Detection of amplicon contamination during PCR exhibiting two different annealing temperatures

Field of the Invention

The invention pertains to the field of molecular diagnostic methods, specifically to methods using the polymerase chain reaction. More specifically, the invention provides a method to detect the presence of amplicon contamination in a polymerase chain reaction.

Background of the Invention

The polymerase chain reaction (PCR) is a method to selectively amplify DNA. The method uses paired sets of oligonucleotides that hybridise to opposite strands of DNA and define the limits of the sequence that is amplified. The oligonucleotides prime multiple sequential rounds of DNA synthesis catalysed by a thermostable DNA polymerase. Each round of synthesis is normally preceded by a melting and re-annealing step. The method can rapidly amplify virtually any DNA sequence (Saiki et al., Science 239:487, 1988).

PCR is widely used for the genetic identification of unique sequences in individual organisms. Uses include: forensic analysis (Gill, (2002) Biotechniques 32, 366-372), diagnosis of genetic disorders and disease susceptibility, neoplastic disease (Raj, (1998) Cancer 82, 1419-1442), detection of infectious diseases (Daxboeck (2003) Clinical Microbiology and Infection 9, 263-273) and food testing (Malorne (2003) Intemational Journal of Food Microbiology 83, 39-48). The DNA of interest is typically amplified from genomic DNA, viral DNA, or from cDNA reverse transcribed from RNA.

In common with other assays, PCR is subject to both 'false negative' and 'false positive' results. False negative results are due to reaction failure. False positive results may be caused by primers annealing to sequences other than the true recognition sequence leading to amplification of spurious products, or by primers annealing to the true recognition sequence present in contaminating DNA derived from a source other than the sample being diagnosed. True recognition sequences contained in the sample being diagnosed will be termed "target" in the following. True recognition sequences contained in contaminating DNA are not classified as target DNA.

The most frequent and potent source of contaminating DNA that causes false positives is previously amplified PCR products (termed amplicons) with recognition sequences identical to those of the primers being used (Rolfs et al., (1992) PCR: clinical diagnostics and research. Springer-Verlag, Berlin). The probability of contamination increases when diagnostic PCR is carried out many times for one DNA sequence, and when the PCR technique has been designed to detect one, or a few, molecules of DNA. Contamination by previous PCR products is called 'carryover' to distinguish it from contamination by DNA from other sources.

Even strict adherence to good laboratory practice and protocols that aim to avoid any contact of amplicon with pre-amplification reagents or samples cannot guarantee the absence of false positives due to amplicon contamination. As a result, a significant part of the cost of diagnostic or forensic PCR assays is caused by the need to include a relatively large number of negative controls and to repeat arrays of assays if the slightest indication of a contamination is found.

Thus, a method to individually ascertain the absence of amplicon contamination in any reaction is highly desirable.

State of the Art

Two approaches can be envisioned to solve the general problem posed by amplicon contamination: either the amplicon can be destroyed subsequent to its detection in order to avoid it contaminating any later reaction, or methods must be found to discriminate amplification product arising from original sample target content as opposed to arising from amplicon contamination. The former idea has been implemented in the method of dUTP incorporation and subsequent destruction of the amplicon by uracil-N-glycosylase (see EP0401037 and references contained therein; Longo et al. (1990), Gene 93, 125-128), a method widely employed today. Other methods have been devised that follow similar rationales (Cimino et at. (1991), Nucleic Acids Research 19, 773-774; Walder et al. (1993), Nucleic Acids Research 21, 4339-434).

Richards (US5650302) discloses a method to incorporate restriction nuclease recognition sites into the primers in order to render amplicon contaminants unamplifiable when digested with the correspondent nuclease prior to amplification.

Destruction of the amplicon after determination of its presence or quantity will reduce the likelihood of downstream contamination, but since the amplicon destruction itself is a process subject to possible failure, it cannot positively rule out false positive results due to amplicon contamination.

