WO1995006753A1

WO1995006753A1 - Method and compositions for primer specific solid-phase detection of pcr products

Info

Publication number: WO1995006753A1
Application number: PCT/US1994/009869
Authority: WO
Inventors: Howard A. Fields; Yury E. Khudyakov
Original assignee: The United States of America, represented by The Secretary, Department of Health & Human Services
Priority date: 1993-09-03
Filing date: 1994-09-02
Publication date: 1995-03-09
Also published as: AU7718594A

Abstract

The invention provides a method of detecting the presence of a nucleic acid in a sample comprising the following steps: a) amplifying a nucleic acid from the sample using a first primer having a known sequence which can be selectively digested when in double stranded form and a second primer having a marker molecule such that a double stranded nucleic acid amplification product is formed having the known nucleotide sequence on one strand and the marker molecule on the other strand; b) digesting the known nucleotide sequence from one strand of the double stranded amplification product to expose a single stranded sequence complementary to the known nucleotide sequence on the strand having the marker molecule; c) hybridizing the single stranded sequence to a complementary nucleic acid bound to a solid support to capture the amplification product; d) removing uncaptured amplification product; and e) detecting the presence of the marker molecule captured on the solid support, the presence of marker molecule indicating the presence of the nucleic acid in the sample. Also provided is a kit comprising a pair of polymerase chain reaction primers, the first primer having a poly U tail on the 5' end and the second primer having a marker molecule linked to the 5' end. The kit can further include uracil DNA glycosylase. The kit can have biotin as the marker molecule and include streptavidin-horseradish peroxidase and a substrate for the horseradish peroxidase to detect the marker.

Description

METHOD AND COMPOSITIONS FOR PRIMER SPECIFIC SOLID-PHASE DETECTION OF PCR PRODUCTS

BACKGROUND OF THE INVENTION

The polymerase chain reaction (PCR) is a recently developed method for specific amplification of DNA fragments. The simplicity and high efficiency of the reaction makes it not only a very powerful research method, but also a very reliable and sensitive diagnostic tool for the detection of nucleic acids of different pathogens. The PCR has been utilized many times in the diagnosis of numerous diseases. However, this reaction, although efficient and simple, has not found a substantial niche in thediagnostic laboratories around the world.

One major reason of this delayed acceptance of the PCR in practical diagnostics is inefficient methods for the detection of the PCR products. The most common way of detection is agarose gel electrophoresis. This method requires relatively large amounts of the amplified DNA. To obtain this large amount of DNA the PCR is usually carried out through many cycles of amplification, which makes the reaction very sensitive to cross-contamination of treated specimens, or increases non-specific products. These non-specific products can lead to misinterpretation of the results. In addition, gel electrophoresis detection of PCR products is not amenable to the needs of routine diagnostic laboratories, which are unlikely to have appropriate equipment.

PCR results are generally interpreted by visual analysis of a band stained with ethidium bromide, which is a subjective method requiring highly qualified staff. As a result, many attempts to design a colorimetric non- isotopic method for the detection of PCR products analogous to immunological reactions for enzyme immunoassay (EIA) have been attempted. Colorimetric reactions are much more sensitive, can be measured by simple photometers, and can be quantitative allowing more reliable and more objective interpretation of the results. The main problem with colorimetric approaches for detection is the unavailability of a specific method to capture the PCR products. There are three different currently available ways to capture PCR products: (1) hybridization with a probe attached to a solid-phase (microtiter well) , (2) antibodies specific to RNA-DNA hybrids, which can be prepared to specifically capture hybrids formed between amplified DNA and specific RNA probes, and (3) specific labeling of the PCR products (usually biotinylation) by using special labeled primers, or nucleotides.

Only hybridization with a probe provides sequence specific capture of the PCR fragments. However, the main disadvantage of hybridization is low efficiency of the process because of high dependence on DNA denaturation conditions. At annealing temperatures or at neutralization conditions after alkali denaturation, DNA forms a double-stranded structure. If the double-stranded DNA is denatured it can hybridize with an oligonucleotide probe and the product can be captured and detected; however, if the DNA is not denatured it cannot be captured, because there is no way for the probe to hybridize with the DNA at annealing conditions. Thus, the usual hybridization techniques are inefficient, since three different competing reactions occur simultaneously when standard annealing conditions are used: (1) probe binding, (2) restoration of the double-stranded form of the PCR fragments, and (3) non-specific burial of the interacting region of the amplified DNA product inside of the macrostructure organized in the DNA.

