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This invention relates to the amplification and detection of nucleic acids and blood samples.
The subject of this invention is a process for the amplification of nucleic acid sequences, DNA or RNA, from blood samples by means of an enzymatic amplification method having a salt concentration. The process is characterized in that no
preparation of the blood sample otherwise necessary to prepurify the nucleic acid sequences to be amplified is performed and the proportion of the sample in the reaction mixture for the amplification process is greater than 5 volume %, preferably greater
than 10 volume %.
BACKGROUND OF THE INVENTION
There is an increasing need in biological research, and more particularly diagnostic medicine, for identifications and characterizations of nucleic acids. By "nucleic acids" there are to be understood in the present case the deoxyribose nucleic
acids (DNA) and the ribose nucleic acids (RNA) in either naturally occurring form or as they can be produced by modern methods of chemical and biological synthesis of substantially any sequence and length.
Conventional methods used in molecular biology to prepare nucleic acids from blood are complex and include steps such as centrifuging, phenol/chloroform extraction of the samples or precipitations of the nucleic acids with organic solvents, which
are useless for rapid and possibly automatable enzymatic amplification of nucleic acids without substantial preparation. A recent compilation of such methods is found in "An efficient and simple method of DNA extraction from whole blood and cell-lines
to identify infectious agents" by V. N. Loparev et al., J. Vir. Methods 34:105-112 (1991) and in "Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material" by P. S. Walsh et al., BioTechniques 10(4):506-513 (1991).
It has been reported in the literature that whole blood inhibits the polymerase chain reaction (PCR) when present even in very small quantities -- i.e., 1 volume % in the reaction mixture. The reason for this inhibition is believed to be due to
heme derivatives consisting of porphyrin rings that are present in blood (R. Higuchi, PCR Technology, Chapter "Simple and rapid preparation of samples for PCR", pages 31-38, H. Ehrlich Ed., Stockton Press, 1989).
According to Higuchi, supra, a method of preparing target DNA molecules in blood samples for PCR is to isolate the mononuclear blood cells (MC) by way of ficoll gradients or to isolate the leucocytes by centrifuging after lysis of the
erythrocytes and to incubate the MCs with proteinase K. After digestion, the proteinase K is inactivated at 95.degree. C. and an aliquot of the sample is used in the PCR process.
Mercier et al. describe PCR amplification of various fragments of chromosomal DNA from fresh blood or frozen blood in a concentration of 1 to 2 volume % in a PCR reaction mixture (Nucleic Acids Research 18:5908 (1990)) in which the amplification
solution containing the blood sample (without Taq polymerase) was repeatedly brought for 3 minutes at a time to temperatures of 95.degree. C. and 55.degree. C., a step which facilitated subsequent amplification by PCR.
Panaccio et al. (Nucleic Acids Research 19:1151 (1991)) describe the amplification of DNA from whole blood using thermostable DNA polymerase from Thermus thermophilus. They show that DNA from 4 .mu.l of blood in 100 .mu.l of reaction mixture (4
volume %) is amplifiable using Thermus thermophilus DNA polymerase. On the other hand, as little as 1 volume % of blood completely inhibits amplification by means of the DNA polymerase from Thermus aquaticus (Taq).
Beutler et al. (BioTechniques 9:166 (1990)) describe in detail the effect of anticoagulants on the amplification by PCR of DNA targets from blood samples. It was impossible to amplify these targets even when the DNA was concentrated from
heparinized blood by means of a nucleic acid extract. Further processes for purifying DNA that facilitate use of PCR with the DNA thus isolated are also described, including the treatment of DNA with heparinase II. There were no problems with
amplifying DNA isolated from EDTA-treated blood.
Israeli et al. (Nucleic Acids Research 19:6051 (1991)) describe the amplification by PCR of RNA which was isolated from frozen heparin-treated whole blood by extraction after conversion of the RNA into eDNA. Israeli demonstrate that the
difficulties in conducting PCR were due to the heparin. Only when the isolated RNA was treated with heparinase before transcribing into eDNA, was the PCR successful.
Franchis et al. (Nucleic Acids Research 16:10355 (1988)) also describe the inhibition of the PCR process using Taq polymerase when amplifying samples of genomic DNA isolated from human blood. The inhibitor, which was not specifically identified
in the article, could be removed by boiling and filtering the DNA.