Shuber (US 6,207,372) discloses a multiple duplex primer PCR method, where a universal primer sequence at the 5' end of various primer pairs allows for uniform amplification conditions for multiple targets.

Shuldiner (WO9115601) discloses a method by which RT-PCR-reactions, where the first step is elongation of a DNA primer on a RNA template, can be made more specific over a background of possible DNA contaminant sequences. This method employs two primers, one of which is a hybrid sequence comprising a target-RNA-specific sequence tract and a tagging-tract. Discrimination is achieved between target RNA and possible contaminating genomic or plasmid DNA on the basis of the different hybridization temperatures of DNA-DNA versus RNA-DNA duplexes. Since RNA-DNA-double strands are more stable and hence, have a higher melting or annealing temperature than DNA-DNA double strands, a reaction temperature can be selected at which the target-specific primer part will only anneal to a RNA target. Subsequent duplication of this first DNA transcript generated from the hybrid primer will result in a DNA strand that is elongated at its 3' end with the complementary tagging sequence, to which the hybrid primer will anneal in all subsequent amplification steps at the elevated temperature. This means that the method disclosed in WO9115601 cannot discriminate between, or exclude from being amplified, DNA amplicon produced in a previous reaction using the same primer set, although it may be a useful tool to exclude amplification from genomic DNA contaminations.

In parallel to any efforts to reduce the occurrence of contamination, it is desired to be able to discern amplification product arising from target sequences in the sample (the true positive result) and the false positive result arising from amplicon contamination.

Shuber (WO 9920798) has disclosed a method to detect contamination by amplicon sequences that relies on the use of two different oligonucleotide primer sets in two different amplification reactions. A first set of primers comprising a target-detection sequence and a contamination-detection-sequence, which is added to the 5' end of the primers, are employed to detect the presence of target sequence in the original sample, a reaction that is termed "first amplification reaction". In a second reaction mixture, a second set of primers comprising only the contamination detection sequence, are used on the original sample, in order to detect contamination of the sample by amplicon molecules produced in previous reactions.

The method disclosed in WO 9920798 achieves detection of amplicon contamination in the original sample, however it does not rule out positively the presence of amplicon contamination in any step that may be specific for the "first" (according to the terminology of WO 9920798) amplification reaction. As one example, the two reactions differ in the primers employed, and thus any amplicon contamination in the solution containing the oligonucleotide primers for the first reaction would not be detected.

Another aspect of the disclosure of WO 9920798 that may be improved upon is the use of two reaction vessels. Although tolerable in some instances, the doubling of the expense in time and material both for the reaction preparation and reagents may offset the advantage in savings from reducing false positives. A method that enables the discrimination of true and false positives without the expense of having to conduct two separate reactions would thus be highly desirable.

In view of this state of the art, it is the object of the present invention to provide a method and suitable otigonudeotide primers in order to enable amplification reactions that will discriminate between amplification signals arising from target sequences contained in the sample (true positives) and amplification signals arising from contamination by amplicon products produced in previous amplification reactions.

The problem outlined above is solved by the invention as defined in the independent claims.

According to the present invention, PCR reactions are performed using a forward primer and a reverse primer for amplification of the target sequence, both primers comprising a target-specific sequence element B on their 3'-end, and a non-target-specific amplicon-specific tag sequence A on their 5' end. The full primer sequence will be contained in and hybridize to all amplicon products resulting from the use of these primers in previous reactions, but only the target-specific 3' sequence tracts will be contained in and hybridize to original sample target sequences.

Due to the difference in length of hybridisable sequence tract, the primers will differ in annealing temperatures on amplicon contamination and true sample target, the latter annealing temperature being substantially lower. According to the present invention, the primer pair containing A and B sequence elements as defined above, is used on the sample, together with all other ingredients normally present in a PCR reaction, most prominently heat-stable polymerase, reaction buffer and deoxyribonucleotides.

