WO1994010342A1 - Method for quantifying nucleic acids - Google Patents

Abstract

The invention relates to methods for quantifying nucleic acid sequences via the use of competitive amplification methods. Competitive nucleic acid sequences are used, which differ in length from targeted regions. These competitive sequences are characterized by containing exogenous sequences.

Description

METHOD FOR QUANTIFYING NUCLEIC ACIDS

FIELD OF THE INVENTION

This invention relates to the field of quantitative analysis. More specifically, it relates to the quantification of nucleic acid molecules in a sample.

BACKGROUND AND PRIOR ART

One of the most powerful tools available for analyzing biological materials is the ability to identify and to analyze specific nucleic acid molecules. The analysis of DNA and RNA is used in the context of diagnostics, forensics, basic biological research, and so forth.

A major drawback to the analysis of nucleic acids is that the sequence of interest, i.e., the "target", may exist in vanishingly small amounts. As a result, many of the well known techniques for analysis, including immunoassays and hybridization probe assays using labelled binding partners for the sequences, are not sensitive enough to be useful.

To overcome the problem caused by such small samples, the art has developed various methodologies to amplify the target sequences, thereby providing larger samples for assaying.

Without question, the most powerful technique to this end is the polymerase chain reaction. U.S. Patent Nos. 4,683,195;

4,683,202 and 4,800,159, the disclosures of which are incorporated by reference, all show this technique. Various modifications of "PCR" are also known, as are other methodologies for nucleic acid amplification, including, e.g., ligand chain reaction or "LCR".

Regardless of the methodology used to amplify a target nucleic acid molecule, there is still an issue in that the artisan must be able to quantify the particular material. The art teaches various methodologies for quantifying nucleic acids, as will be seen, e.g., in Gilliand et al., Proc. Natl.

Acad. Sci. USA 87: 2725-2790 (April 1990), the disclosure of which is incorporated by reference. Choi, Clin. Chem. 37(11):

1893-94 (1991) , discusses the importance of determining quantities of a particular nucleic acid sequence in a sample, as well as the many problems which can arise in carrying out an amplification assay.

Li et al., J. Exp. Med. 174: 1259-1262 (11-91), the disclosure of which is incorporated by reference, teach an improved form of competitive PCR assay. The technique, while useful and valuable, can and has been improved.

The purpose of the invention described herein is to present an improved methodology for quantifying nucleic acids of a known sequence in a particular sample. The invention involves the use of competitive sequences, and will be explained in more detail in the examples which follow. BRIEF DESCRIPTION OF THE INVENTION

The invention involves a method for quantifying a nucleic acid in a sample, based upon a competitive amplification reaction. Especially preferred are competitive polymerase chain reaction, or "PCR" methodologies.

The methods involve the use of a competitive nucleic acid molecule, i.e. , one which competes with the target or template of interest for amplification by nucleotide primers added to the sample. This competitive molecule, described in greater detail infra. is of a length which differs from the target sequence - i.e., it is shorter or longer. By adding known amounts of the competitor to samples and setting up a series of values therefrom, one can quantify the target nucleic acid sequence.

It is especially preferred that the competitive molecule be constructed in one of several ways.

The first approach is to add an exogenous nucleotide sequence to the amplified region of choice. The exogenous sequence is one which does not appear in the target nucleic acid sequence. Preferably, it is less than 100 and more than

20 bases in length.

A second way in which to make the competitor involves splicing out, or deleting, a portion of the sequence to be amplified. As a result, the competitor sequence has a different size than the target sequence.

A third methodology combines the first and second - i.e. , a portion of the sequence to be amplified is deleted, and an exogenous sequence is added. With respect to the exogenous sequence in this method, the criterion in the first method applies. Preferably, the deleted sequence and the added sequence are of different lengths.

A fourth, and especially preferred method, referred to as the "universal competitor construction method", involves elements of the first methodology, but also involves adding short "tail" of anywhere from 2-8 nucleotide bases to each complement of two primers used in the amplification protocol, and then an exogenous or endogenous sequence. The "tail", referred to supra. is chosen to create a desired restriction endonuclease site in the competitor. A special advantage of this approach is that, contrary to standard competitive systems, one need not be concerned about the existence of a defined, recognizing restriction site in the region of amplification. As a result, constraints which exist in other systems regarding choice and location of primer are moot.