Ravaggi et al. (PCR Methods and Applications 4:291-292 (1992)) describe the amplification of HCV RNA from human serum by means of PCR. The RNA was transcribed directly from the serum into eDNA with reverse transcriptase without previous
purification. An aliquot containing approximately 3 volume % of DNA then was introduced into a PCR mix.
One of the goals of this invention is to overcome the difficulties noted in the prior art described above in order to use increased quantities of blood directly in an enzymatic amplification process for nucleic acids, such as, for example, PCR,
and more particularly, when the blood is treated with anticoagulants.
SUMMARY OF THE INVENTION
The subject of this invention is a process for the amplification of target nucleic acid sequences, RNA or DNA, present in blood by means of an enzymatic amplification method, said amplification method using at least one salt. This process is
advantageous in that the blood sample need not be treated prior to amplification to isolate or purify the target nucleic acid sequences. The amount of sample in the amplification reaction mixtures is greater than or equal 5 volume %, preferably greater
than or equal to 10 volume %.
This invention may also be used to determine genetic sequences, for example, of humans, from blood and to identify foreign nucleic acids of microorganisms in the blood, including nucleic acids from bacteria, DNA or RNA viruses or eucaryotic
nucleic acids. The claimed process is particularly useful in the detection of small quantities of an infectious microorganism in a blood sample.
DETAILED DESCRIPTION OF THE INVENTION
The term "blood sample" is used herein to denote any kind of sample whose origin can be derived from blood. It can be, for example, liquid blood, such as fresh whole blood with all its constituents, or plasma. It also includes dried blood such
as is present, for example, in blood stains, to coagulated blood or the serum obtained therefrom, and to blood stabilized by vitrification as is described by R. Ramanujan et al. (Clin. Chem. 39:737 (1993)).
Blood in its natural form consists of a liquid component, the plasma, and a corpuscular component, the blood cells (erythrocytes, leucocytes, thrombocytes etc.). The "plasma" is that portion of the anticoagulated blood which remains after
centrifuging. It is a clear light yellow liquid which contains in addition to albumins, coagulants and plasma proteins, sugar, minerals and other products of metabolism. The "serum" is the liquid part of the blood which is obtained by centrifuging
after coagulation and lacks the coagulant factors such as fibrinogen.
The whole blood of adults excluding leucocytes, consists of 55% plasma with 141.7 to 148.8 mM sodium and potassium and 45% erythrocytes with 90.4 to 106 mM sodium and potassium. The average value of sodium and potassium is of about 124 mM
(Documenta Geigy 1973, pages 560-564).
The term "monovalent ions" used herein denotes only ions that are present in blood having a single positive charge -- i.e., mainly Na+ and K+. The chloride content is disregarded. Correspondingly, the term "bivalent ions" denotes ions carrying
two positive charges -- i.e., mainly Mg.sub.2 + and Ca.sub.2 +.
Fresh blood is typically treated with anticoagulants to prevent premature coagulation. The best known anticoagulants are heparin, the salts of citronic acid (citrates), and the salts of ethylenediamine-tetraacetic acid (the salt hereafter
abbreviated EDTA).
According to the information provided by some manufacturers of containers for blood sample collection, for example, Sherwood Medical, EDTA, citrate and heparin are typically added to blood samples as follows:
EDTA is added to blood as a tripotassium salt (0.1 ml of a 15% solution per 10 ml of blood). This results in an additional concentration of approximately 10 mM of added potassium of the blood sample.
Citrate is added to the blood as a trisodium salt (normally 0.5 ml of a 3.8% solution to 4.5 ml of blood). This results in the addition of approximately 36 mM of sodium to the blood sample.
Heparin is added as lithium salt in a final concentration of 14.3 USP units per ml of blood. This corresponds to approximately 0.1 mg of hepaxin per ml of blood or 5-15 .mu.M of salt.
Based on the foregoing, typical blood sample preparations have approximately the following final concentrations of monovalent ions: (a) EDTA treated blood: 135 mM; (b) citrate treated blood: 160 mM; and (c) heparin treated blood: 124 mM. Further
particulars on recommended salts and their concentration as anticoagulants are given in "N.S. Evacuated Tubes for Blood Sample Collection", Third Edition (1991), Vol. 11 (No. 9):6 (NCCLS Document A1-A3).
