WO2002018616A1

WO2002018616A1 - Adjusting the efficiency of nucleic acid template amplification by attenuated pcr with template-mimic oligonucleotides

Info

Publication number: WO2002018616A1
Application number: PCT/US2001/027287
Authority: WO
Inventors: Song-Hua Ke
Original assignee: Hitachi Chemical Co., Ltd.; Hitachi Chemical Research Center, Inc.
Priority date: 2000-09-01
Filing date: 2001-08-30
Publication date: 2002-03-07

Abstract

The invention relates to modified template-mimic oligonucleotides (TMOs) used to adjust the amplification efficiency of an abundant target without affecting the amplification of other targets in a DNA synthesis reaction. The invention may be used in PCR or any other primer dependent DNA amplification technology.

Description

ADJUSTING THE EFFICIENCY OF NUCLEIC ACID TEMPLATE AMPLIFICATION BY ATTENUATED PCR WITH TEMPLATE-MIMIC

OLIGONUCLEOTIDES

Field of the Invention

The invention relates to nucleic acid template amplification, and particularly to a method for adjusting the amplification efficiency of an abundant target without affecting the amplification of other targets in a DNA synthesis reaction. The invention may be used in PCR or any other primer dependent DNA amplification technology.

Background of the Invention

Multiplex RT-PCR is a powerful tool for multiple gene expression analysis, both qualitatively and quantitatively. Endogenous gene transcripts such as β-actin, glyceraldehyde-3 -phosphate dehydrogenase (GAPDH), or 18S rRNA are usually used as internal controls for quantification of gene expression to prevent variations in the final detection from tube to tube, or from experiment to experiment (BioTechniques, 2000,

29:332-337). However, the high abundance of these transcripts prevents their use as a control in regular PCR for co-amplification with low abundance gene transcripts. Several methods have been reported to address the problem. United States Patent No. 6,057,134 to Ambion Inc. has introduced a "Competimer" technology. In this technology, 3' end modified primers, named 'competimer', that are modified at the 3' end to prevent DNA synthesis, are introduced in PCR reactions to compete with unblocked primers for hybridization to a target sequence and subsequently to modulate the amplification efficiency of a target sequence. However, the competimer design is complicated and the inhibition efficiency of competimers is not high enough to accommodate large differences between any two genes.

Summary of the Invention

The present invention provides a new attenuated PCR method for adjusting the efficiency of target template nucleic acid amplification by controlling the ratio of templatelike oligos (template-mimic oligos, TMOs) to amplification primers. Unlike the competimers having sequences complementary to target templates, the TMOs have sequences complementary to amplification primers. In hybridization steps of PCR, the TMOs bind to amplification primers and prevent them from binding to templates for polymerization synthesis. In an embodiment, the amplification efficiency of target template can be easily adjusted by controlling the ratio of template-mimic oligos to amplification primers with a 100% inhibition capacity.

In an embodiment, the present invention provides a method of modulating amplification efficiency of a target sequence in primer-dependent polyrnerase-mediated DNA synthesis, comprising: (a) adding a template-mimic oligonucleotide to a primer- dependent polyrnerase-mediated DNA synthesis reaction mixture including primers, in an amount effective to block a primer effective to amplify a target sequence in the mixture from hybridizing to the target sequence, said template-mimic oligonucleotide having a sequence complimentary to said primer, said template-mimic oligonucleotide being inactive on primer-dependent polyrnerase-mediated DNA synthesis; and (b) conducing primer-dependent polyrnerase-mediated DNA synthesis reaction.

In the above, the template-mimic oligonucleotide may have a 3' terminal modified to prevent its extension by DNA polymerase. The 3' terminal modification includes chemically modification; the addition of a dideoxynucleotide, phosphate, biotin, digoxygenin, fluorescein, an amine, a thiol, an azo (N₃) group, or fluorine; and modification by phosphorylation or biotinylation.