The PCR reaction is then conducted for several cycles at an elevated temperature, at which the AB primers will only anneal to any eventual contamination, and for several cycles at a lower temperature, at which the B sequence of the primers can anneal to target sequences contained in the sample.

The inventive method is preferably performed in a real time PCR machine so that product can be detected by fluorescence throughout the reaction. For example, the first 30 cycles are performed at an annealing temperature of 72degC and the second set of cycles at <58degC.

If the PCR reaction mix is contaminated with previously tagged amplicon(s) a product is obtained after the first 30 cycles at an annealing temperature of 72degC.

In a preferred embodiment, methods of the invention comprise the utilisation of optimal primer construction for PCR and a single PCR amplification reaction performed first at a higher and then at a lower annealing temperature. Accordingly in a highly preferred embodiment the method uses two chimeric primers that have a 3' B region that has substantial sequence similarity and is complementary to target

DNA and a 5' A region(s), or tag(s), that is unrelated to and non-complementary to the target DNA. The primers are designed such that amplification of target DNA will occur at the lower annealing temperature but not at the higher annealing temperature, whereas the product generated at the lower annealing temperature will be amplified at the higher annealing temperature. PCR amplification is then performed in a single closed reaction vessel first at a higher and then at a lower annealing temperature. It is preferred that the higher and the lower annealing temperatures differ by at least 5 degrees Celsius.

Since amplification at the higher annealing temperature is performed before amplification at the lower annealing temperature, the presence of product after high temperature annealing amplification indicates the PCR was contaminated with previously amplified product (carryover contamination), and the results should be discarded. A lack of amplified product after high temperature annealing amplification indicates the PCR was not contaminated with previously amplified product. Presence of PCR product following the low temperature annealing but not high temperature annealing cycles indicates a positive result for the sample that is not due to carryover contamination.

The 5' A, or tag, region non-complementary to the target DNA may be any sequence (irrespective of length) that does not hybridise to a given target DNA sequence under stringent conditions (conditions that remove all primer DNA except that bound by specific complementary base pairing to a substantial length of DNA sequence).

A variant of the preferred embodiment uses one chimeric primer and one non-chimeric primer completely complementary to target DNA throughout its length. As before the chimeric primer is designed such that the melting temperature of a hybrid between the 3' B region and its complement on target DNA is lower than the melting temperature of a hybrid between the full length chimeric primer and its complement. The non-chimeric primer has a melting temperature between itself and its complement on target DNA that is higher (preferably by an amount of 5 deg Celsius or more) than the melting temperature of a hybrid between the 3' B region of the chimeric primer and its complement. Amplification with the primers for a number of cycles is undertaken first at a higher and then at a lower annealing temperature.

A further variant of the preferred embodiment uses two chimeric primers and two additional primers that are complementary to the 5'A region(s), or tag(s), of the chimeric primers. The chimeric primers comprise a 3'B region that has substantial sequence similarity and is complementary to target DNA and a 5'A region (or tag) that is unrelated to and non-complementary to the target DNA. The primers are designed such that the melting temperature of a hybrid between the 3' B region and its complement on target DNA is lower (preferably by an amount of 5 deg Celsius or more) than the melting temperature of a hybrid between the 5'A region(s) and its complementary sequence. Amplification with the four primers is undertaken first at a higher and then at a lower annealing temperature, the difference between the two annealing temperatures being greater than or equal to 5 degree Celsius. At the lower annealing temperature hybridisation between the target DNA specific 3' A regions of the primers and the target DNA generates amplified product. The product of this reaction incorporates the full-length chimeric primers (AB) including the non-complementary 5' A tag. At the higher annealing temperature, hybridisation of the 2 chimeric primers and the 2 primers complementary to the 5'A region(s) of the chimeric primers and consequent DNA amplification will only occur if previously formed product incorporating the chimeric primers is present. Since amplification at the higher annealing temperature is performed before amplification at the lower annealing temperature, the presence of product after the high temperature annealing amplification cycles indicates the PCR was contaminated with previously amplified product (carryover contamination).