The present invention provides a method for the capture and detection of PCR products utilizing a primer specific hybridization capture of the desired DNA, combined with colorimetric detection ar-d quantitation, which overcomes the above problems.

SUMMARY OF THE INVENTION

The invention provides a method of detecting the presence of a nucleic acid in a sample comprising the following steps: a) amplifying a nucleic acid from the sample using a first primer having a known sequence which can be selectively digested when in double stranded form and a second primer having a marker molecule such that a double stranded nucleic acid amplification product is formed having the known nucleotide sequence on one strand and the marker molecule on the other strand; b) digesting the known nucleotide sequence from one strand of the double stranded amplification product to expose a complementary single stranded sequence on the strand having the marker molecule; c) hybridizing the single stranded sequence to a complementary nucleic acid bound to a solid support to capture the amplification product; d) removing uncapturec. amplification product; and e) detecting the presence of the marker molecule captured on the solid support, the presence of marker molecule indicating the presence of the nucleic acid in the sample.

The invention also provides a kit comprising a pair of polymerase chain reaction primers, the first primer having a poly U tail on the 5' end and the second primer having a marker molecule linked to the 5' end. The kit can further include uracil DNA glycosylase. The kit can have otin as the marker molecule and include streptavidin-horseradish peroxidase and a substrate for the horseradish peroxidase to detect the marker. DETAILED DESCRIPTION OF THE INVENTION

The invention provides a method of detecting the presence of a nucleic acid in a sample comprising the following steps: a) amplifying a nucleic acid from the sample using a first primer having a known sequence which can be selectively digested when in double stranded form and a second primer having a marker molecule such that a double stranded nucleic acid amplification product is formed having the known nucleotide sequence on one strand and the marker molecule on the other strand; b) digesting the known nucleotide sequence from one strand of the double stranded amplification product to expose a complementary single stranded sequence on the strand having the marker molecule; c) hybridizing the single stranded sequence to a complementary nucleic acid bound to a solid support to capture the amplification product; d) removing uncaptured amplification product; and e) detecting the presence of the marker molecule captured on the solid support, the presence of marker molecule indicating the presence of the nucleic acid in the sample. The method is also referred to herein as primer specific solid-phase detection or PS-SPD.

Amplification methods can include PCR as provided in the Examples, or other methods. For example, such methods include amplification by the ligase chain reaction. (Barany, F. "Genetic diseases detection and DNA amplification using cloned thermostable ligase". PNAS 88:189-193, 1991) Any chain amplification method based on utilization of primers for DNA polymerase activity will be applicable for this method of detection. For example, NASBA™ (nucleic acid sequence-based amplification) , SDA, strand displacement amplification and the QBeta replicase system are other alternative amplification methods

(Kievits et al. "NASBA™ isothermal enzymatic in vitro nucleic acid amplification optimized for the diagnosis of HIV-1 infection", J. Viroloσical Methods 35:273-286, 1991; Walker et al. "Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system" PNAS 89:392- 396, 1992; and Lizardi et al. Bio. Technology 6:1197-1202, 1988) . The sequence of the primers used will be selected based on the nucleic acid to be detected (i.e., amplified) and will be modified according to the present invention.

In some instances the known nucleotide sequence used in the method described above is on the 5' end of the first primer and the marker molecule is linked to the 5' end of the second primer. For example, in the above method, the known nucleotide sequence on the 5' end of the first primer can be a poly U tail and the digesting step is accomplished with uracil DNA-glycosylase (UDG) . The poly U tail should be linked to the 5' end of the primer because otherwise the additional sequence can disturb primer extension during the amplification process. Digestion with UDG is accomplished according to the known methods and as further described herein.

In some embodiments of the above method, the known nucleotide sequence is positioned other than on the 5' end of the first primer and the marker molecule is positioned other than on the 5' end of the second primer. For example the known nucleotide sequence could be on the 3' end of the first primer and the marker molecule is linked to the 3' end of the second primer. The known nucleotide sequence should not be added to the 3' -end of primers if it is not complementary to the amplified sequence, because it may interfere with the amplification process.