In an embodiment, the template-mimic oligonucleotide is an RNA, or a DNA RNA chimera. Further, if the template-mimic oligonulceotide is exclusively complementary to the primer, amplification of other sequences, if any, in the primer-dependent polyrnerase- mediated DNA synthesis is not affected. In an embodiment, a template of the target sequence may be a β-actin, 18S, 28S, or 5S ribosomal, or glyceraldehyde 3-P phosphate dehydrogenase template.

The primer-dependent polyrnerase-mediated DNA synthesis reaction includes an end-point RT-PCR reaction, an end-point PCR reaction, a real-time RT-PCR reaction, and a real-time PCR reaction, for example. In another embodiment, the present invention provides a method of multiplex thermal cycling, primer-dependent, polyrnerase-mediated DNA synthesis, comprising: (a) providing a primer-dependent polyrnerase-mediated DNA synthesis reaction mixture including at least one primer pair effective to amplify sequences including a target sequence; (b) placing at least one template-mimic oligonucleotide in said reaction mixture in an amount effective to modulate amplification efficiency of the target sequence by a primer, each template-mimic oligonucleotide having a sequence complimentary to said primer, said template-mimic oligonucleotide being inactive on primer-dependent polyrnerase-mediated DNA synthesis; and (c) conducing multiplex thermal, primer- dependent, polyrnerase-mediated DNA synthesis reaction.

In still another embodiment, the present invention provides a method of attenuated PCR, comprising: (a) providing a PCR mixture; (b) placing at least one template-mimic oligonucleotide in said mixture in an amount effective to modulate amplification efficiency of a target sequence by a primer, each template-mimic oligonucleotide having a sequence complimentary to said primer, said template-mimic oligonucleotide being inactive on PCR synthesis; and (c) conducing PCR.

In yet another embodiment, the present invention provides a method of attenuated PCR, comprising: (a) providing a PCR mixture including a standard sequence, primers to amplify the standard sequence, at least one target sequence, and primers to amplify the target sequence; (b) placing a template-mimic oligonucleotide having a sequence complimentary to said primer for the standard sequence in said mixture in an amount effective to modulate amplification efficiency of the standard sequence, said template- mimic oligonucleotide having a non-extendable 3' terminal; and (c) conducing PCR while maintain amplification of said standard sequence in a linear range concurrently with linear amplification of said target sequence.

The present invention can be applied to an attenuated PCR kit comprising (a) an amplification primer and (b) a template-mimic oligonucleotide having a sequence complimentary to said primer, said template-mimic oligonucleotide having a non- extendable 3' terminal. For purposes of summarizing the invention and the advantages achieved over the prior art, certain objects and advantages of the invention have been described above. Of course, it is to be understood that not necessarily all such objects or advantages may be achieved in accordance with any particular embodiment of the invention. Thus, for example, those skilled in the art will recognize that the invention may be embodied or carried out in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objects or advantages as may be taught or suggested herein.

Further aspects, features and advantages of this invention will become apparent from the detailed description of the preferred embodiments which follow.

Brief Description of the Drawings

These and other features of this invention will now be described with reference to the drawings of preferred embodiments which are intended to illustrate and not to limit the invention.

Figures la and lb show schematic outlines of attenuated PCR and standard PCR, respectively. In attenuated PCR, both amplification primers and template-mimic oligos (TMOs) are used in the reaction. During hybridization step, TMOs compete with templates for binding of amplification oligos. Only the templates hybridized with amplification primers will be amplified.

Figure 2 shows electrophoresis bands of β-actin and Caspase 9, indicating the effect of β-actin TMO on template amplification in an embodiment. The concentrations of the template-mimic oligo are as follows: Lane 1 - 2000 nM, Lane 2 - 1600 nM, lane 3 - 1200 nM, lane 4 - 800 nM, Lane 5 - 600 nM, lane 6 - 400 nM, Lane 7 - 300 nM, Lane 8 - 200 nM, Lane 9 - 150 nM, Lane 10 - 100 nM, Lane 11 and 12 - 0 nM. Total 30 PCR cycles; β- Actin template - 50 pg/μl; Caspase 9 template — 5 pg/μl; amplification primers for each template - 200 nM. Figure 3 shows the relationship between band intensity (unit) and concentration of

TMO, indicating the effect of β-actin TMO on template amplification in an embodiment. (•), β-actin amplification: amplification primer - 200 nM; template - 50 pg/μl. (°), Caspase 9 amplification: amplification primer - 200 nM; template - 5 pg/μl. 30 cycles PCR; 30 μl reaction. Figure 4 shows the relationship between percentage of inhibition (%) and TMO concentration, indicating inhibition efficiency of TMO in an embodiment. Beta-Actin PCR with 200 nM primer. Template - 50 pg/μl; 30 cycles PCR; 30 μl reaction.