These methodologies, as well as other features of the invention are described in the disclosure which follows. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the construction of a competitive sequence for human TGF-β (hTGF-β) .

Figure 2 depicts results obtained following cleavage of the hTGF-β competitor with restriction endonuclease Msel.

Figure 3A shows results of competitive PCR to determine hTGF-β on control T cells.

Figure 3B parallels Figure 3A, but using T cells stimulated with cross linked CD2 and CD3 monoclonal antibodies.

Figure 3C parallels Figure 3B, using T cells treated with cyclosporin and cross-linked CD2 and CD3 mAbs.

Figure 4 shows construction of a competitor for human IL- 2. Figures 5A, 5B and 5C correspond to Figures 3A, 3B and

3C, except that the former represent measurement of IL-2 RNA.

Figure 6 shows construction of a competitor for human gamma interferon.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Example 1

The quantitative methodology of the invention was used in a competitive PCR for the human TGF-β ("hTGF-β") gene. The sequence of this gene is disclosed by Derynck et al., Nature 316: 701-705 (1985).

In order to create a competitive template, two primers were used which correspond to nucleotide bases 2155-2174 and 2381-2400 of the gene, i.e.:

PI: 5^,-CTG CGG ATC TCG GTG TCA TT-3' P2: 5^,-CTC AGA GTG TTG CTA TGG TG-3'

(SEQ ID NOS: 1 and 2, respectively). These two primers were combined with total cDNA from normal CD2 antigen positive T cells. A constant amount (2 ul) of total cDNA was combined with the primers (200 nM each) together with a 50 ul reaction mixture of IxTaq buffer, 1 U Taq DNA polymerase, and 40 uM of each dNTP. This mixture was amplified using 32 cycles of PCR, after which the PCR products were resolved on 2% agarose gel. The PCR products, which correspond to a 246 base pair long fragments, could be visualized using ethidium bromide staining and photography.

The resulting PCR products were then used to create the competitive template. The 246 base pair fragment contains an Msel recognition site 210 base pairs from the 5' end, i.e:

The 246 base pair fragment was digested with Msel to yield fragments of 210 and 36 base pairs. These fragments were then combined with a 44 base pair insert, constructed so as to complement the restriction site products. This phosphorylated fragment was then ligated with the 210 and 36 base pair fragments, using E. coli DNA ligase. Following ligation, the product was isolated on 2% low melting point agarose gel, eluted and purified. The product is a 290 base pair fragment. The 290 base pair competitor was then combined with total cDNA, following the same protocol as described supra, with the exception that several runs were carried out using known, but varying concentrations of the fragment. The same methodology as is described supra was carried out to identify the products.

Authentication was also carried out, by treating the resolved, 290 base pair products of the competitive reaction with Msel. If the experiment was successful, subfragments of

210, 44 and 36 base pairs, in length would be produced. This was shown to be the case. See figure 2.

The methodology described was used to show variation in the level of expression of hTGF-β as a result of different stimuli. Figure 3 shows these results. To elaborate, figure 3A shows unstimulated T cells tested using the construct. Figure 3B shows the results obtained when the T cells were stimulated with anti-CD2 and anti CD-3 mAb, and figure 3C following stimulation with both cyclosporin and CD2-CD3 mAbs (100 mg/ml) . The scanning, and computation of the levels based upon standard techniques yields the following values: Unstimulated T cells: 1038 amol CD-2/CD-3 mAbs: 1992 amol Cyclosporin: 9903 amol

In another set of experiments, T cells were either unstimulated, or stimulated with SN-1,2 dioctanoylglycerol (10.0 ug/ml) plus ionomycin (1.0 urn), or these two materials plus cyclosporin. Again, competitive PCR was carried out, using the methodology discussed supra. The results are presented at the end of Example 2, which follows. Example 2

Levels of human IL-2 mRNA were studied in the same way, using the same methodology as in Example 1. The sequence of the human IL-2 gene is known from, e.g., Fujita et al., Proc. Natl. Acad. Sci. USA 80: 7437-7441 (1983). Primers 1 and 2 were prepared, corresponding to bases 3113-3132 and 5087-5106 of the gene, i.e.:

PI: 5'-CCT CTG GAG GAA GTC CTA AA-3'

P2: 5^,-ATG GTT GCT GTC TCA TCA GA-3'

(SEQ ID NOS: 3 and 4, respectively). When amplification was carried out using the two primers, the resulting PCR product is 149 base pairs in length. When this product was digested with Msel, three fragments of 46, 15 and 88 base pairs were generated. The 15 base pair fragment was discarded, and the two larger pieces, together with the same 44 base pair insert of Example 1 resulted in a 178 base pair length competitor. The competitive PCR methodology was carried out in the same manner described supra. Quantitation of the results, shown in figures 5A, 5B and 5C, which correspond to figures 3A, 3B and 3C, was as follows:

Unstimulated: not detectable CD-2/CD-3 mAbs: 17,170

Cyclosporin: not detectable.