The known nucleotide sequence on the first primer can be a oligoribonucleotide sequence in which case the digesting step would be accomplished with an RNase. The oligoribonucleotide sequence may be introduced into any site of a primer: 3' -end, center, or 5'-end. Alternatively, the primer may be completely composed of ribonucleotides if desired. In this case, the ribonucleotides should be complementary to the DNA being primed. The primary concern in the design and placement of the oligoribonucleotide is that the secondary structure of the single-stranded region that results from removal of the known sequence from the double-stranded PCR product should avoid strong secondary structure because such structure could interfere with capture of amplified nucleic acids by the complementary probe attached to a solid-phase. Probes attached to a solid-phase can be obtained commercially as described in the Examples or readily obtained by standard methods. For example, biotinylated albumin can be adsorbed to the solid phase. Then streptavidin-biotin attached to the nucleotide capture probe can be added and the streptavidin will bind to biotin on albumin.

In the method using polyribonucleotides, one primer is chemically synthesized to contain a stretch of ribonucleotides incorporated at any position. The length of this region should be enough for strong complementary capture with a probe on a solid-phase (usually more than 7 nt) . After the completion of an amplification reaction, the DNA fragment is treated with RNAse (e.g., RNAse A, Pharmacia Biotech Inc., Piscataway, NJ) in the amplification reaction buffer for 1-10 min. Because RNAse A is highly active, the RNA portion of the amplification product can be efficiently removed. After amplification, the digestion, capture and detection reactions can be accomplished directly in the same microtiter well. The steps for capture and detection are as described for the use of UDG.

The method described above can also be accomplished using a primer having any known deoxyribonucleotide sequence that can be digested with a 5'→3' DNA exonuclease. In case of application of 5' -exonuclease, as with the RNAse method there is no need to add any additional moiety to the primers used for the amplification step. In this embodiment the primer sequence itself will be modified, for example by inserting a stop signal capable of preventing digestion of the entire amplified sequence by the exonuclease. More specifically, the primer should contain at the distance of at least 4 or more (preferably about 10) nucleotides from the 5' -end a modification in internucleotide bonds such as thio-groups, etc. that significantly inhibits the exonuclease reaction, but does not prevent the DNA polymerase reaction. A primary consideration is that the modification of the primer stops exonuclease digestion. In this regard, a stop signal can also be positioned at the 5' end of the second primer to prevent digestion. Thus, as with the method using the poly(dU) tail, a PCR product will result that is a double-stranded DNA with 3'- protrusion of sufficient length for the capture by a probe attached to a solid-phase and with a marker molecule that can be detected to indicate the presence of the amplified product.

The complementary sequence bound to the solid support can be poly(dT) in the case where poly(dU) is the known sequence. Although the best results will be obtained if the sequence of the capture probe is complementary to the exposed single stranded sequence, it will be understood that some insubstantial number of mismatched bases can be present and the method still be effective. Thus, the term "complementary" is used herein to describe both exactly matching nucleotide sequences, as well as those having some mismatched nucleotides that do not prevent selective hybridization. The term "selective hybridization" is used herein to describe hybridization that permits the selective capture and identification of the target DNA in a sample over background. Marker molecules may be inserted at any position in the primer or in the amplified fragment between primers during amplification process. Methods of attaching marker molecules to nucleotide sequences are well known in the art, and kits for performing the attachment are available. For introduction of biotin, there are kits available from companies providing reagents and protocols (for example, Clonotech, USA). See also Leary et al. "Rapid and sensitive colorimetric method for visualizing biotin- labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose:bio-blots" PNAS 80:4045-4049, 1983. The position of the marker molecule in the primer is not important except to the extent that its position should not interfere with the amplification process. The effect of the marker molecule on amplification using the primer can be readily determined by testing it in an amplification process.

The method of the invention can utilize a number of marker molecules. For example, where the marker molecule is biotin, detecting the marker molecule can be by the following steps: a) binding streptavidin-horseradish peroxidase to the biotin on the captured amplification product; b) removing unbound streptavidin-horseradish peroxidase; c) contacting the captured DNA with a sufficient amount of a substrate to react with the horseradish peroxidase; and d) detecting the reaction of the substrate and horseradish peroxidase (HRP) , the reaction indicating the presence of the nucleic acid in the sample. The substrate can be diaminobenzidine-nickel chloride-hydrogen peroxide, among the other known and readily available substrates.