Figure 5 shows electrophoresis bands of β-actin, indicating the effect of β-actin Competimer on template amplification in an embodiment. The concentrations of the β-actin

Competimer are as follows: Lane 1 - 1600 nM, lane 2 and 9 - 1200 nM, lane 3 and 10 - 800 nM, lane 4 and 11 - 400 nM, lane 5 and 12 - 200 nM, lane 6 and 13 - 100 nM, lane 7 and 14 - 0 nM. Lanes 1-7: β-Actin PCR with 200 nM amplification primers; lanes 9-14: β- actin PCR with 61 nM amplification primers. Template - 50 pg/μl; 30 cycles PCR; 20 μl reaction.

Figure 6 shows the relationship between band intensity (unit) and Competimer concentration, indicating the effect of β-actin Competimer on β-actin PCR amplification in an embodiment. (•), β-actin PCR with 200 nM amplification primers, (o), β-actin PCR with 61 nM amplification primers. Template - 50 pg/μl; 30 cycles PCR; 20 μl reaction. Figure 7 shows the relationship between percentage of inhibition and Competimer concentration (nM), indicating inhibition efficiency of Competimer in β-actin PCR amplification. (•), β-actin PCR with 200 nM amplification primers, (o), β-actin PCR with 61 nM amplification primers. Template - 50 pg/μl; 30 cycles PCR; 20 μl reaction.

Figure 8 shows electrophoresis bands of p53 and β-actin, indicating dynamic range of attenuated PCR. Lanes 1-7: 600 nM β-actin template-mimic oligo; lanes 8 - 14: no β- actin template-mimic oligo; M - 100 bp DNA marker. Lane 1 and 8: 1 ng/μl of β-actin template and 0.01 ng/μl of p53 template; lane 2 and 9: 0.1 ng/μl of β-actin template and 1 pg/μl of p53 template; lane 3 and 10: 0.01 ng/μl of β-actin template and 0.1 pg/μl of p53 template; lane 4 and 11: 1 pg/μl of β-actin template and 0.01 pg/μl of p53 template; lane 5 and 12: 0.1 pg/μl of β-actin template and 1 fg/μl of p53 template; lane 6 and 13: 0.01 pg/μl of β-actin template and 0.1 fg/μl of p53 template; lane 7 and 14: 1 fg/μl of β-actin template and 0.01 fg/μl of p53 template. Amplification primers - 200 nM, 40 PCR cycles, 30 μl reaction.

Figure 9 shows the relationship between DNA band intensity (unit) and log (picogram/μl) of initial template in each amplification, indicating a dynamic range of attenuated PCR amplification of β-actin and p53. (•), β-actin amplification with 600 nM β- actin TMO; (o), β-actin amplification without β-actin TMO; (T), p53 amplification with 600 nM β-actin TMO; (Δ), p53 amplification without β-actin TMO.

Figures 10a and 10b show the relationship between PCR base line subtracted RFU and cycles, indicating effectiveness of attenuated real-time PCR (Figure 10b), where 200 nM of each β-actin TMO was used in each reaction, compared to standard real-time PCR (Figure 10a). (o) -0.2 ng/μl of β-actin template; (•), 0.02 ng/μl of β-actin template; (■), 2 pg/μl of β-actin template; (□), 0.2 pg/μl of β-actin template; (0), 0.02 pg/μl of β-actin template. Figure 11 shows the relationship between threshold cycle (Ct) and log (template amount, ng), indicating standard curves for β-actin cDNA from regular real-time PCR (•) and attenuated real-time PCR (o) reactions. These curves are generated from the data of figure 10 by plotting the C_t values against the logarithm of the initial template concentration (ng/μl). The C_t value is inversely proportional to the log of the initial initial template concentration. For the same amount of template, the C_t values are increased from the attenuated real-time PCR comparing with the values from the regular real-time PCR. The results suggest that TMO can attenuate the real-time PCR amplification efficiency.