When experiments were carried out with the stimuli described in Example 1, and mRNA levels measured, results were obtained as set forth in the following table 1. The table also includes the results secured for hTGF-β, using the competitive methodology. T«ble 1. Differential Regulation of TGP-0 mRNA and R.-2 mRNA in T Cells

* Highly purified T cells (10⁶ cells/ml) were incubated with the agents shown for 1, 4, 16, or 40 h. Total RNA was then isolated, reverse tra icribed into cDNA, and amplified by PCR. The amount of TGF-/3 mRNA was quantified by using the 290-bp TGF-0 competitor DNA constru and the IL-2 mRNA with the 178-bp H.-2 competitor DNA construct in the competitive PCR. DAG, 10.0 μg/ml; ionomycin, 1.0 μM; CsA. 100 ng/'m

Example 3 Competitor sequences for use in quantifying the human gamma interferon ("hIFN-7" hereafter) gene were prepared. The sequence of the gene is provided by Gary et al., Nature 298: 859-863 (1982). Two sets of experiments were carried out, and in both, the competitive sequences were based upon nucleotides 1831-1850, and 2083-2102. These sequences are as follows: SEQ ID NO: 5 (bases 1831-1850) :

5^,-GCA GGT CAT TCA GAT GTA GC-3' SEQ ID NO: 6 (bases 2102-2083):

5'-TTC CTT GAT GGT CTC CAC AC-3' These sequences served as the 5' and 3' primers, respectively, in PCR experiments described herein.

The sequences are selected from exons 2 and 3 of the hlFN-γ gene, the structure of which is well known, as will be seen in figure 6. Exons 2 and 3 flank a small intron, referred to as "intron B".

Complementary primers for each of PI and P2 were prepared, following standard base complementation rules, and each had a four nucleotide base tail "GATC" added to its 5- 'end.

To create double stranded competitive nucleic acid molecules in the first set of experiments, a 92 base pair sequence was inserted in between one of the primers, and the complement of the second primer. This inserted sequence of 88 base pairs corresponded to bases 475-562 of the hIFN-7 gene, together with the four nucleotide sequence "GATC" ligated to the 5' end. Thus, the double stranded competitor molecule may be represented as:

5'-PjGATC(475-562)GATCCP₂ 3'-CP-CTAG(475-562)CTAGP₂ where "P_!" is primer 1, "P₂" is "primer 2", and "C" stands for complement. In order to prepare the competitor, the primers, their complements, and the inserts (both strands) were combined in 10 M TrisΗCl (pH 7.8), 100 mM NaCl, 2 mM EDTA, and 26.5 uM of each of the elements listed herein. The mixture was treated at 94.5°C for 5 minutes, and then for 10 minutes at each of 75°C, 70°C, 68°C, 62°C, and 60°C in succession, followed by 30 minutes at 57°C, which results in annealing. The resulting, annealed (i.e., double stranded) materials were purified using low melting point agarose gel, and then ligated to each other to yield a 136 base pair competitor. Ligation conditions were 50 mM Tris-HCl (pH 7.4), 10 mM MgCl₂, 10 mM DTT, 1 mM spermidine, ImM ATP, 100 ug/ml bovine serum albumin, mixed with the double stranded DNA at 4°C for 12-16 hours.

In the second set of experiments, a competitor was prepared based upon intron B. As is indicated in figure 6, intron B is known to constitute a sequence of 95 base pairs. This competitor is thus identical to nucleotides 1831- 2102, inclusive, of the hlFN-γ gene. Example 4

Once the competitors were prepared, competitive PCR was carried out. The assay involved coamplification of a constant amount of IFN-7 cDNA (2 ul) using known, but varying amounts of competitor. The cDNA was prepared from total mRNA of human T cells, following well known techniques. As presented in Table ₂, precise dilutions of competitor were prepared, using amounts ranging from 10 pg to 10 ag.