A further variant of the preferred embodiment uses two chimeric primers and one additional primer complementary to the 5'A region of one of the chimeric primers.

The chimeric primers comprise a 3'B region that has substantial sequence similarity and is complementary to target DNA and a 5'A region (or tag) that is unrelated to and non-complementary to the target DNA. One chimeric primer is designed such that the melting temperature of a hybrid between the 3' B region and its complement on target DNA is greater than or equal to 5 degree C less than the melting temperature of a hybrid between the 5'A region(s) and its complementary sequence. The other chimeric primer is designed such that the melting temperature of a hybrid between the 3' B region and its complement on target DNA is greater than or equal to 5 degree Celsius less than the melting temperature of a hybrid between the full length chimeric primer and its complement. Amplification with the three primers is undertaken first at a higher and then at a lower annealing temperature, the difference between the two annealing temperatures being greater than or equal to 5 degree Celsius. The reaction and interpretation of product formation is analogous to the one outlined in the preceding alternative.

In each of the aforementioned embodiments the number of cycles at the different annealing temperatures may vary and will require optimisation for individual assays. For example a few amplification cycles at the higher annealing temperature followed by 25 cycles at the lower annealing temperature may permit discrimination between true positives and false positives due to carryover contamination.

If the maximum concentration of target DNA and minimum number of PCR cycles (e.g. 15) needed to observe it by real time PCR were known, amplification cycles at the higher temperature could be undertaken to a point at which, when lower temperature amplification began, if product is observed after fewer cycles (eg in this case 10 cycles) this could only be due to amplicon contamination.

Both primer annealing temperatures and the number of of high and low temperature cycles will require optimisation for individual assays. More than two different annealing temperatures may be used in a given reaction.

Methods of the invention comprise PCR primers that have a 5' A region, or tag, non-complementary to the target DNA that may be any sequence (irrespective of length) that does not hybridise to a given target DNA sequence under stringent conditions. The 3' B region of the primers must hybridise to the target DNA under stringent conditions but does not need to be an exact match to the target sequence. The 5'A non-complementary regions, of the chimeric forward and reverse primers may be identical or different.

In a preferred embodiment the amplification reaction is either PCR, reverse transcriptase PCR or qPCR (quantitative PCR) or chip based PCR. The sample containing nucleic acid to be amplified can be from any source containing biological material. The method may be particularly useful in the context of multiplex PCR, as appropriately designed tags may help normalise the amplification of multiple DNA targets (US6207372).

In another preferred embodiment, the method according to the invention is performed conducting the low temperature and the high temperature reaction simultaneously. Conventional PCR machines only allow one temperature being selected for a reaction vessel at any one time, so the reaction would have to be performed in two different vessels, which is one possible embodiment of the present invention. With the arrival of chip-based micro-fluidics technologies, it would be possible to design a reaction chamber that allows conducting two reactions from the same PCR reaction mix on the same sample at two different temperatures, simultaneously.

In conventional high throughput PCR the likelihood of detecting sporadic carryover contamination is depends on the proportion of negative controls included. In the method of the invention every sample acts as a negative control for carryover contamination. Therefore the present invention will enable monitoring of the frequency of carryover contamination and permit appropriate remedial action to taken to reduce such contamination. It will also enable facile identification of which component of the PCR is contaminated with previous amplicons.

The invention is compatible with other PCR technologies including, but not exclusively: nested PCR, multiplex PCR, hot start PCR, touchdown PCR, mimic templates, dUTP/UNG, molecular beacons/LUX fluorogenic primers, microfluidic chip based devices for PCR, and ELISA PCR. Furthermore, unlike other methods to control amplicon contamination the invention works effectively regardless of G+C content or amplicon length, and does not modify the DNA in such a way that it is incompatible with downstream applications such as cloning or sequencing.