The final concentration of monovalent ions in the reaction mixture (mainly potassium and sodium) for amplification of the target nucleic acids depends upon the buffer, the volume of the sample and the kind of anticoagulant in the whole blood.
Note that inasmuch as the concentration of anticoagulants may vary with different makers, in the following description of the concentration of monovalent ions, only the amounts of salts from the blood alone (approximately 124 mM) and the added amounts
from the addition of solutions having defined salt concentrations, will be considered. The monovalent ion concentration due to the anticoagulants will be disregarded. In the case of heparin, this value is insignificant in any event.
The target nucleic acids to be amplified by the processes of the present invention can be present in the blood cells (e.g. genomic DNA, mRNA), in plasma, and in serum.
In plasma or serum, the nucleic acids can be the cell's own DNA or RNA which are liberated by cell lysis, or they can be foreign nucleic acids that are introduced by bacteria or viruses. Some RNA viruses do not transcribe DNA. In these cases,
the first amplification can take place after transcription of the RNA into DNA. Further amplification cycles can be performed on the DNA stage alone or by way of new RNA intermediate stages, as will be described by way of example hereinafter.
As previously stated, identifying particular nucleic acids from blood samples containing a mixture of other nucleic acids is presently difficult. Even when an amplification process that can act specifically in the presence of very large
quantities of different sequences is used, certain target nucleic acids, for example, those of a non-repetitive gene, must be concentrated before amplification is carried out. For purposes of diagnosis, the amplification process must selectively amplify
only the desired nucleic acid sequence, for example, a 100 Bp long DNA fragment which occurs in every human cell only once in a total of 31 million other sequences of the same magnitude (the human genome consists of 3.1.times.10.sup.9 base pairs or
3.4.times.10.sup.-12 g of DNA). Presently, in order to use the polymerase chain reaction (PCR), the best known enzymatic amplification process, it is necessary to perform a relatively elaborate process to prepare the samples prior to amplification. The
aim of these additional steps, which normally have to be used, is to remove or neutralize suspected amplification inhibitors in the blood by prepurification of the nucleic acids, and thus facilitate unimpeded amplification of the nucleic acid sequences
of interest. Sample preparation procedures for the target nucleic acids to be amplified differ according to whether the sample is from cells, plasma/serum, or from whole blood.
Before this invention, PCR amplification typically was inhibited by excessive salt concentrations in samples having a large blood component. Calculations show that blood with an average natural concentration of monovalent ions of approximately
124 mM will have an adverse affect on amplification carried out using Taq polymerase. Having regard to conventional PCR buffers, for example, the 10 X buffer, which is designed to give a final concentration in the prepared reaction mixture of 50 mM K+,
the concentration of monovalent ions (K+, Na+) in the reaction mixture having a 10 volume % blood content totals approximately 63 mM, and approximately 113 mM when the blood content is 50 volume %. As was previously stated, these values do not include
the salt contribution of any anticoagulant used in the sample.
On the other hand, the DNA polymerase of Thermus aquaticus (Taq polymerase), which is the polymerase most used in PCR, has optimum synthesis around approximately 50 mM KCl (PRC Technology 1989; Chapter 2: Taq DNA Polymerase, by D. H. Gelfand).
This is why normal PCR buffers have the KCl concentration mentioned in the reference. No activity for Taq polymerase can be detected in conventional sequencing reactions at more than 75 mM KCl, or in 10-minute incorporation assays at more than 200 mM
KCl.
We have surprisingly found that if overall salt concentration is controlled -- i.e., adjusted in the reaction mixture -- nucleic acids can be amplified directly from untreated blood samples. An increase of initial nucleic acids is obviously
available in higher volume samples, for example, .gtoreq.10 volume %, of a blood sample in the PCR reaction mixture. Knowing the salt concentration of a blood sample, that is of monovalent and bivalent ions contributed by the blood to the reaction
mixture in which the amplification is performed, the overall salt concentration in the reaction mixture can be maintained in a predetermined range and adapted to the requirements of the enzyme to be used by the utilization, inter alia, of an
appropriately concentrated salt solution. By way of example, the salts of the elements of the first and second groups of the periodic table can be used for this purpose, with Na+, K+and Mg2+ being preferred.