Table _2 Dilution series of Human IFN-y competitors of 272bp and 136bp

The first step of the assay involved roughly titrating the cDNA of interest against broad ranges of dilutions of the competitor using logarithmic increments. Accuracy was then determined using narrow ranges of competitor. This step is not necessary when a range of applicable concentrations for the target molecule is known.

A series of tubes were prepared, each of which contained 10 ul of sample competitor at known concentration, together with 2 ul of total cDNA. A 38 ul sample of master PCR reaction mixture was added, leading to final concentrations of 200 nM primers, and 40 um dNTPs per unit/reaction of Taq polymerase. The mixture was overlayed with a drop of mineral oil, and then subjected to amplification (preheat at 94°C for 30 seconds, denature at 94°C for 30 seconds primer anneal at 57°C for 30 seconds, extend at 72°C for 30 seconds, followed by incubation at 72°C for an additional five minutes after 25 to 30 cycles of amplification. Negative and positive controls wee also run (negative: no cDNA; positive: no competitor) . Following the amplification, aliquots of each sample were subjected to electrophoretic analysis on a 3% agarose gel (2 parts Nusieve; 1 part agarose) , and products were visualized by ethidium bromide staining and photography. Absorbance was determined by analyzing the negatives using laser densitometry. The ratios of absorbances of cDNA to competitor were plotted against competitor DNA concentration. Example 5

It was necessary to prove the validity of the protocol described in Example 4. This was accomplished by carrying out restriction endonuclease studies; indeed, the "GATC" tail was used to facilitate restriction endonuclease analysis, as the enzymes Sau3Al and Msel would be expected to create fragments of a specific size. Specifically, Sau3Al recognizes GATC and Msel recognizes TTAA. When the aforementioned endonucleases were added to PCR products, the appropriate sized fragments were found (for Sau3Al: two 20, one 96 base pair fragment; for Msel, one 53 and one 83 base pair fragment). Example 6

Once the efficacy of the methodology was proven, comparison was carried out between the two competitors. These will be referred to as the "natural" competitor (bases 1831- 2102) , and the "constructed" competitor (the 136 base pair fragment) .

Equal amounts of the competitors were analyzed electro- phoretically on 2.5% agarose (Nusieve: agarose of 2:1). The agarose has been prepped in TAE buffer (40 mM Tris acetate/1 mM EDTA) containing 0.5 ug/ml of ethidium bromide. Following electrophoresis, the gel was photographed using a polaroid camera, and the negative analyzed using laser densito etry. The results indicated that the size of the competitor did not affect the straining of the DNA or its visibility. Example 7

The efficiency of amplification of the competitors were tested. Equal amounts of each competitor were placed in separate tubes, and amplified following the PCR protocols discussed supra. Analysis of the PCR products, using the agarose methodology discussed supra. showed that efficiencies were the same. Example 8

Following the experiments supra. wherein efficiency of amplification was found to be identical, a competitive assay was carried out wherein equal amounts of both competitors were placed in the same tube, and amplified in accordance with the previously described experiments. Following agarose gel analysis, the smaller competitor was found to have a higher amplification efficiency. If a known amount of natural competitor was used as sample, and the constructed competitor used as competitor, detected values would be lower than the actual values. The converse is also true, leading to the extrapolation of a correction coefficient:

N =

wherein (bp)s is the length of target DNA, and (bp)c is the length of competitor. Corrected values of target DNA were obtained by multiplying the detected value by the correction sufficient.

Example 9

In view of the development of the correction coefficient discussed supra. the two competitors were again used to quantify levels of IFN-7 mRNA in human T cells. The methodology was as above. When the correction coefficient was used, experimental results converged on the same values.

The foregoing examples describe a method for quantifying the amount of a particular nucleic acid molecule or sequence in a sample. If one knows the base sequence in question, primers can be prepared for use in an amplification assay. The synthesis of such primers has become a routine part of the analytical arts and requires no further elaboration. In the invention as described herein, the primers that are chosen may also used for the preparation of the competitive molecule to be used in the assay. This competitive molecule is combined with the sample of interest as well as any other reagents necessary to carry out amplification. At a minimum, these include nucleotide triphosphates ("dNTPs") , and a catalyst, such as a polymerase, for joining the bases together. The mixture of materials is then subjected to parameters under which the amplification may be carried out. The particulars of the amplification may vary, but in general they include a heating step to separate double stranded ("ds") target and competitor, and changing of the temperature at various points to optimize the synthesis of complementary strands.