Additional embodiments use primers that hybridise to the 5'A tag regions or include some sequence that matches the 3'B region and target DNA.

The primers used in the present invention could be chemically modified by means well known to those skilled in the art, including but not exclusively by (i) conjugation to fluorophores, biotin, enzymes etc., (ii) introduction of non-standard DNA bases e.g. a 3' terminal ribose residue or dUTP.

The present invention is suitable for use with any samples that may contain a particular target DNA molecule, in particular biomedical samples.

The methods of the invention are especially suitable for high throughput applications and diagnostic kits, including but not exclusively, for inherited diseases, infectious diseases, and clinical assays e,g for cancer detection.

Figure legends:

Figure 1

Schematic representation of a chimeric primer. The B region is complementary to target DNA whereas the B region is not complementary to target DNA.

Figure 2

Schematic representation of methodology using chimeric primers and outcome with and without carryover contamination.

In this hypothetical example, two chimeric primers are used (forward [FW] and reverse [REV]). Each has a unique 5' sequence (dashed line, A) that is unrelated to the target template sequence to be amplified and a 3' sequence (solid line, B) that matches the target template sequence to be amplified. PCR tags the product (amplicon) with unique 5' sequences. The annealing temperature of the FW and REV primers for the tagged amplicon is more than 72degC. The annealing temperature of the FW and REV primers for the target template is less than 58degC.

Example 1

An oligonucleotide corresponding to a 70bp region of Ebola virus was synthesised and used as target DNA. The sequence of the oligonucleotide corresponded to base pairs 721-790 of the Entsiami strain of the Zaire ebolavirus spike glycoprotein gene (GI accession number AY526102):<img file="EP1598429A1_D0001.tif" /> The forward (F) and reverse (R) primers used for amplification were 5'-GTA CGG ATC CAG ACA GAG TAT TTG TTCG-3' (SEQ ID NO 2) and GTT CGC TAC CAA CTG TGG TGT GAA TCT TG-3' (SEQ ID NO 3) respectively. The first ten bases are non-complementary to the Ebola target DNA.

Stocks of 1ng/ml target DNA and 0.5µg/ml F and R primers were made up in ddH₂O. PCR product was generated in a 100µl reaction comprising 1µl target DNA stock, 3µl 10 mM dNTPs, 2µl of each of the F and R primers, 10µl 10X REDTaq PCR reaction buffer, 3µl REDTaq DNA polymerase and 79µl ddH₂O. A Hybaid TouchDown PCR thermocycler was used. Cycling parameters were 1X 94°C 4 min, 30X 94°C 1 min, 48.5°C 45 s, 72°C 1 min, and 1X 72°C 5 min. The PCR product was purified using a Qiagen kit, visualised as a single discrete band on a 2% agarose gel, and quantitated by spectrophotometry.

A stock of 1ng/ml PCR product was made up in ddH₂O.

PCR reaction mixtures comprised 3µl 10 mM dNTPs, 10µl 10X REDTaq PCR reaction buffer, 3µl REDTaq DNA polymerase and the following: <ul id="ul0001" list-style="none" compact="compact"><li>1) 1µl target DNA stock, 2µl of F and R primer stock, 81µl ddH₂O</li><li>2) 1µl target DNA stock, 1µl PCR product stock, 2µl of F primer stock, 83µl ddH₂O</li><li>3) 1µl target DNA stock, 1µl PCR product stock, 2µl of R primer stock, 83µl ddH₂O</li><li>4) 1µl target DNA stock, 1µl PCR product stock, 2µl of F and R primer stock, 80µl ddH₂O</li></ul>