Depending upon the amount of blood sample in the overall reaction mix, the salt concentration in the reaction mix can readily be controlled by an appropriately concentrated or dilute salt solution. This means that if there is a high blood
proportion in the reaction mixture, no further monovalent salts are added -- i.e., if the blood proportion is high enough the buffer capacity and the salt content of monovalent or bivalent ions of the sample may in some circumstances be sufficient to
facilitate a specific amplification -- i.e., it is unnecessary to use a further salt solution.
The salt solution can contain, in addition to salts, buffers and other components necessary and/or advantageous for the particular amplification process. These components can be, for example, Tricine (N-(Tris(hydroxymethyl)-methyl-glycin), Tris
(Tris-(hydroxymethyl)-aminomethane-hydrochloride), ionic or non-ionic detergents, such as, for example, Triton X-100 (alkylphenylpolyethyleneglycol) or Tween (polyoxyethylenesorbitol monolaureate), and salts of other elements such as, for example, salts
of Mn or Co and so on, as are important for the activity of the particular enzymes used in the particular amplification.
This invention is applicable to any enzymatically based amplification process. A number of enzymatic amplification processes are described in the literature. One process such process, the ligase chain reaction (LCR), as described, inter alia,
in EP-A 320 308 or EP-A 336 731. Further explanations and applications of this method have been described by Wu and Wallace, Genomics 4:560-569 (1989).
Other enzymatic amplification processes are TAS and 3SR. These are described in EP-A 310 229 and EP-A 373 960, respectively. Other descriptions can be found in Guatelli et al., Proc. Natl. Acad. Sci. US 87: 1874-1878 (1990) and Kwok et al.,
Proc. Natl. Acad. Sci. USA 86:1173-1177 (1989), respectively. In these processes, a number of enzymes are used either simultaneously or consecutively in the amplification process, for example, a DNA polymerase and an RNA polymerase and other enzymes
are used.
Another amplification process is based on the use of the replicase of the RNA bacteriophage Q.beta.. The operation of this process has been described e.g. in EP-A 361 983 or in Lizardi et al., TIBTECH 9, 53-58 (1991).
Another enzymatic amplification process, and that which is preferred in the application of the present invention, is the polymerase chain reaction (PCR), which is one of the processes described in U.S. Pat. Nos. 4,683,195 and 4,683,201. In a
preferred embodiment, the nucleic acids are amplified by means of a thermostable polymerase. Useful polymerases from various thermostable bacteria include Thermus aquaticus (U.S. Pat. Nos. 4,889,818 and 5,079,352), Thermus thermophilus (WO/9108950),
Pyrococcus furiosus (WO 92/9688) and Thermococcus litoralis (EP-A 455 430). These enzymes are useful in either their purified natural or recombinant form and are commercially available. Polymerases which can be isolated from the genus Thermus are
preferred. The thermostable DNA polymerase from Thermus aquaticus ("Taq") is particularly preferred in the present invention.
The PCR process can be used to amplify and detect RNA based nucleic acid targets by first transcribing the RNA into DNA. Such a process is described, for example, in WO 91/09944. The so-called reverse transcriptases can, for example, also be
used as enzymes in this case. The reverse transcriptase activity of a DNA polymerase such as the Taq polymerase, can also be used for this purpose. The corresponding presence of other bivalent ions (in this case e.g. of Mn2+) should, where applicable,
be ensured to make full use of this activity.
The practice of the process according to the present invention when carried out with PCR or TAS, is used with varying temperatures and is conveniently performed in an automated system in which the temperature for denaturing, the hybridization of
the primers and the polymerization reaction can be accurately controlled. An appropriate device for this purpose is described in U.S. Pat. No. 5,038,852. Apparatuses of this kind are also commercially available.
The oligonucleotides for carrying out the enzymatic amplification of the nucleic acids and the subsequent detection thereof can be prepared by known methods, for example, by solid phase synthesis (Oligonucleotide Synthesis: A practical Approach,
IRL Press, Oxford, UK, Ed. M. J. Gasit (1984)). Many such oligonucleotides are also commercially available.