Figure 12 shows electrophoresis bands of Casepase9 and β-actin, indicating RT- PCR detection of Caspase 9 gene. Lane 1-3: 75,000 Hs578T cells/well; Lane 4-5: 300,000 U937 cells/well. Amplification primers - 200 nM; β-actin TMO - 1.1 μM; 30 cycles PCR.

Detailed Description of the Preferred Embodiment

The new attenuated PCR method for adjusting the efficiency of target template nucleic acid amplification by controlling the ratio of template-mimic oligos (TMOs) to amplification primers will be described. The TMOs have sequences complementary to amplification primers. In hybridization steps of PCR, the TMOs bind to amplification primers and prevent them from binding to templates for polymerization synthesis.

The invention includes applying TMOs to extend the dynamic linear range over several logs, perferably over 4 to 5 logs, of the initial input for a target template allowing easier quantification for both single and multiplex PCR. The invention also includes applying TMOs to real-time PCR quantification. The present invention also provides evidence that the existence of the TMOs in a multiplex PCR only affects the efficiency of the target sequence amplification without changing the amplification efficiency of other templates co-existing in the reaction. The TMO-assisted attenuated PCR technology provides a convenient and efficient method for simultaneous detection of high and low- abundant genes in multiplex RT-PCR. The utility of this new strategy is demonstrated in quantitative detection of target genes such as β-actin, p53 and caspase-9. Current method has also been compared with the Ambion's Competimer Technology. The method of the preferred embodiment is more potent and easier to control than the "Competimer" method. The present invention may include the following steps: selecting internal control genes; designing amplification primers; designing TMOs which have sequences complementary to the amplification primers, the 3' end of each sequence being non- extendable; selecting target genes and amplification primers therefor; and conducting attenuated PCR, wherein the TMOs are used at a concentration effective to coordinate the efficiency of target gene amplification and the efficiency of internal control gene amplification.

Amplification primers can be designed using various types of primer-designing software including technology disclosed in U.S. patent application No. 07/974,409 filed November 12, 1992, entitled "Method and Reagent for Measuring Messenger RNA" which is incorporated herein by reference. TMOs of the present invention have sequences complementary to amplification primers. The length of each TMO may be 5 bases shorter to 50 bases longer than the complementary amplification primer (preferably 0-10 bases longer, more preferably 1-5 bases longer). The minimum length of a TMO may be a hexamer. Preferably, the extra base sequence of the TMO is different from the corresponding template gene sequence. Further, the 3' end of each TMO may not be extendable, e.g., modified to block extension by DNA polymerase (adding a blocker or adding random sequences or any different sequences from the template).

The weight ratio of TMOs to amplification primers may be 0.1 to 15, preferably 1 to 7 (as measured when the TMO has a sequence for optimum hybridization to the primer), depending on the required level of inhibition of amplification efficiency. Schematic outline of attenuated PCR

Figures la and lb describe the method developed here to adjust the amplification efficiency of a target without affecting other templates amplification efficiencies. In the attenuated PCR reaction, two kinds of oligonucleotides are applied to a nucleic acid template. One is a pair of amplification oligonucleotides or amplification primers, whose function is to serve as primers during hybridization and enzymatic synthesis process to amplify templates with DNA polymerases. The two amplification oligonucleotides or primers are the regular oligonucleotides used in a standard PCR reaction. The other kind is a pair of special oligos, which are not used in a standard PCR reaction. They are called template-mimic oligos (TMOs). Their function is to provide binding sites for the amplification oligos in the hybridization steps, but the hybridization does not provide enzymatic synthesis sites.

