Process for controlling contamination of nucleic acid amplification reactions

Abstract: An amplification procedure is performed on a first sample in which one or more of the four normal ribonucleoside triphosphates(rNTPs) or deoxyribonucleoside triphosphates (dNTPs) is replaced with an exo-sample nucleotide that is not present in nucleic acid to be amplified. After amplification, any contaminating amplified product that may be remaining is subjected to a physical, chemical, enzymatic, or biological treatment which renders nucleic acid containing the exo-sample nucleotide substantially unamplifiable. The treatment may be done as a separate step or it may be done in the presence of a second sample containing nucleic acid sequences to be amplified. The amplified nucleic acid sequences derived from the first sample which contaminate the second sample are not further substantially amplified during amplification of nucleic acid sequences of the second sample.

Description: The present invention relates to processes which amplify nucleic acid sequences. In particular, the present invention discloses a means for eliminating the products of an execution of a nucleic acid amplification process that contaminate subsequent executions of the amplification process. The polymerase chain reaction (PCR) procedure amplifies specific nucleic acid sequences through a series of manipulations including denaturation, annealing of primers, and extension of the primers with DNA polymerase (Mullis KB et al ., US 4,683,202, US 4,683,195; Mullis KB, EP 201,184; Erlich H, EP 50,424, EP 84,796, EP 258,017, EP 237,362; Erlich H, US 4,582,788; Saiki R et al ., US 4,683,202; Mullis KB et al . (1986) in Cold Spring Harbor Symp. Quant. Biol. 51 :263; Saiki R et al : (1985) Science 230 :1350; Saiki R et al . (1988) Science 231 .487; Loh EY et al . (1988) Science 243 :217). These steps can be repeated many times, potentially resulting in large amplifications of the number of copies of the original specific sequence. It has been shown that even single molecules of DNA can be amplified to produce hundreds of nanograms of product (Li H et al . (1988) Nature 335 :414). Other known nucleic acid amplification procedures include the transcription-based amplification system of Kwoh D et al . (1989) Proc.Natl.Acad.Sci.USA 86 :1173. "Uracil DNA glycosylase" (UDG), a term of art, refers to an enzyme which cleaves the glycosidic bond between the base uracil and the sugar deoxyribose, only when the monomeric nucleotide dUTP is incorporated into a DNA molecule, resulting in incorporation of a deoxyuridine moiety (Duncan B (1981) in The Enzymes 14 :565, ed.: Boyer P). The enzyme does not act upon free dUTP, free deoxyuridine, or RNA (Duncan, supra ). A consequence of amplification processes such as PCR is that the amplification products themselves can be substrates for subsequent PCR procedures. Furthermore, because the quantities of the amplification products can be large, the dispersal of even an extremely small fraction of a reaction such as a PCR reaction into the laboratory area potentially can lead to contamination of later attempts to amplify other samples. The present invention seeks to provide an improvement upon in vitro nucleic acid amplification procedures in general by making amplification products distinguishable from naturally occurring nucleic acid such as DNA. Accordingly, such products may be rendered inactive as templates for further amplification prior to the start of the succeeding amplification reaction. Thus in a first aspect of the present invention there is provided a process for amplifying nucleic acid, the process comprising: (a) hybridizing an oligonucleotide primer to the nucleic acid; (b) synthesising nucleic acid which incorporates deoxyuridine from the primer, that is substantially complementary to the nucleic acid; (c) separating the nucleic acid and the synthesised nucleic acid; (d) optionally when required, repeating (a) using the nucleic acid and the synthesised nucleic acid as templates for the primer; and (e) subjecting the synthesised nucleic acid to uracil DNA glycosylase treatment to render it unable to serve as a template for synthesising further nucleic acid. Suitably the original nucleic acid will remain unaffected, i.e. still able to act as a template in further amplification. The nucleic acid is preferably DNA. The present invention also encompasses a process for amplifying one or more nucleic acid sequences, the process comprising: (a) incorporating deoxyuridine into the nucleic acid of a first sample during amplification of the nucleic acid sequences; and (b) subjecting the nucleic acid of a second sample to uracil DNA glycosylase treatment which renders nucleic acid containing deoxyuridine substantially unamplifiable and which does not substantially affect amplification of nucleic acid that does not contain deoxyuridine; whereby amplified nucleic acid sequences derived from the first sample which contaminate the second sample are not further substantially amplified during amplification of the nucleic acid sequences of the second sample. The process may further comprise: (c) amplifying nucleic acid sequences of the second sample. Alternatively or in addition, the process may further comprise: (d) terminating the treatment of step (b), for example by heating. In this specification "amplifying" or "amplification" is advantageously a process