All the enzymatic amplification processes hereinbefore mentioned use specific salt concentrations and/or buffers for optimum matching to the requirements of the particular enzyme used. In each of the various amplification processes, the DNA or
RNA sought to be amplified is often isolated from other components in the sample by a pre-purification step or is at least highly concentrated. The enzymatic buffer does not then need to be adapted to any of additives that are introduced by the sample.
However, if such pre-purification is performed, then correspondingly elaborate preparation steps are also necessary, inter alia. because of the risk of contamination of the sample.
For purposes of describing how the invention operates, it is herein demonstrated in conjunction with PCR to amplify target nucleic acids in blood samples.
A special feature of this invention is in amplification of nucleic acids from heparinized blood. It is presently known that PCR amplification of nucleic acids in blood samples is inhibited when the samples are previously treated with heparin as
an anticoagulant. This fact is consistent with earlier findings in enzymology wherein heparin was used as a specific inhibitor of DNA-binding proteins because the latter have a greater affinity for heparin than for their substrate, the nucleic acids (T.
A. Bickle et al., Nucleic Acid Res. 4:2561, 1977; J. Leis et al., Methods in Enzymology XXIX:153, 1974). Such prior findings would be expected to instill in a skilled artisan the prejudice that amplification of DNA from heparin-treated blood samples by
means of PCR in the reaction mixture is impossible. However, we found, unexpectedly, that the amplification of DNA is possible in reactions with high (e.g. .gtoreq.10 volume %) to very high blood content (e.g. .gtoreq.50 volume %). This is possible
even with the use of conventional PCR buffers -- that is, no special adaptation of the KCl concentration in the conventional PCR buffer (approximately 50 mM of KCl) to the salt concentration introduced by the sample components is needed (see, e.g.,
Example 2). Another alternative is to neutralize the added amount of the salt with buffers when the salt content of the sample is very high. The salt concentration of monovalent ions in the reaction mixture is approximately 10-160 mM. Preferably, the
monovalent ion concentration is approximately 10-90 mM.
Another special feature of this invention is the amplification of nucleic acids from EDTA treated blood. In this kind of blood, no specific DNA amplification can be performed with elevated amounts of blood sample (upwards from about 30 volume %)
when the conventional concentration of 50 mM of KCl in the PCR buffer is used. This corresponds to a maximum salt concentration of monovalent ions of approximately 135 mM (see Table 1, Example 1 and Table 2, Example 2, respectively).
The effect of the salt on the PCR enzyme is also noted even when DNA is used as substrate which has been purified in order to prevent the influence of blood compounds. Specific amplification of desired target is possible only above 10 mM of KCl
in the reaction mixture (Example 1, Table 1). Correspondingly, no addition of KCl to the reaction mixture is necessary, for example, upwards from 20 volume % of EDTA-treated blood component because the sample itself is providing approximately 25 mM of
monovalent ions (Table 1). Consequently, for the amplification of DNA or RNA from EDTA-treated blood, the salt concentration of monovalent ions in the reaction mixture should be between 10 and 135 mM, preferably between 30 and 80 mM.
Yet another object of this invention is the amplification of nucleic acids from citrated blood. When blood treated with citrate as an anticoagulant is used as sample material for enzymatic amplification, different results are obtained depending
on the sample concentrations. If, for example, the conventional buffer containing 50 mm of KCl is used in the PCR, amplification is possible with a sample concentration of up to approximately 20 volume % in the reaction mixture. However, a higher KCl
concentration is usually advisable in the case of optimized magnesium concentrations and can be supplied, for example, by way of the PCR buffer. For example, an additional KCl concentration of 100-150 mM in the reaction mixture is suitable for
amplifying blood samples having concentrations equal to and greater than 50 volume % (Example 2). This also corresponds approximately to a concentration equal to or higher than 70 mM of monovalent ions in the sample. A salt concentration of monovalent
ions in the reaction mixture of between 30 and 200 mM is therefore suggested for enzymatic amplification. Preferably, a salt concentration of monovalent ions from about 60 to about 150 mM is used.
Also, it is found with citrated blood that it may, in some circumstances, be advantageous to adapt the concentration of bivalent ions. For efficient PCR, for example, the amount of Mg2+ should be raised above the otherwise normal figure of 1.5-2
mM in the reaction mixture, especially when very high blood concentrations (e.g. .gtoreq.20 volume %) are present in the reaction mixture. A concentration of more than 3 mM of Mg2+ is typically necessary and can go as high as 40 mM without damage.