The two TMOs have sequences complementary to two amplification oligos separately. The 5' end base of each TMO is complementary to the 3' end base of the corresponding amplification oligo. The 3' end of each TMO is about 5 bases (or more or less bases) longer or shorter than the corresponding amplification primer. The optimal template-mimic oligos are 1 to 5 bases longer than the amplification primers at their 3' end. The content of the extra base sequence in the TMO is different from the original template sequence. Ideally, the 3 ' end of the TMOs has been modified by any means in a way that blocks the TMO from extension by DNA polymerase, such as 3 ' addition of phosphate, biotin, a dideoxynucleotide, or a 3 '-3' linked 5' support blocker (which is commercially available from Operon Technologies or Glen Research). The TMO can be a DNA, it also can be an RNA, an RNA/DNA chimera, or a PNA. The TMO may be used not only in a PCR reaction, but also in other primer-dependent nucleic acid synthetic reactions, such as Self-Sustained Sequence Amplification (3SR) (PNAS, 1990, 87:1874-1878), or Nucleic

Acid Sequence-Based Amplification (NASBA) (Nature, 1991, 350:91-92).

Effects of TMOs and Ambion's "Competimer" on DNA Amplification.

Figures 2, 3 and 4 show the inhibition effect of β-actin TMO on β-actin DNA amplification. In this reaction, caspase 9 was co-amplified with β-actin. The top band at 580 bp position is caspase 9 PCR product in figure 2. When β-actin TMO concentration increases from 0 to 2000 nM, the intensity of caspase 9 DNA bands remains approximately constant, except at the low TMO concentration (less than 200 nM) where the caspase 9 products are lower than products from reactions at the high concentration of TMO (because of the reagents limitation caused by the consumption of β-actin template amplification). These clearly suggest that the inhibition effect of β-actin TMO is not because of general inhibition of Taq DNA polymerase, but because of the template-specific amplification inhibition.

Figures 5, 6 and 7 show the inhibition effect of β-actin "Competimer" on β-actin DNA amplification. The Competimer can inhibit the β-actin template amplification as demonstrated in figure 5 and 6. But the maximum inhibition efficiency is less than 60% as shown in figure 7. At 800 nM of Competimer, the inhibition efficiency reaches the plateau stage. Contrary to the phenomena, TMO can have inhibition efficiency up to 100% as demonstrated in Figure 4. The two methods are compared and summarized in table 1. Table 1. Comparison of TMO Method with "Competimer" Technology

Notes: *The potency of Ambion's "Competimers" as modulators is affected not only by their concentration, but also by their length, and the structure of the 3' modified group, since the competimers have to compete with the unblocked amplification primers for hybridization to target sequences in a template. In the TMO method, these two factors have less effect on the potency of TMOs as modulators.

** IC₅₀ is defined as the inhibition concentration of TMO at which 50% of maximum amplification efficiency is inhibited. Dose-Dependent Target Specific Amplification of Attenuated PCR with TMOs

Figures 8 and 9 show PCR amplification with serially 10-fold diluted β-actin and p53 plasmid template DNAs. In each reaction, β-actin plasmid template concentrations are 100-fold greater than p53 plasmid templates. In the experiment, 600 nM of β-actin TMOs and 200 nM amplification primers for each template are employed. For the control experiment, only 200 nM amplification primers are used. Figure 9 suggests that the final products of attenuated PCR amplification depend on the initial template concentration. The linear range of initial template concentration for attenuated PCR is about 4 to 5 logs of the concentration of input DNAs, while a regular PCR has 2.5 to 3 logs of input DNAs. For p53 target amplification, it is essentially the same for both PCR reactions. Attenuated Real-time PCR.

Figures 10a, 10b, and 11 show that for the same amount of template, the C_t values are increased from the attenuated real-time PCR comparing with the values from the regular real-time PCR. The results suggest that TMO can attenuate the real-time PCR amplification efficiency. Attenuated RT-PCR.