The competitor and target sequence both hybridize to the primers, and as a result there will be two amplification products. As the target sequence and competitor are different in size from the sequence separating sequences PI and P2 in the target molecule, the products will be different in size and will resolve at different points on, e.g., a gel. Upon resolution, the signal on a gel is directly proportional to how much of that material is present in the sample. As such, one can compare results obtained with a particular sample to controls, thereby quantifying the amount of target sequence in a sample.

The methodology described herein may be used to quantify any nucleic acid molecule such as gene sequences. In addition to those sequences described herein, any of the various growth factors, cytokines, lymphokines, enzymes, receptors, and so forth with which the art is familiar may be quantified. In addition, sequences of interest from prokaryotes, Viruses, funghi, and any other life form of interest may be quantified. The quantification may be of one sequence or, if the competitors chosen are of sufficiently different sizes, quantification of more than one sequence may also be carried out.

Other modifications of the invention as described herein will be apparent to the skilled artisan and do not require further enable ent.

The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, it being recognized that various modifications are possible within the scope of the invention.

Claims

We claim:

1. Method for quantifying a known nucleic acid molecule or portion of a nucleic acid molecule in a sample, comprising:

(a) contacting said sample with a first primer and a second primer, each of which is complementary to a portion of said known nucleic acid molecule,

(b) adding a competitor nucleic acid molecule to said sample, wherein said competitor nucleic acid molecule consists of two portions of said nucleic acid molecule to be amplified, separated by an exogenous nucleic acid sequence,

(c) incubating said sample under conditions favoring hybridization of said first and second primers to both said known nucleic acid molecule and said competitor and amplification thereof, (d) determining amount of amplified competitor, and

(e) comparing said amount of amplified competitor to a control value, to determine amount of said nucleic acid molecule.

2. Method of claim 1, wherein said known nucleic acid molecule is RNA.

3. Method of claim 1, wherein said known nucleic acid molecule is DNA.

4. Method of claim 2, wherein said RNA is mRNA.

5. Method of claim 3, wherein said DNA is cDNA.

6. Method of claim 1, wherein said exogenous nucleic acid sequence is more than 20 and less than 100 nucleotide bases in length.

7. Method for quantifying a known nucleic acid molecule or portion of a nucleic acid molecule in a sample, comprising: (a) contacting said sample with a first primer and a second primer, each of which is complementary to a portion of said known nucleic acid molecule,

(b) adding a competitor nucleic acid molecule to said sample, wherein said competitor nucleic acid molecules contains part but not all of the nucleotide sequence amplified by said first and second primers and an exogenous nucleotide sequence, (c) incubating said sample under conditions favoring hybridization of said first and second primers to both said known nucleic acid molecule and said competitor and amplification thereof, (d) determining amount of amplified competitor, and

(e) comparing said amount of amplified competitor to a control value, to determine amount of said nucleic acid molecule.

8. Method of claim 7, wherein said known nucleic acid molecule is RNA.

9. Method of claim 7, wherein said known nucleic acid molecule is DNA.

10. Method of claim 8, wherein said RNA is mRNA.

11. Method of claim 9, wherein said DNA is cDNA.

12. Method for quantifying a known nucleic acid molecule or portion of a nucleic acid molecule in a sample, comprising:

(a) contacting said sample with a first primer and a second primer, each of which is complementary to a portion of said known nucleic acid molecule, (b) adding a competitor nucleic acid molecule to said sample, wherein said competitor nucleic acid molecule comprises a nucleotide sequence which differs in length from the nucleic acid molecule amplified by said first and second primers, and contains at least one restriction endonuclease site, not found in said known nucleic acid molecule,

(c) incubating said sample under conditions favoring hybridization of said first and second primers to both said known nucleic acid molecule and said competitor and amplification thereof, (d) determining amount of amplified competitor, and

(e) comparing said amount of amplified competitor to a control value, to determine amount of said nucleic acid molecule.

13. Method of claim 12, wherein said known nucleic acid molecule is RNA.

14. Method of claim 12, wherein said known nucleic acid molecule is DNA.

15. Method of claim 13, wherein said RNA is mRNA.

16. Method of claim 14, wherein said DNA is cDNA.