acids Res. 19:1151). However, essentially all organisms possess SSBs with compositions unique to each organism. Other SSBs which have been characterized biochemically include one from a filamentous bacteriophage (Brayer and McPherson, 1984, Biochemistry. 23:340-349), a family of sequence-homologous proteins from plant virus (Saito et al., 1988, Virology 167:653-656, and Citovsky etal., 1990, Cell 60:637-647), and one from Agrobacterium tumefaciens (Citovsky et al., 1989, Proc. Natl. Acad. Sci. USA 86:1193-1197). SSBs possess enough structural similarity to suggest that DNA binding is associated with a consensus structure of alternating aromatic amino acids (phenylalanine, tyrosine, and tryptophan) and charged amino acids (glutamate, aspartate, lysine, and arginine) (Prasad and Chiu, 1987, J. Mol. Biol. 193:579-584) such that artificial polypeptides might be created which function as well as the biological SSBs in improving PCR specificity and yield. In addition, enough is known about SSB structure and function to suggest ways to improve function by genetic engineering.
Although the three basic tactics of PCR specificity enhancement (Hot Start.TM. methods, amplified DNA restriction, and SSB addition to the reaction mixture) each can serve alone to improve specific amplification, combinations of the three approaches may have special benefits. For example, whereas Hot Start.TM. methods block only that nonspecificity resulting from reactant incubation at ambient temperature before cycling is started, SSB s may reduce mis-priming which arises during thermal cycling. On the other hand, SSB used without a manual or wax-mediated Hot Start.TM. process occasionally will trigger massive primer dimerization which interferes with specific amplification. The combination of the two methods optimally reduces mis-priming and primer dimerization.
The preceding background art has dealt with conventional PCR, wherein test sample nucleic acids are extracted from a biological source in a way which destroys target sequence association with individual cells or subcellular structures. So-called in situ nucleic acid hybridization methods have evolved to detect target sequences in the cells or organelles where they originated (for a review of the field, see Nagai et 1987, Intl. J. Gyn. Path. 6:366-379). Typically, in situ hybridization entails (1) preparation of a histochemical section or cytochemical smear, chemically fixed (e.g., with formaldehyde) to stabilize proteinaceous subcellular structures and attached to a microscope slide, (2) chemical denaturation of the nucleic acid in the cellular preparation, (3) annealing of a tagged nucleic acid probe to a complementary target sequence in the denatured cellular DNA, and (4) localized detection of the tag annealed to target, usually by microscopic examination of immobilized nonisotopic (absorbance or fluorescence staining) or isotopic (autoradiographic) signals directly or indirectly generated by the probe tag. However, conventional in situ hybridization is not very sensitive, generally requiring tens to hundreds of copies of the target nucleic acid per cell in order to score the presence of target sequence in that cell.
Recently, the sensitivity enhancement associated with PCR amplification of target sequence has been combined with the target localization of in situ hybridization to create in situ PCR, wherein PCR is performed within chemically fixed cells, before (Haase et al., 1990, Proc. Natl. Acad. Sci. USA 87:4971-4975, incorporated herein by reference) or after (Nuovo et al., 1991, Amer. J. Pathol. in press, incorporated herein by reference) the fixed cells have been attached to a microscope slide; the amplified nucleic acid is located by microscopic examination of autoradiographs following isotopically tagged probing (Haase et al., supra) or stained patterns directly deposited on the microscope slide following enzyme-linked detection of biotin-tagged probes (Nuovo et al., supra). The cells may be suspended (Haase et al., Supra) or may be part of a tissue section (Nuovo et al., supra) during in situ amplification.
In situ PCR requires a delicate balance between two opposite requirements of PCR in a cellular preparation: the cell and subcellular (e.g., nuclear) membranes must be permeabilized sufficiently to allow externally applied PCR reagents to reach the target nucleic acid, yet must remain sufficiently intact and nonporous to retard diffusion of amplified nucleic acid out of the cells or subcellular compartments where it is made. In addition, the amplified nucleic acid must be sufficiently concentrated within its compartment to give a microscopically visible signal, yet remain sufficiently dilute that it does not reanneal between the denaturation and probe-annealing steps. Haase et al., supra, relied on paraformaldehyde fixation of cells to have created sufficient but not excessive permeability. Nuovo et al., supra, also employed a single, commercially available, proteinase treatment to improve permeability.
Both Haase et al., supra, and Nuovo et al., supra, used a series of PCR primer pairs to specify a series of overlapping target sequences within the genome of the targeted organism to improve retention of amplified target nucleic acid within the cellular compartment where it was made. The resulting PCR product was expected to be so large (greater than 1,000 base pairs) that its diffusion from site of origin should be greatly retarded. However, the use of multiple primer pairs severely reduces the practicality of in situ PCR, not just because of the expense associated with producing so many synthetic oligonucleotides, but even more seriously because many PCR target organisms, especially pathogenic virus, are so genetically plastic that it is hard to find even a few short sequences which are sufficiently invariant to make good primer and probe sites. Other important target sequences, such as activated oncogenes, inactivated tumor suppressor genes, and oncogenic chromosomal translocations, involve somatic point mutations and chromosomal rearrangements which can be distinguished from the parental sequence if relatively short PCR products are amplified from single primer pairs. Multiple primer pairs and long structures would frustrate attainment of the specificity often needed to distinguish cancerous cells from their normal neighbors. Multiple primer pairs jeopardize PCR in a different way as well; they promote primer dimerization and mis-priming, reducing sensitivity and specificity and increasing the likelihood of false-negative results because nonspecific amplification radically reduces the yield of amplified target sequence. Reinforcing the tendency of multiple primer pairs to enhance nonspecific amplification are the rather high primer concentrations preferred for in situ PCR (Nuovo et al., supra).
One useful variant of conventional PCR detects target RNA sequences in test samples by creating complementary DNA (cDNA) sequences with the catalytic mediation of added reverse transcriptase; the cDNA then is subjected to standard PCR amplification (Kawasaki et al., 1988, Proc. Natl. Acad. Sci. USA 85(15):5698, and Rappolee et al., 1989, J. Cell. Biochem. 39:1-11). Recently, such RNA PCR has been streamlined by using a thermostable DNA polymerase which, depending on exact chemical conditions, also shows strong reverse transcriptase activity. This enzyme and its optimized application to RNA PCR are subject of PCT U.S. patent application Ser. No. US90/07641, filed Dec. 21, 1990, incorporated herein by reference. Adaptation of in situ PCR to RNA targets will realize the full potential of the method to differentiate among neighboring cells in a histochemical or cytochemical preparation with respect to somatic mutation, pathogenic infection, oncogenic transformation, immune competence and specificity, state of differentiation, developmental origin, genetic mosaicism, cytokine expression, and other characteristics useful for understanding both normal and pathological conditions in eukaryotic organisms.
The present invention increases the convenience, sensitivity, and specificity of in situ PCR, also eliminating any need for multiple primer pairs to detect a single target sequence. In doing so, it also allows in situ PCR to discriminate among alleles and increases the practicality of in situ PCR analysis of RNA targets.