Another example of a marker molecule is digoxigenin (DIG) . This marker can be added to the primer according to the methods provided herein (Kessler, C. "The digoxigenin: anti-digoxigenin (DIG) technology - a survey on the concept and realization of a novel bioanalytical indicator system" Mol. Cell. Probes 5:161-205, 1991). For the detection, the captured product is contacted with an antibody to DIG conjugated with HRP or other enzyme (e.g., alkaline phosphatase, etc.). An appropriate substrate for the enzyme is then added to detect the captured product using the standard protocols known for the enzyme, such as that described herein for HRP. The enzyme-substrate reaction can be de σted and quantified colorimetrically as described in the Examples.

The P£ SPD reaction is a very flexible process and can be easily modified to make it more sensitive and specific. For example, both PCR primers can be provided with different U-rich sequences at the 5' -ends. After removal of dU's from the termini of the PCR fragments by UDG, two single-stranded protrusions will be exposed for binding. One protrusion may be used for the capture of the fragment to a solid-phase. The other protrusion may be bound by another oligonucleotide covalently linked to a reporter enzyme (e.g., horseradish peroxidase, alkali phosphatase, etc.) directly, or to a protein (for example, GSA, synthetic peptides, etc.). In the case of direct conjugation of HRP with a oligonucleotide, the HRP will be attached only to the PCR fragment and will not interact with primers, a situation that occurs with primers labeled with biotin. This modification will prevent competition between primers an. PCR fragments for HRP resulting in a more sensitive assay. When an oligonucleotide is attached to another protein, rather than to HRP, HRP may then be conjugated to antibody specific for the protein. This modification represents one of the many possible sensitive approaches for the detection of PCR fragments because an additional amplification step is involved in the reaction, namely, interaction of many antibody molecules to a protein. A key to the advantages of the present invention is primer specific capture of the PCR fragments to a solid support. Basically, this process in the PS-SPD is a hybridization step carried out under unique conditions such that only the probe and a region of the PCR fragment responsible for the interaction with the probe are exposed in single-stranded form. This allows both sequences to be hybridized without competition with any other nucleotide sequences. When performing the PS-SPD, the PCR fragment maintains double-stranded form, and only the region to be bound to the probe is exposed in single-stranded form resulting in process of hybridization that is very specific and efficient compared with other hybridization capture methods. Thus, the opportunity for the probe to capture PCR fragments is much higher in the PS-SPD compared with usual hybridization techniques.

The invention also provides a kit comprising a pair of polymerase chain reaction primers, the first primer having a poly U tail on the 5' end and the second primer having a marker molecule linked to the 5' end. The kit can further include uracil DNA glycosylase. The kit can have biotin as the marker molecule and include streptavidin-horseradish peroxidase and a substrate for the horseradish peroxidase.

Also provided is a kit comprising a pair of polymerase chain reaction primers, the first primer having a known polyribonucleotide sequence on the 5' end and the second primer having a marker molecule linked to the 5' end. As described above the oligoribonucleotide sequence can be positioned elsewhere in the primer and should be of sufficient length to permit capture of the product after digestion with the ribonuclease. The above kit can further comprise a ribonuclease. The kit can have biotin as the marker molecule and include streptavidin- horseradish peroxidase and a substrate for the horseradish peroxidase.

The simplicity, specificity, and sensitivity of the PS-SPD make this method a highly reliable diagnostic tool for the detection of nucleic acids of different pathogens. This method needs no additional equipment and the test can be conducted in any immunodiagnostic laboratory.

The following examples are intended to illustrate, but not limit, the invention. While they are typical of those that might be used, other procedures known to those skilled in the art may be alternatively employed.

EXAMPLES

Detection of the PCR Fragment Specific for the Hepatitis C Virus (HCV) Genome

Oligodeoxynucleotide Synthesis.

Deoxyoligonucleotides were synthesized using an automatic synthesizer (Applied Biosystem) and purified by electrophoresis in 10% PAGE containing 7M urea with TBE buffer (0.045M Tris-borate, 0.001 EDTA, ph8.3). Oligonucleotides were recovered from the gel by electroelution. This process can be used to obtain both the primers with the poly U tails and the poly T for capture probes.