There is therefore considerable freedom in the choice of the Mg2+ concentration provided it is above a critical minimum.
Disregarding the Mg.sup.2 + concentrations in blood the Mg2+ concentration in the reaction mixture is very important when EDTA is present in the blood sample as an anticoagulant. Approximately 0.35-0.4 mM of Mg2+ is bound by 10 volume % of
EDTA-treated blood, approximately 1.75-2.0 mM of Mg2+ is bound by 50 volume % of EDTA-treated blood, and approximately 3.5-4.0 mM of Mg2+ is bound by 80 volume % of EDTA-treated blood. The quantities of Mg2+ bound to EDTA are therefore always equimolar. However, since a free Mg2+ concentration is necessary for successful amplification, the bound Mg2+ in the reaction mix must be taken into consideration. For an optimum PCR, more than 1 mM of free Mg2+ should be present. If the free Mg2+ concentration
is 1 mM or less, at most only 50% of the maximum possible amplification yield is obtained. The top limit is approximately 20 mM of free Mg2+. Higher values produce suboptimal amplification yields. For an optimum PCR, an Mg2+ concentration of 1.4 mM
has been found to be the necessary minimum concentration in the presence of 10 volume % of EDTA-treated blood. 3.0 mM of Mg2+ was found to be the necessary minimum concentration in the presence of 50 volume % of EDTA-treated blood and 5.0 mM of Mg2+ was
found to be the necessary minimum concentration at 80 volume % of EDTA-treated blood. What is particularly surprising about these figures is that the free Mg2+ concentration of EDTA-treated blood quantity of concentrations as high as 80 volume % can be
used as a reaction mix for amplification without any pretreatment.
Additional KCl concentrations (in mM) supplied by the PCR buffer to the reaction mixture in the case of optimized MgCl.sub.2 concentrations for the amplification of DNA in reactions having a concentration of 10 to 50 volume % of unpurified blood
samples treated with various anticoagulants led to the results given in the following Table. The Table below gives an overview of how, for example, with increasing sample concentration in the reaction mixture, the optimum concentration of monovalent
ions (here, for example, by means of KCl) of the added PCR buffer varies in the manner as discussed above.
______________________________________ Sample concentration (volume %) in the reaction solution 10% 20% 30% 40% 50% ______________________________________ EDTA blood 50 25 0 0 0 Heparin blood 70 50 10 0 0 Citrated blood 100 100 80 50 50
______________________________________
As an additional step to ensure efficient amplification, the blood sample can be frozen before use if this has not already been done by virtue of the nature of sample storage. Another possible way of enhancing amplification is first to denature
the sample alone or the prepared reaction mixture in a thermocycler a few times by heating and cooling. About 5-20 such cycles with heating to approximately 85.degree.-95.degree. C. and cooling to approximately 40.degree.-60.degree. C. are sufficient. The temperature need be maintained only briefly at the respective levels, 1 to 2 minutes being sufficient. However, the time period may be longer or shorter. The mix with the components for amplification (primers, triphosphates, enzyme) can already be
included, or to protect the enzyme, can be added after these denaturing cycles (e.g. as PCR mix).
Addition of the enzyme after denaturation of sample is necessary when isothermal amplification processes such as, TAS, are used in which the enzymes are heat-sensitive. This kind of preparation before actual amplification has already been used
in various similar ways in the prior art. For example, Mercier et al., supra, writes that prior repeated thermal denaturing improves the subsequent PCR. This denaturing can be effected simply by some additional cycles during the general amplification
reaction when, in any event, the enzymatic process (such as PCR and LCR) requires the denaturing step for DNA amplification. This variation of pretreatment of the sample or reaction mixture is generally applicable to all blood samples.
It is believed that the increase efficiency of amplification due to prior freezing or heating of the samples occurs not because of the neutralization of PCR inhibitors, but because of improved lysis of the cells or viruses. The target nucleic
acids, are therefore, liberated before the start of PCR and are thus more readily accessible to the amplification reagents.