The results presented in figure 12 show the competitive interference from β-actin amplification by regular RT-PCR reduce the amplification of caspase-9 gene. TMO application in the multiplex attenuated RT-PCR can prevent the competitive interference from high-copy number genes such as β-actin gene in figure 12. EXAMPLE Taq DNA polymerase and dNTP were purchased from Promega. β-Actin cDNA, Caspase-9 cDNA and p53 cDNA plasmids were constructed by ligation of their cDNA fragments with pSP64 poly(A) vector (from Promega) or pCR2.1 (from Invitrogen). DNA oligonucleotides were from Operon Inc. (Alameda, CA). Oligos used for the experiments presented in figures 2 through 9 are shown in Table 2.

Table 2. Oligo Sequences, Function and Their PCR Product Sizes

Notes: * 3' teπriinator is a 5 '-support blocker which is an unmodified deoxynucleoside

(dA, dC, dG or T) that is attached to the rest of the oligo through a 3 '-3' linkage. PCR amplification and detection

PCR was carried out in a thermal cycler with a hot-lid from MJ. Research (PTC- 200). In a regular PCR, the PCR conditions were similar to the protocol recommended by Promega. 30 μl reaction mixtures contained 0.03 fg -30 ng of plasmid DNAs, 61 nM or 200 nM of amplification primers, and 200 μM of each dNTP in a buffer of 10 mM Tris-HCl

(pH 9.0 at 25°C), 50 mM KC1, 2.5 mM MgCl and 0.1% Triton X-100. The temperature cycles were 94°C for 50 sec (except for a 2 minute first cycle), 50°C for 1 min and 72°C for 1 minute with a final extension step for 6 min at 72°C. For attenuated PCR, the experimental condition was the same as regular PCR except 50 nM -2000 nM TMOs were added into the reaction. 6 μl of each reaction was examined for size and purity on a 1.5% agarose or 4-20 % gradient PAGE (Novex, San Diego, CA). The band intensity of each reaction was quantified with a densitometer (Molecular Dynamics) and plotted with Sigmaplot software.

Real-time PCR. TaqMan® Universal PCR Master Mix was purchased from Applied Biosystems. β-

Actin cDNA plasmids were constructed by ligation of β-actin cDNA fragments with pSP64 poly (A) vector (From Promega) in the lab. The plasmids were used as templates in realtime PCR. Pure dye calibration solution for iCycler iQ real-time PCR detection system was from Bio-Rad. DNA oligonucleotides and probes were from Operon Inc. (Alameda, CA). The oligonucleotide used as TaqMan probe was purified with HPLC.

The sequence of the TaqMan probe is:

5' FAM-CTGGACTTCGAGCAAGAGATGGCCAC-BHQ1 3'. (SEQ ID NO: 13)

The forward primer sequence is: 5' TGCGTGACATTAAGGAGAAG 3'. (SEQ ID NO: 14) The reverse primer is: 5' GCTCGTAGCTCTTCTCCA 3'. (SEQ ID NO: 15)

The TMO sequence for the forward primer is:

5' CTTCTCCTTAATGTCACGCATTGA 3'-dC-5'. (SEQ ID NO: 16)

The TMO sequence for the reverse primer is:

5' TGGAGAAGAGCTACGAGCCATA 3'-dC-5'. (SEQ ID NO: 17) The [dC-5'] in each TMO represents a reversed base connecting to each oligo 3' end through a 3 '-3' linkage.

PCR was carried out with a real-time PCR detection system named iCycler from Bio-Rad. 25 μl reaction mixtures containing 0.5 pg - 5.0 ng of template DNAs, 5 pmoles of each amplification primers, and 6 pmoles of the TaqMan probe were applied to the wells of a 96-well thin-wall PCR plate. For TMO-assisted attenuated PCR, 5 pmoles of each TMO were applied with other components for a regular PCR. The plate was covered with a piece of optical-quality sealing tape. PCR programs were 7 min at 95 °C, followed by 40 cycles of 30 sec at 95°C and 45 sec at 60°C. The fluorescent detection was set at 60°C step. The PCR reaction data was analyzed with the software provided by Bio-Rad and SigmaPlot and presented in figures 10A, 10B and 11.