for amplifying at least one specific nucleic acid sequence contained in a nucleic acid or a mixture of nucleic acids wherein each nucleic acid consists of two separate complementary strands, of equal or unequal length, which comprises: (1) treating the strands with two oligonucleotide primers, for each different specific sequence being amplified, under conditions such that for each different sequence being amplified an extension product of each primer is synthesized which is complementary to each nucleic acid strand, wherein the primers are selected so as to be sufficiently complementary to different strands of each specific sequence to hybridize therewith 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; (2) separating the primer extension products from the templates on which they were synthesized to produce single-stranded molecules; and (3) treating the single-stranded molecules generated from (2) with the primers of (1) under conditions that a primer extension product is synthesized using each of the single strands produced in (2) as a template. The present invention thus contemplates a process for amplifying one or more nucleic acid sequences, comprising the steps of: (A) with a first sample containing a nucleic acid sequence or a mixture of nucleic acid sequences, wherein each nucleic acid sequence has two separate complementary strands of equal or unequal length: (a) treating the strands with two oligonucleotide primers, for each different specific sequence being amplified, under conditions such that for each different sequence being amplified an extension product of each primer is synthesized which is complementary to each nucleic acid strand and which incorporates an exo-sample nucleotide, wherein the primers are selected so as to be sufficiently complementary to different strands of each specific sequence to hybridize therewith 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; (b) separating the primer extension products from the templates on which they were synthesized to produce single-stranded molecules; (c) treating the single-stranded molecules generated from (b) with the primers from (a) under conditions such that a primer extension product is synthesized using each of the single strands produced in (b) as a template; (d) repeating (a) through (c) at least once, thereby amplifying the specific nucleic acid sequence contained by the first sample; and (B) with a second sample containing a nucleic acid sequence or a mixture of nucleic acid sequences, wherein each nucleic acid sequence has two separate complementary strands of equal or unequal length and wherein amplified nucleic acid sequences from (A) may be present in the second sample: (e) subjecting the nucleic acid of the second sample to uracil DNA glycosylase treatment which renders nucleic acid containing deoxyuridine substantially unamplifiable and which does not affect amplification of nucleic acid that does not contain deoxyuridine; whereby any primer extension products of the first sample, amplified in (A) and present in the second sample, are not further substantially amplified in (B). The process may further comprise, during (B): (f) repeating (a) through (c) at least once, thereby amplifying any of the specific nucleic acid sequence contained by the second sample. The process may also further comprise, during (B): (g) terminating the uracil DNA glycosylase treatment. The termination of the uracil DNA glycosylase treatment is preferably accomplished by heating. The present invention involves a process for incorporating deoxyuridine into DNA or RNA during amplification procedures. The invention eliminates the products of previous amplification(s) from further amplification by means of uracil DNA glycosylase treatment that leaves (original) nucleic acid from the sample unaffected in its ability to be amplified. This treatment may greatly reduce a major problem associated with amplification of nucleic acids, namely contamination of starting materials with end products of previous amplification processes. In other words, the present invention provides a process of discriminating against amplification products, and in favour of nucleic acids normally found in nature, prior to the start of succeeding amplification reactions. More specifically, the invention encompasses in vitro procedures which utilize enzymes to amplify specific nucleic acid sequences. One example of such a procedure is known as the polymerase chain reaction (PCR). A serious limitation of the PCR procedure and other similar procedures is contamination of the laboratory environment with the amplified nucleic acid end products of individual reactions. Such contamination commonly results in amplification not only of authentic nucleic acid which may be present in the sample of interest, but also of the contaminating end products from previous reactions. The present invention may provide a process for removing possible contamination of this type, without affecting the desired amplification of authentic nucleic acids. The present invention may involve first performing amplification procedures in which one or more of the four normal ribonucleoside triphosphates (rNTPs) or deoxyribonucleoside triphosphates (dNTPs) is replaced with dUTP that is normally absent from or present very rarely in nucleic acids found in the samples whose amplification is desired. The RNA or DNA produced during such