|||||||||||||| USOO5142O33A United States Patent (19) (11) Patent Number: 5,142,033 Innis (45) Date of Patent: Aug. 25, 1992 54) STRUCTURE-INDEPENDENT DNA y AMPLIFICATION BY THE POLYMERASE FOREIGN PATENT DOCUMENTS CHAIN REACTION 0237362 9/1987 European Pat. Off. 75) Inventor: Michael A. Innis, Moraga, Calif. w 025801717 3/19889 European Pat. OffA 73) Assignee: Hoffmann-La Roche Inc., Nutley, OTHER PUBLICATIONS N.J. Barr et al., 1986, Bio Techniques 4(5):428-432. Saiki et al., 1988, Science 239:476-49. (21) Appl. No.: 738,324 Promega advertisement and certificated of analysis 22 Filed: Jul. 31, 1991 dated Aug. 9, 1988 "Tag Track Sequencing System". Heiner et al., 1988, Preliminary Draft. Related U.S. Application Data McConlogue et al., 1988, Nuc. Acids Res. 16(20):9869. 63) continuation of ser, No. 248,556, sep. 23, 1988, Pat. Inset a 1988, Proc. Natl. Acad. Sci. USA No. 5,091,310. 85:9436-9440. & Mizusawa et al., 1986, Nuc. Acids Res. 14(3):1319-1324. 51) Int. C. ....................... C07H 21/04; SES 6. Simpson et al., 1988 Biochem. and Biophys. Res. Comm. 151(1):487-492. 52) U.S. C. .......................................... 536/27; 435/6; Chait, 1988, Nature 333:477-478. 435/15; 435/91; 435/83; 435/810; 436/501; 436/808; 536/28: 536/29; 530/350; 530/820; Primary Examiner-Margaret Moskowitz 935/16: 935/17, 935/18: 935/78; 935/88 Assistant Examiner-Ardin H. Marschel 58) Field of Search ....................... 435/6, 91, 15, 810, Attorney, Agent, or Firm--Kevin R. Kaster; Stacey R. 435/183; 436/501, 808: 536/27-29; 935/16, 17, Sias 18, 78, 88: 530/820, 350 (57) ABSTRACT 56) References Cited Structure-independent amplification of DNA by the U.S. PATENT DOCUMENTS polymerase chain reaction can be achieved by incorpo 4,683, 95 7/1987 Mullis et al. ............................ as ration of 7-deaza,2'-deoxyguanosine-5'-triphosphate 4,68302 7/1987 Mullis ................ so into the amplified DNA. 4,795,699 M1989 Tabor et al. ..... 435/5 4.92,794 5/1990 Tabor et al. ........................., 435/9 9 Claims, 2 Drawing Sheets 1 2 3 4 5 tee U.S. Patent Aug. 25, 1992 Sheet 1 of 2 5,142,033 O V CO CN ve i s : U.S. Patent Aug. 25, 1992 Sheet 2 of 2 5,142,033 1 2 3 4 5 er geis C FIG. 2 5,142,033 1. 2 nucleic acid helicase can also be used for this purpose. STRUCTURE-INDEPENDENT DNA However, this heating process can destroy the activity AMPLIFICATION BY THE POLYMERASE CHAN of the agent for polymerization, typically a DNA poly REACTION merase, that is used in PCR. Consequently, DNA poly merase must be added to PCR reaction mixtures after This application is a continuation of application Ser. each heat-denaturation step unless a thermostable poly No. 07/248,556, filed Sept. 23, 1988, now U.S. Pat. merase is employed. European Patent Publication No. No.5,091,310. 258,017 discloses a number of such thermostable polymerases, of which the polymerase from Thermus BACKGROUND OF THE INVENTION 10 aquaticus, Taq polymerase, is most preferred for use in 1. Field of the Invention PCR, because the enzyme can survive repeated cycles The present invention relates to a process for ampli of heating and cooling in PCR. fying a specific segment of nucleic acid. Methods for Saiki et al., Jan. 19, 1988, Science 239:487-49, de amplifying nucleic acid can be used to facilitate the scribe methods for using Taq polymerase in PCR. Saiki cloning of DNA and the characterization of both DNA 15 et al. also describe the amplification of a specific DNA and RNA sequences and so are useful methods for pur segment present only once in a sample of 10 to 106 cells. poses of recombinant DNA technology and the study of Methods for purifying Taq polymerase and for the molecular biology. In addition, the amplification of recombinant expression of the enzyme are disclosed in specific nucleic acid segments greatly facilitates the Ser. No. 143,441, now abandoned, filed Jan. 12, 1988, detection of pathogens and of disease states associated 20 which is a CIP of Ser. No. 063,509, filed Jun. 17, 1987, with the presence of particular nucleic acid sequences, which issued as U.S. Pat. No. 4,889,818, which is a CIP so the present invention is also of importance in medical Ser. No. 899,241, now abandoned, filed Aug. 22, 1986, diagnostic procedures. The detection of specific nucleic each of which is incorporated herein by reference. acid sequences is also useful for purposes of forensic Instruments for performing automated PCR are dis medicine and paternity and individuality determination. 