The four reactions were amplified using cycling parameters of 1X 94°C 4 min, 30X 94°C 1 min, 72°C 45 s, 72°C, 1 min, and 1X 72°C 5 min. A 5µl aliquot was removed from each reaction and thermocycling of the same reaction continued at 30X 94°C 1 min, 48,5°C 45 s, 72°C, 1 min, and 1X 72°C 5 min. At the end of the reaction a second 5µl aliquot was taken and both aliquots from each reaction were visualised on a 2% agarose gel. <tables id="tabl0001" num="0001"><table frame="all"><tgroup cols="5" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="31.50mm" /><colspec colnum="2" colname="col2" colwidth="31.50mm" /><colspec colnum="3" colname="col3" colwidth="31.50mm" /><colspec colnum="4" colname="col4" colwidth="31.50mm" /><colspec colnum="5" colname="col5" colwidth="31.50mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="left"><b>Annealing temperature</b></entry><entry namest="col3" nameend="col3" align="left"><b>Contamination</b></entry><entry namest="col4" nameend="col4" align="left"><b>Primers</b></entry><entry namest="col5" nameend="col5" align="left"><b>PCR outcome</b></entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" /></row><row><entry namest="col1" nameend="col1" align="left">1</entry><entry namest="col2" nameend="col2" align="left">72°C</entry><entry namest="col3" nameend="col3" align="left">Amplicon</entry><entry namest="col4" nameend="col4" align="left">F and R</entry><entry namest="col5" nameend="col5" align="left">Product</entry></row><row><entry namest="col1" nameend="col1" align="left">2</entry><entry namest="col2" nameend="col2" align="left">72°C</entry><entry namest="col3" nameend="col3" align="left">Amplicon</entry><entry namest="col4" nameend="col4" align="left">F only</entry><entry namest="col5" nameend="col5" align="left">No product</entry></row><row><entry namest="col1" nameend="col1" align="left">3</entry><entry namest="col2" nameend="col2" align="left">72°C</entry><entry namest="col3" nameend="col3" align="left">Amplicon</entry><entry namest="col4" nameend="col4" align="left">R only</entry><entry namest="col5" nameend="col5" align="left">No product</entry></row><row><entry namest="col1" nameend="col1" align="left">4</entry><entry namest="col2" nameend="col2" align="left">72°C</entry><entry namest="col3" nameend="col3" align="left">No contamination</entry><entry namest="col4" nameend="col4" align="left">F and R</entry><entry namest="col5" nameend="col5" align="left">No product</entry></row><row><entry namest="col1" nameend="col1" /></row><row><entry namest="col1" nameend="col1" align="left">1</entry><entry namest="col2" nameend="col2" align="left">72°C then 48.5°C</entry><entry namest="col3" nameend="col3" align="left">Amplicon</entry><entry namest="col4" nameend="col4" align="left">F and R</entry><entry namest="col5" nameend="col5" align="left">Product</entry></row><row><entry namest="col1" nameend="col1" align="left">2 72°C</entry><entry namest="col2" nameend="col2" align="left">then 48.5°C</entry><entry namest="col3" nameend="col3" align="left">Amplicon</entry><entry namest="col4" nameend="col4" align="left">F only</entry><entry namest="col5" nameend="col5" align="left">No product</entry></row><row><entry namest="col1" nameend="col1" align="left">3</entry><entry namest="col2" nameend="col2" align="left">72°C then 48.5°C</entry><entry namest="col3" nameend="col3" align="left">Amplicon</entry><entry namest="col4" nameend="col4" align="left">R only</entry><entry namest="col5" nameend="col5" align="left">No product</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">4</entry><entry namest="col2" nameend="col2" align="left">72°C then 48.5°C</entry><entry namest="col3" nameend="col3" align="left">No contamination</entry><entry namest="col4" nameend="col4" align="left">F and R</entry><entry namest="col5" nameend="col5" align="left">Product</entry></row></tbody></tgroup></table></tables><img file="EP1598429A1_D0002.tif" /><img file="EP1598429A1_D0003.tif" /><img file="EP1598429A1_D0004.tif" />