amplification processes can be differentiated from sample nucleic acids. Thus, one may be able to discriminate against nucleic acids produced during amplification processes in favour of sample DNA or RNA prior to or during succeeding amplification processes, such that previously amplified nucleic acid can no longer be amplified, while sample DNA or RNA remains amplifiable. It is believed that since the invention of the various nucleic acid amplification methods no one has disclosed a means for eliminating contamination of input nucleic acid by the products of previous cycles of amplification. The term "amplifying", as used herein, refers to any in vitro process for increasing the number of copies of a nucleic acid or nucleotide sequence or sequences. Nucleic acid amplification results in the incorporation of nucleotides into DNA or RNA. The terms "amplify" and "amplification" are to thus be construed accordingly. When a nucleic acid is referred to as being unamplifiable, this may be considered as the nucleic acid being incapable of allowing synthesis of substantially complementary nucleic acid, and thus incapable of acting as a template. "Nucleotide" is a term of art that refers to a base-sugar-phosphate combination. Nucleotides are the monomeric units of nucleic acid polymers, i.e. of DNA and RNA. The term includes ribonucleoside triphosphates, such as rATP, rCTP, rGTP, or rUTP, and deoxyribonucleoside triphosphates, such as dATP, dCTP, dGTP, or dTTP. "Nucleoside" is a term of art referring to a base-sugar combination, i.e. a nucleotide lacking a phosphate moiety. It is recognised in the art that there is a certain interchangeability in usage of the terms nucleoside and nucleotide. For example, the nucleoside deoxyuridine triphosphate, dUTP, is deoxyribonucleoside triphosphate. After incorporation into DNA, it serves as a DNA monomer, formally being deoxyuridylate, i.e. dUMP or deoxyuridine monophosphate. One may say that one incorporates dUTP into DNA even though there is no dUTP moiety in the resultant DNA. Similarly, one may say that one incorporated deoxyuridine into DNA even though that is only part of the substrate molecule. Deoxyuridine may be incorporated into the nucleic acid that is synthesised during amplification, such as by conducting the amplification process in a medium which contains dUTP. Although the triphosphate form of deoxyuridine, dUTP, is present in living organisms as a metabolic intermediate, it is rarely incorporated into DNA. When dUTP is accidentally incorporated into DNA, the resulting deoxyuridine is promptly removed in vivo by normal processes, e.g processes involving the enzyme UDG. Thus, deoxyuridine occurs rarely or never in natural DNA. It is recognised that some organisms may naturally incorporate deoxyuridine into DNA. For nucleic acid samples of those organisms, deoxyuridine would not be considered a suitable nucleotide. The presence of deoxyuridine may be determined readily using methods well known to the art. The term "incorporating" refers to becoming part of a nucleic acid polymer, or sequence. The term "terminating" refers herein to causing uracil DNA glycosylase treatment to stop. The term includes means for both permanent and conditional stoppages. Both permanent heat denaturation and lack of enzymatic activity due to a temperature outside the enzyme's active range would fall within the scope of this term. In the process according to this invention, an amplification procedure is performed on a first sample in which one or more of the four normal ribonucleoside triphosphates (rNTPs) or deoxyribonucleoside triphosphates (dNTPs) is replaced with dUTP. After amplification, any contaminating amplified product that may be remaining is subjected to uracil DNA glycosylase treatment which renders nucleic acid containing deoxyuridine substantially unamplifiable. The treatment may be done as a separate step, or preferably, may be done in the presence of a second sample containing nucleic acid sequences to be amplified. The amplified nucleic acid sequences derived from the first sample which contaminate the second sample are not further substantially amplified during amplification of nucleic acid sequences of the second sample. The deoxyribonucleoside triphosphate dUTP may be conveniently incorporated into an enzymatic DNA amplification procedure, exemplified herein by PCR, thereby resulting in deoxyuridine-containing DNA. The DNA products of such a reaction will normally contain many uracil bases. Discrimination between natural DNA and the resultant, deoxyuridine-containing products of amplification procedures may be obtained with the enzyme uracil DNA glycosylase (UDG). Treatment of DNA containing uracil bases with uracil DNA glycosylase results in cleavage of the glycosidic bond between the deoxyribose of the DNA sugar-phosphate backbone and the uracil base. The loss of the uracil creates an apyrimidinic site in the DNA, which blocks DNA polymerase from using the DNA strand as a template for the synthesis of a complementary DNA strand (Schaaper R et al . (1983) Proc. Natl. Acad. Sci. USA 80 :487). The presence of substantial numbers of apyrimidinic sites in each DNA target molecule interferes with amplification procedures which use DNA polymerase to synthesize copies of target DNA. As exemplified herein, the basic amplification protocol is the well known PCR method. PCR may be modified in three ways: (1) dUTP was substituted for dTTP; (2) UDG was added to the initial PCR reaction mixture; and (3) an initial incubation period was added to allow UDG to destroy contaminating products of prior PCR reactions. The UDG itself was either permanently inactivated by high temperature in the first PCR cycle or was not active at the high temperatures used with Taq polymerase in the currently preferred PCR protocol. This inactivation prevents UDG from destroying newly-synthesized PCR products. Nucleic acid amplification protocols that do not eliminate UDG activity usually will require an extra UDG-inactivation step. While termination of the uracil DNA glycosylase treatment that renders nucleic acid containing deoxyuridine resistant to the amplification process is preferred (as exemplified herein, heat inactivation of UDG), the invention also includes embodiments lacking a termination step. For example, one might use amounts of the enzyme and durations of treatment high enough to eliminate expected contamination of starting materials but insufficient to keep up with the rate of amplification. In other words, treatment might be able to destroy contaminating nucleic acid but an amplification process might still be able to produce new nucleic acid faster than the treatment could destroy the newly synthesized nucleic acid. A second aspect of the present invention relates to a process for preventing or reducing contamination of a second sample comprising nucleic acid by a first sample comprising nucleic acid, the process comprising; (a) incorporating deoxyuridine into a nucleic acid during amplification of nucleic acid to produce a first sample; (b) when the second sample is suspected of being contaminated by the first sample, subjecting the second sample to uracil DNA glycosylase treatment to render nucleic acid containing deoxyuridine substantially unamplifiable. Preferred features and characteristics of the first aspect are as for the second aspect mutatis mutandis . The invention will now be described by way of example which is intended to be merely illustrative of the present invention, and is not to be construed as being limiting. COMPARATIVE EXAMPLE A polymerase chain reaction (PCR) was performed to amplify a region of the human papilloma virus type 16 (HPV 16) DNA (Durst M et al . (1983) Proc. Natl. Acad. Sci. USA 80 :3812). The sequences of the primers used were 5'GGTCGATGTATGTCTTGTTG3' and 5'GTCTACGTGTGTGCTTTGTAC3'. HPV 16 DNA was excised from a full length plasmid clone, pT7HPV16 (for the purposes of this invention, equivalent to the pUC8 plasmids described by Seedoff K et al . (1985) Virol. 145 :181) with the restriction enzyme Bam H I. The linear DNA (10 picograms) was added to PCR reactions containing 50 microliters of 25 mM Tris HCl pH 8.3, 5 mM MgCl₂, 50 mM NaCl, 0.01% gelatin, 0.2 mM each dATP, dGTP, dCTP, 0.2 mM either dUTP or dTTP, 1 micromolar of each primer, and 12.5 units of thermostable DNA polymerase from Thermus aquaticus (Cetus/Perkin-Elmer). The reactions were amplified in a thermal cycler (Cetus/Perkin-Elmer) using the following temperature profile: 5 minutes at 94°C, then 30 cycles of 1 minute at 94°C (denaturation), two minutes at 55°C (annealing), and 3 minutes at 72°C (primer extension). After completion of the temperature cycles, a final extension of 10 minutes at 72°C was done. Amplification of the 284 base pair HPV 16 DNA fragment was confirmed by agarose/ethidium bromide gel electrophoresis (Maniatis T et al . (1982) Molecular Cloning , Cold Spring Harbor Laboratory) of the PCR reaction products (5 microliters of each reaction per lane). All reactions showed substantial amplification. Negative control reactions to which no HPV 16 DNA was added did not produce any DNA products. The concentration of the PCR amplification products was estimated from the agarose gel. New PCR reactions were contaminated with ten femtogram quantities of the amplification products that contained either deoxythymidine, resulting from incorporation of dTTP, or deoxyuridine, from dUTP-containing reactions. Positive control reactions contained 10 picograms of linear HPV 16 DNA. Negative control reactions did not receive any target DNA. The new PCR reactions contained dUTP instead of dTTP, and either 5 nanograms of UDG (Van de Sande J, University of Calgary; also available from Duncan Laboratories, 19 E. Central Ave., Paoli, PA 19301 USA) or no UDG. All reactions were incubated for 15 minutes at 37°C to allow the UDG to act on deoxyuridine-containing DNA, and then were taken through the same thermal cycling protocol as above. Aliquots of each reaction were analyzed by agarose/ethidium bromide gel electrophoresis. The agarose gel analysis showed that without UDG treatment the deoxyuridine-containing PCR products could be re-amplified to give a DNA product indistinguishable in size, as evidenced by gel electrophoresis, from the products obtained by amplifying the normal HPV 16 DNA. Reactions in which the deoxyuridine-containing DNA was incubated with UDG prior to PCR did not give any visible products on the agarose gel. PCR amplification products that contained deoxythymidine were successfully amplified whether or not they had been incubated with UDG. This experiment showed that UDG substantially abolished amplification of PCR products containing deoxyuridine, but had no substantial effect on the amplification of DNA containing deoxythymidine.