Polymerase Chain Reaction.

For PCR, nested primers were designed as follows: (1) external primers

YK-104 5' -GGTGCACGGTCTACGAGACCT YK-105 5'-CTGTGAGGAACTACTGTCTTC; and (2) internal primers

YK-103U 5' -UUUUUUUUUUUUUCAGAAAGCGTCTAGCCATGGCGTT YK-106B 5' -BIOTIN-CCCTATCAGGCAGTACCACAA

One primer has a short oligo(dU) sequence at the 5' - terminus, which is not complementary to the sequence of the target DNA. During the polymerase reaction, however, this stretch of dU is used to synthesize an oligo (dA)- sequence. The second PCR primer is labeled with biotin and is used for the detection of the attached PCR fragments.

The PCR reaction was conducted using nested primers with internal primers as described above. Briefly, PCR was carried out in a volume 100 μl with 1 μM of each primer, 200 μM of each dNTP, 2U Taq polymerase, in 10 mM Tris-HCl, pH 8.5, 50 mM KC1, 1.5 mM MgCl₂, 0.01% gelatin. All components were mixed in a reaction cocktail, followed by 30 cycles of PCR as follows: 94°C for 45 sec, 65^βC for 20 sec, and 72^βC for 1 min.

Selective digestion of PCR product.

Aliquots of the amplification reaction mixture, usually 4 μl, were treated with 1 unit of UDG (Gibco BRL, Great Island, NY) and 1 μl of HRP-SA (Gibco BRL, Great Island, NY) in lxUDG buffer containing 30 mM tris-HCl, pHδ.O, 50 mM KC1, 5 mM magnesium chloride for 10 min at 37 C. Uracil DNA-glycosylase is a very stable and efficient enzyme. Usually, 5 μl of the PCR mixture was treated with 1 unit of UDG, however, titration of the enzyme shows that there is no real difference in PS-SPD efficiency when the mixture was treated for 10 min with 1 unit or 0.001 unit of UDG. The reaction was stopped by washing the plates with binding buffer. Capture and detection of the HCV specific PCR fragment.

Aliquots of 1-5 μl of the reaction mixture treated with UDG were then transferred into microtiter wells covered with oligo(dT)20 (Cambridge Biotech, Bethesda, MD) and incubated in 100 μl of the binding buffer (10 mM tris- HCl, pH7.5, 0.5 m sodium chloride, 0.5% sodium dodecyl sulphate) for 10-30 min at room temperature. The oligo(dT) was attached to the wells, using known methods, by Biotech Research Laboratories (Rockville, Maryland) . The wells were thoroughly washed 5 times with binding buffer. Bindinc 3f the PCR fragment to the microtiter well is very efficient and the bound complex of DNA-HRP-SA can be detected after only 1-5 min incubation with binding buffer. The binding reaction is more efficient at 20^βC than at 37^βC.

PCR products captured to the surface of the wells were detected by the addition of an appropriate substrate for HRP, O-phenylenediamine (OPD) and hydrogen peroxide 5- 10 min. The reaction was stopped with acid and read at an optical density (OD) value of 439 nm (AutoReader II, ORTHO Diagnostic System, Raritan, NJ) .

PCR mixtures prepared from a normal human serum, or obtained using unmodified PCR primers were utilized as a negative control. Optical density values of the negative controls were between 0.005-0.03. Positive samples were very reactive with one microliter of the PCR mixture yielding OD values greater than 1.5. Simultaneously, different aliquots of the PCR mixture were analyzed with agarose gel electrophoresis. Direct comparison demonstrated that PS-SPD is approximately 100-times more sensitive than gel electrophoresis. As little as 0.001 μl of the PCR mixture could be detected with PS-SPD.

Because of the high efficiency and sensitivity of the PS-SPD, it is feasible to use fewer cycles of amplification for the PCR. Usually, to detect PCR fragments by gel electrophoresis, the reaction was carried through 30 cycles of amplification with internal primers. It was found that the PCR product could be detected by PS- SPD after only 10 cycles of amplification, while no product could be detected by gel electrophoresis. Moreover, the product of the amplification reaction after 30 cycles in the presence of both external and internal primers in the same mixture was undetectable by gel electrophoresis Jμt readily detectably by PS-SPD.