The foregoing description of the invention has been made with particular preference for use with PCR as the amplification process for the target nucleic acids and with the use of Taq polymerase as the enzyme. The target-containing sample may be
whole blood, plasma or serum. The present invention also contemplates use of polymerases (DNA or RNA) other than Taq, such as one of the thermostable polymerases previously mentioned from Thermus thermophilus, Thermococcus litoralis or Pyrococcus
furiosus. Individual adaptations of the concentration of monovalent and/or bivalent ions in the reaction mixture may be necessary in each case, depending, inter alia, on the enzyme. Particulars about the procedure for certain other enzymes are given
infra in Example 6. The range of operation of the enzymes tested is at both high (e.g. .gtoreq.10 volume %) and very high blood concentrations (e.g. .gtoreq.40 volume %) in the concentration range of approximately 10 to 160 mM of monovalent ions (which
was also the concentration described previously with Taq polymerase) and using heparinized samples. In view of the present disclosure, ascertaining the specific limits for other polymerases is a relatively straight forward process for a skilled artisan. Similar considerations as discussed supra. apply to other enzymes, such as, the various reverse transcriptases that are used when the target is RNA and the amplifying process is PCR or TAS.
The following examples are provided by way of illustrating the invention and do not limit the scope of the invention.
The efficient amplification provided by the novel process is demonstrated in Examples 3 and 11, and its reproducibility is demonstrated in Example 4.
Evidence for other possible uses of the novel process is provided by the analysis of very large volumes of blood in Example 5 and of dried blood samples in Example 8. Example 12 provides evidence of the amplification of RNA-target by the PCR
process from unpurified serum and plasma samples.
EXAMPLES
General observations regarding PCR conditions used and reported herein:
Unless otherwise specified, all the reactions were performed in a total volume of 50 .mu.l, the so-called "reaction mix". Because of the high sample concentrations therein, correspondingly highly concentrated 10 X PCR buffers containing the
necessary salts were used (for the composition of specific PCR buffers, see Point 2 below).
Also, a standard PCR mix containing the nucleoside triphosphates, the primers, buffers and polymerase was used. Unless otherwise specified, the DNA polymerase from Thermus aquaticus was used.
1 ) The 50 .mu.l reaction mixture was composed as follows:
(a) 4.5 .mu.l of a 10 x concentrated PCR buffer
(b) 5.0 .mu.l of the PCR mix
(c) The required sample volume (.mu.l)
(d) Autoclaved H.sub.2 O bidest. q.s. to 50 .mu.l.
2) Composition of PCR buffer (A)
(1) 10 X PCR buffers of the so-called "L" series contained:
-- 50 mM of Tricine (pH 8.8) (25.degree. C.)
-- 15 mM of MgCl2
-- 0.5% Tween 20 (polyoxyethylene-sorbitol-monolaurate)
-- and various concentrations of KCl as follows:
Buffer 10 X L0=0 mM KCl
Buffer 10 X L1=100 mM KCl
Buffer 10 X L2=200 mM KCl and so on up to
Buffer 10 X L15=1,500 mM KCl.
(2) 10 X buffer of the LxM series corresponded to the L series but contained 150 mM of MgCl.sub.2 instead of 15 mM. For example:
10 X PCR buffers of the L.times.M series contained:
50 mM of Tricine, pH 8.8 (25.degree. C.),
150 mM of MgCl.sub.2
0.5 % Tween 20
and the following different concentrations of KCl:
Buffer 10 X L0=0 mM KCl
Buffer 10 X L1=100 mM KCl
Buffer 10 X L2=200 mM KCl and so on up to
Buffer 10 X L15=1,500 mM KCl.
(3) 10 X T-solution consisted of:
-- 15 mM of MgCl.sub.2
-- 0.5 % Tween 20
(no Tricine, Tris or KCl)
3) Composition of PCR mix (B) for amplification:
0.5 .mu.l of each dNTP (dATP, dCTP, dGTP, dTTP) as 10 mM solution
0.5 .mu.l of the corresponding 10 X PCR buffer
0.5 .mu.l of each primer (50 .mu.M)
1.25 to 2.5 units of DNA polymerase
Water q.s. for a final volume of 5 .mu.l.
Unless otherwise specified, DNA polymerase from Thermus aquaticus was used.