Claims: A process for amplifying nucleic acid, the process comprising: (a) hybridizing an oligonucleotide primer to the nucleic acid; (b) synthesising nucleic acid which incorporates deoxyuridine from the primer, that is substantially complementary to the nucleic acid; (c) separating the nucleic acid and the synthesised nucleic acid; (d) optionally when required, repeating (a) using the nucleic acid and the synthesised nucleic acid as templates for the primer; and (e) subjecting the synthesised nucleic acid to uracil DNA glycosylase treatment to render it unable to serve as a template for synthesising further nucleic acid. A process for preventing or reducing contamination of a second sample comprising nucleic acid by a first sample comprising nucleic acid amplified by a process as claimed in claim 1, the process comprising subjecting the second sample to uracil DNA glycosylase treatment to render nucleic acid containing deoxyuridine substantially unamplifiable, when the second sample is suspected of being contaminated by the first sample. A process for amplifying one or more nucleic acid sequences, the process comprising: (a) incorporating deoxyuridine into nucleic acid during amplification of a first sample of the nucleic acid sequence(s) amplified by a process as claimed in claim 1; and (b) subjecting nucleic acid of a second sample to uracil DNA glycosylase treatment which renders nucleic acid containing deoxyuridine substantially unamplifiable and which does not substantially affect amplification of nucleic acid that does not contain deoxyuridine; whereby amplified nucleic acid sequences derived from the first sample which contaminate the second sample are not further substantially amplified during amplification of the nucleic acid sequences of the second sample. A process as claimed in claim 3 further comprising: (c) amplifying nucleic acid in the second sample. A process as claimed in any one of claims 1 to 4 further comprising: (d) terminating the treatment in (b), such as by heating. A process as claimed in any one of claims 1 to 5 wherein the amplifying is a process for amplifying at least one specific nucleic acid sequence contained in a nucleic acid or a mixture of nucleic acids wherein each nucleic acid consists of two separate complementary strands, of equal or unequal length, which comprises: (e) treating the strands with two oligonucleotide primers, for each different specific sequence being amplified, under conditions such that for each different sequence being amplified an extension product of each primer is synthesized which is complementary to each nucleic acid strand, wherein the primers are selected so as to be sufficiently complementary to different strands of each specific sequence to hybridize therewith 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; (f) separating the primer extension products from the templates on which they were synthesized to produce single-stranded molecules; and (g) treating the single-stranded molecules generated in (f) with the primers from (e) under conditions that a primer extension product is synthesized using each of the single strands produced in (f) as a template. A process as claimed in claim 1, the process comprising: (A) with a first sample containing a nucleic acid sequence or a mixture of nucleic acid sequences, wherein each nucleic acid sequence has two separate complementary strands of equal length: (a) treating the strands with two oligonucleotide primers, for each different specific sequence being amplified, under conditions such that for each different sequence being amplified an extension product of each primer is synthesized which is complementary to each nucleic acid strand and which incorporated deoxyuridine, wherein the primers are selected so as to be sufficiently complementary to different strands of each specific sequence to hybridize therewith 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; (b) separating the primer extension products from the templates on which they were synthesized to produce single-stranded molecules; (c) treating the single-stranded molecules generated from (b) with the primers from (a) under conditions such that a primer extension product is synthesized using each of the single strands produced in (b) as a template; (d) repeating (a) through (c) at least once, thereby amplifying the specific nucleic acid sequence contained by the first sample; and (B) with a second sample containing a nucleic acid sequence or a mixture of nucleic acid sequences, wherein each nucleic acid sequence has two separate complementary strands of equal or unequal length and wherein amplified nucleic acid sequences from (A) may be present in the second sample: (e) subjecting the nucleic acid of the second sample to uracil DNA glycosylase treatment which renders nucleic acid containing deoxyuridine substantially unamplifiable and which does not affect amplification of nucleic acid that does not contain deoxyuridine; whereby any primer extension products of the first sample, amplified in (A) and present in the second sample, are not further substantially amplified in (B).

Inventors: HARTLEY JAMES L, HARTLEY, JAMES L.
Applicants: LIFE TECHNOLOGIES INC, LIFE TECHNOLOGIES INC.
Application year: 1990
Main classification: C12Q1
IPC codes: C12Q1, C07H21