Combined digestion, capture and detection.

In addition, to simplify the detection of the PCR fragments, the PS-SPD was performed without separating the digestion step of treatment with UDG and the detection step of treatment with HRP-SA from the step of binding of the PCR fragment to the solid phase. In this case, 1 μl of the PCR mixture was transferred directly into microtiter wells filled with either 100 μl of the binding buffer or IxUDG buffer with 1% bovine serum albumin, both containing UDG and HRP-SA. After incubation for 10 min at room temperature, the wells were washed with binding buffer and the product captured onto the solid-phase was detected by the addition of substrate.

Reactions performed in the UDG-buffer gave a strong response with positive/negative ratios (P/N) greater than 50 (background OD values approximately 0.08). However, reactions performed in binding buffer gave P/N values less than 2.5. Thus, digestion and binding can be performed in the same microtiter well in only 15-20 min.

The amplification step can also be performed in the same vessel as the subsequent steps. To this end, the reaction can be carried out in a microtiter plate or other vessel designed for performance of PCR that can be coated with the capture probe.

Claims

What is claimed is:

1. A method of detecting the presence of a nucleic acid in a sample comprising: a) amplifying a nucleic acid from the sample using a first primer having a known sequence which can be selectively digested when in double stranded form and a second primer having a marker molecule such that a double stranded nucleic acid amplification product is formed having the known nucleotide sequence on one strand and the marker molecule on the other strand; b) digesting the known nucleotide sequence from one strand of the double stranded amplification product to expose a complementary single stranded sequence on the strand having the marker molecule; c) hybridizing the single stranded sequence to a complementary nucleic acid bound to a solid support to capture the amplification product; d) removing uncaptured amplification product; and e) detecting the presence of the marker molecule captured on the solid support, the presence of marker molecule indicating the presence of the nucleic acid in the sample.

2. The method of claim 1, wherein the known nucleotide sequence is on the end of the first primer and is an oligoribonucleotide sequence and the digesting step is accomplished with RNAse.

3. The method of claim 1, wherein the known nucleotide sequence is on the 3' end of the first primer and the marker molecule is linked to the 3' end of the second primer.

4. The method of claim 1, wherein the known nucleotide sequence is on the 5' end of the first primer and the marker molecule is linked to the 5' end of the second primer.

5. The method of claim 4, wherein the known nucleotide sequence on the 5' end of the first primer is a poly(dU) sequence and the digesting step is accomplished with uracil DNA-glycosylase.

6. The method of claim 4, wherein the known nucleotide sequence on the end of the first primer is a deoxyribonucleotide sequence that can be digested with a 5'→3' DNA exonuclease.

7. The method of claim 1, wherein the marker molecule is biotin and the detecting step comprises: a) binding streptavidin-horseradish peroxidase to the biotin on the captured amplification product; b) removing unbound streptavidin-horseradish peroxidase; c) contacting the captured DNA with a sufficient amount of a substrate for the horseradish peroxidase to react with the horseradish peroxidase; and d) detecting the reaction of the substrate and horseradish peroxidase, the reaction indicating the presence of the nucleic acid in the sample.

8. The method of claim 1, wherein the amplification is by the polymerase chain reaction.

9. The method of claim 1, wherein the amplification is by the ligase chain reaction.

10. A kit comprising a pair of polymerase chain reaction primers, the first primer having a poly(dU) sequence on the 5' end and the second primer having a marker molecule linked to the 5' end.

11. The kit of claim 10, further comprising uracil DNA- glycosylase.

12. The kit of claim 10, wherein the marker molecule is biotin and further comprising streptavidin-horseradish peroxidase and a substrate for the horseradish peroxidase,

13. A kit comprising a pair of polymerase chain reaction primers, the first primer having a known polyribonucleotide sequence on the 5' end and the second primer having a marker molecule linked to the 5' end.

14. The kit of claim 13, further comprising a ribonuclease.

15. The kit of claim 13, wherein the marker molecule is biotin and further comprising streptavidin-horseradish peroxidase and a substrate for the horseradish peroxidase.