Advantageously, the amplification according to Step 1) was performed with 4.5 .mu.l of 10 X PCR buffer (A) and the sample volume (1(c)) was filled up with the autoclaved H.sub.2 O (1(d)) to 45 .mu.l. The sample was thermally denatured, when
necessary. The 5 .mu.l of the PCR mix, step (b), was added shortly before the start of the PCR. The reaction mixture was covered by two drops, corresponding to 30 to 40 .mu.l, of mineral oil before the denaturing.
4) The thermocycler conditions, per cycle, for amplification of DNA were as follows:
Step 1: 30 sec at 93.degree. C. (separation of the DNA strands)
Step 2: 30 sec at X.degree. C. (hybridization of the primers)
Step 3: 90 sec at 72.degree. C. (polymerase reaction)
20 seconds were interposed between each of the above temperature changes (steps 1-3). A complete cycle, therefore, lasted 3 min and 30 sec. A total of about 35-40 cycles were usually run.
In many experiments the samples were thermally denatured before running PCR. The thermocycler conditions for a denaturing cycle were:
Step 1: 90 sec at 90.degree. C.
Step 2: 90 see at 50.degree. C.
20 cycles were typically run for denaturing, but in some cases, only 5 cycles were run. After the denaturing cycles, the samples were then brought to room temperature, the PCR mix added, and the target nucleic acid was amplified as described.
The following hybridization temperatures (X.degree. C. in Step 2 above) were used with the primers described in Point 5) below:
HLA primer GH26/27: 60.degree. C.
Factor IX primer JR3/JR4: 55.degree. C.
Hepatitis B primer MD 122/MD 123: 50.degree. C.
Rubella primer Ru2/Ru3: 60.degree. C.
5) Sequences of the pCR Primers and Amplified Fragment Size
a) HLA DQ alpha gene (242 base pairs) GH26: GTG CTG CAG GTG TAA ACT TGT ACC AG (SEQ ID NO. 1) GH 27: CAC GGA TCC GGT AGC AGC GGT AGA GTT G (SEQ ID NO. 2)
(for primers and sequence See H. Ehrlich et al., PCR Protocols (Acad. Press., 261-271, 1990).
b) Factor IX gene (234 base pairs) JR 3: AGG ACC GGG CAT TCT AAG CAG TTT A (Exon D) (SEQ ID NO. 3) JR 4: CAG TTT CAA CTT GTT TCA GAG GGA A (SEQ ID NO. 4)
(for primers and sequence see J. Reiss et al., Blut 60:31-36, 1990).
c) Hepatitis B (151 base pairs) MD 122: CTC TCA ATF TTC TAG GGG GA (SEQ ID NO. 5) MD 123: AGC AGC AGG ATG AAG AGG AA(SEQ ID NO. 6)
These primers amplify a 153 bp long fragment of the hepatitis B virus. Primer No. MD122 is at bp 267-286, No. 123 at Bp 401-420 of the HBV genome (sequence numbering according to H. Okamoto et al., J. Gen. Virol. 67:2305-2314, 1986).
d) Rubella (321 base pairs) Ru 2: TGC TTT GCC CCA TGG GAC CTC GAG (bp 1990-2013) (SEQ ID NO. 7) Ru 3: GGC GAA CAC GCT CAT CAC GGT (bp 2290-2310) (SEQ ID NO. 8)
(for sequence of primers see Eggerding F. et al., J. Clin. Microb. 29:945-952, 1991).
6) DNA and RNA polymerases used
a) Taq polymerase from Thermus aquaticus: Super Taq (Stehelin, Switzerland) as 5 units/.mu.l in 20 mM of Tris (pH 8.0), 1 mM of EDTA, 1 mM of DTT and 50% glycerin.
b) DNA polymerase from Thermococcus litoralis: Vent (Trademark of New England Biolabs), 1000 units/ml in 100 mM of KCl, 0.1 mM of EDTA, 10 mM of Tris-HCl (pH 7.4), 1 mM of DTT, 0.1% of Triton-X-100, 100 .mu.g/ml of BSA and 50% glycerol.
c) Pfu DNA polymerase from Pyrococcus furiosus (Stratagen) 2500 units/ml in 50 mM of Tris HCl (pH 8.2), 1 mM of DTT, 0.1 mm of EDTA, 0.1% of Tween-20, 0.1% of NP-40 and 50% of glycerol.
d) rTth DNA polymerase from Therm | | |