25 closed in Ser. No. 899,061, filed Aug. 22, 1986, which is 2. Description of Related Disclosures a CIP of Ser. No. 833,368, filed Feb. 25, 1986, now U.S. Pat. No. 4,683,202 discloses a process for ampli abandoned. Structure-independent amplification of fying a specific nucleic acid segment by the polymerase DNA can also be useful in the DNA sequencing meth chain reaction (PCR). PCR involves the use of two ods disclosed in Ser. No. 249,367, filed Sep. 23, 1988. oligonucleotide primers, an agent for polymerization, a 30 The present invention can be applied in the generation target nucleic acid template, and successive cycles of of single-stranded DNA by the method termed "asym denaturation of nucleic acid and annealing and exten metric PCR" disclosed in Ser. No. 248,896, filed Sep. sion of the primers to produce a large number of copies 23, 1988. The disclosures of these related patents and of a particular nucleic acid segment. The segment cop applications are incorporated herein by reference. ied consists of a specific sequence of nucleotides from 35 PCR methodologies offer enormous practical advan the target template. This specific sequence is defined by tages over any other known way to amplify nucleic acid regions on the template that can hybridize to the prim sequences. On occasion, however, a given pair of PCR ers and the nucleic acid sequence between those re primers does not yield an amplification product, even gions. PCR methodology has had a profound impact in though the target sequence is present in the reaction the fields of molecular biology, recombinant DNA 40 mixture. When faced with such a "no amplification' technology, forensic medicine, and medical diagnostic result, the practitioner must do extensive testing of technology. reagents and operating techniques to determine the U.S. Pat. No. 4,683, 195 discloses a variety of methods origin of the problem. Sometimes such testing reveals for using PCR to amplify and then detect or clone a the cause of the problem to be the particular primers given nucleic acid segment. European Patent Publica- 45 used in the amplification, because primers that hybrid tion No. 237,362 further illustrates the utility of PCR in ize to another region of the target sequence can provide the detection of specific nucleic acid sequences by dis efficient amplification. This problem of "no amplifica closing that PCR can be used in conjunction with la tion" occurs infrequently but, prior to the present in beled probes and "dot-blot" methodology to detect the vention, unpredictably. The problem is compounded by presence of a nucleic acid sequence initially present in 50 the difficulty in determining whether the primers are extraordinarily small amounts. the reason for the inefficient amplification results. Methods for performing PCR are disclosed in Ser. Scientists working in areas not involving the poly No. 063,647, filed Jun. 17, 1987, which issued as U.S. merase chain reaction have observed that certain nu Pat. No. 4,965,188, and which is a continuation-in-part cleic acid sequences can form stable secondary struc (CIP) of Ser. No. 899,513, filed Aug. 22, 1986, now 55 tures, such as palindromic hairpin loops or compressed abandoned, which is a CIP of Ser. No. 828,144, filed regions. Because the presence of such structures can Feb. 7, 1986, which issued as U.S. Pat. No. 4,683, 195, lead to anomalous migration patterns during gel electro and which is a CIP of Ser. No. 791,308, filed Oct. 25, phoresis, i.e., as in DNA sequencing, researchers at 1985, which issued as U.S. Pat. No. 4,683,202, and tempted to find means for preventing the formation of which is a CIP of abandoned Ser. No. 76,975, filed 60 secondary structures in nucleic acids. Barr et al., 1986, Mar. 28, 1985, all of which are incorporated herein by Bio Techniques 4(5):528-532, reported that use of 7 reference. deaza-2'-deoxyguanosine-5'-triphosphate (c"dGTP) in European Patent Publication No. 258,017 discloses a dideoxy-sequencing reaction mixtures helped to to re thermostable DNA polymerase that significantly sim solve abnormal and compressed regions in the sequenc plifies PCR methodology. PCR involves successive 65 ing gels. rounds of denaturation or "melting" of double-stranded The present invention results from a synthesis of nucleic acid. The denaturation process is often carried these two arts and provides a method for structure out by heating the sample of nucleic acid, although a independent amplification of DNA by PCR. 5,142,033 3 4. the single strand copies of the target segment prevent SUMMARY OF THE INVENTION ing priming (annealing of primers to template) and ex The present invention provides a method for struc tending of the primers. ture-independent amplification of DNA by the poly The present invention provides a simple method for merase chain reaction. The method comprises: overcoming this unrecognized problem.