End-point RT-PCR for samples from cultured cellsmRNA Express Kit for the purification of mRNA was from RNAture, Inc. Irvine CA. U937 human lymphoma cells and Hs578T human breast cancer cells were cultured for the mRNA isolation. 3x10^s U937 cells or 7.5x10⁴ Hs578T cells were applied to each well in a 96-well GenePlate from the kit. The procedure for mRNA isolation recommended by RNAture was followed. The RT- reaction for cDNA production was conducted at 42 °C for 50 min in 20 μl reactions with 100 units of M-MLV Reverse Transcriptase (from Promega). After washing the wells with 10 mM Tris buffer (pH 7.5), PCR was carried out in a thermal cycler with a hot-lid from M.J. Research (PTC-200). In a regular PCR, the PCR conditions were similar to the protocol recommended by Promega. The temperature cycles were 94°C for 40 sec (except for a 2 minute first cycle), 55°C for 50 sec. and 72°C for 50 sec. with a final extension step for 6 min at 72°C. For attenuated PCR, the experimental conditions were the same as regular PCR except 50 nM -2000 nM template-mimic oligos (TMO) were added into the reaction. 6 μl of each reaction was examined for size and purity on a 1.5% agarose or 4-20

% gradient PAGE (Invitrogen, San Diego, CA) (Figure 12).

It will be understood by those of skill in the art that numerous and various modifications can be made without departing from the spirit of the present invention. Therefore, it should be clearly understood that the forms of the present invention are illustrative only and are not intended to limit the scope of the present invention.

Claims

WHAT IS CLAIMED IS:

1. A method of modulating amplification efficiency of a target sequence in primer-dependent polyrnerase-mediated DNA synthesis, comprising: adding a template-mimic oligonucleotide to a primer-dependent polymerase- mediated DNA synthesis reaction mixture including primers, in an amount effective to block a primer effective to amplify a target sequence in the mixture from hybridizing to the target sequence, said template-mimic oligonucleotide having a sequence complimentary to said primer, said template-mimic oligonucleotide being inactive on primer-dependent polyrnerase-mediated DNA synthesis; and conducing primer-dependent polyrnerase-mediated DNA synthesis reaction.

2. The method according to Claim 1, wherein said template-mimic oligonucleotide has a 3' terminal modified to prevent its extension by DNA polymerase.

3. The method according to Claim 2, wherein said 3' terminal is chemically modified.

4. The method according to Claim 1, wherein said template-mimic oligonucleotide has a 3' terminal modified by 3' addition of a dideoxynucleotide, phosphate, biotin, digoxygenin, fluorescein, an amine, a thiol, an azo (N₃) group, or fluorine.

5. The method according to Claim 1, wherein said template-mimic oligonucleotide has a 3' terminal modified by phosphorylation.

6. The method according to Claim 1, wherein said template-mimic oligonucleotide has a 3' terminal modified by biotinylation.

7. The method according to Claim 1, wherein said template-mimic oligonucleotide is an RNA, or a DNA/RNA chimera.

8. The method according to Claim 1, wherein said sequence of said template-mimic oligonulceotide is exclusively complementary to said primer, whereby amplification of other sequences, if any, in the primer-dependent polyrnerase-mediated DNA synthesis is not affected.

9. The method according to Claim 1, wherein said template-mimic oligonucleotide is longer in length than the primer.

10. The method according to Claim 1, wherein said primer-dependent polyrnerase-mediated DNA synthesis reaction is an end-point RT-PCR reaction, an end- point PCR reaction, a real-time RT-PCR reaction, or a real-time PCR reaction.

11. The method according to Claim 1, wherein a template of said target sequence is a β-actin, 18S, 28S, or 5S ribosomal, or glyceraldehyde 3-P phosphate dehydrogenase template.

12. The method according to Claim 1, wherein said mixture further comprises other primers effective to amplify other sequences, and said target sequence is a sequence of an internal standard whose amplification is used as an index of amplification of other sequences amplified by the other primers.

13. The method according to Claim 12, wherein the ratio of said primer to said template-mimic oligonucleotide is controlled to maintain amplification of said internal standard in a linear range concurrently with linear amplification of said other sequences.

14. The method according to Claim 12, wherein said target sequence is an endogenous RNA standard, and said primer-dependent polyrnerase-mediated DNA synthesis reaction is a Semi-Quantitative RT-PCR.

15. The method according to Claim 1, wherein the ratio of said primer to said template-mimic oligonucleotide is in the range of 1/15 to 10/1 by weight.

16. The method according to Claim 1, wherein the ratio of said primer to said template-mimic oligonucleotide is in the range of 1/7 to 1/1 by weight.

17. A method of multiplex thermal cycling, primer-dependent, polymerase- mediated DNA synthesis, comprising: providing a primer-dependent polyrnerase-mediated DNA synthesis reaction mixture including at least one primer pair effective to amplify sequences including a target sequence; placing at least one template-mimic oligonucleotide in said reaction mixture in an amount effective to modulate amplification efficiency of the target sequence by a primer, each template-mimic oligonucleotide having a sequence complimentary to said primer, said template-mimic oligonucleotide being inactive on primer-dependent polyrnerase-mediated DNA synthesis; and conducing multiplex thermal, primer-dependent, polyrnerase-mediated DNA synthesis reaction.

18. A method of attenuated PCR, comprising: providing a PCR mixture; placing at least one template-mimic oligonucleotide in said mixture in an amount effective to modulate amplification efficiency of a target sequence by a primer, each template-mimic oligonucleotide having a sequence complimentary to said primer, said template-mimic oligonucleotide being inactive on PCR synthesis; and conducing PCR.

19. The method according to Claim 18, wherein said PCR is an end-point

RT-PCR reaction, an end-point PCR reaction, a real-time RT-PCR reaction, or a realtime PCR reaction.

20. The method according to Claim 18, wherein a template of said target sequence is a β-actin, 18S, 28S, or 5S ribosomal, or glyceraldehyde 3-P phosphate dehydrogenase template.

21. The method according to Claim 18, wherein said mixture includes sequences other than the target sequence and primers to amplify the other sequences, wherein said target sequence is a sequence of an internal standard whose amplification is used as an index of amplification of the other sequences amplified by the other primers.

22. A method of attenuated PCR, comprising: providing a PCR mixture including a standard sequence, primers to amplify the standard sequence, at least one target sequence, and primers to amplify the target sequence; placing a template-mimic oligonucleotide having a sequence complimentary to said primer for the standard sequence in said mixture in an amount effective to modulate amplification efficiency of the standard sequence, said template-mimic oligonucleotide having a non-extendable 3' terminal; and conducing PCR while maintain amplification of said standard sequence in a linear range concurrently with linear amplification of said target sequence.

23. The method according to Claim 21, wherein said PCR is an end-point RT-PCR reaction, an end-point PCR reaction, a real-time RT-PCR reaction, or a realtime PCR reaction.

24. The method according to Claim 21, wherein a template of said standard sequence is a β-actin, 18S, 28S, or 5S ribosomal, or glyceraldehyde 3-P phosphate dehydrogenase template.

25. The method according to Claim 21, wherein said standard sequence is an endogenous RNA standard, and said PCR is a Semi-Quantitative RT-PCR.

26. The method according to Claim 21, wherein the ratio of said primer to said template-mimic oligonucleotide is in the range of 1/15 to 10/1 by weight.

27. The method according to Claim 21, wherein the ratio of said primer to said template-mimic oligonucleotide is in the range of 1/7 to 1/1 by weight.

28. The method according to Claim 21, wherein said template-mimic oligonucleotide has a length which is 5 bases shorter to 50 bases longer than said primer.

29. The method according to Claim 21, wherein said template-mimic oligonucleotide has a length which is 0-10 bases longer than said primer.

30. An attenuated PCR kit comprising (a) an amplification primer and (b) a template-mimic oligonucleotide having a sequence complimentary to said primer, said template-mimic oligonucleotide having a non-extendable 3' terminal.

31. The attenuated PCR kit according to Claim 30, wherein said primer has a sequence hybridizable to a β-actin, 18S, 28S, or 5S ribosomal, or glyceraldehyde 3-P phosphate dehydrogenase template.