FR2669347A1 - Process which makes it possible to carry out at least two successive reactions, while avoiding the risks of contamination, which is especially applicable to techniques for the amplification of DNA or RNA - Google Patents

Abstract

The process which makes it possible to perform at least two successive steps which are necessary in a molecular biology experiment in the same tube, of the Eppendorf tube type, consists in: - depositing in the tube a volume of the first reaction medium (A), - layering the medium (A) with an oil, - depositing the second reaction medium (B) directly over an oil, - mixing, after performing the reaction in the medium (A) and/or (B), the media (A) and (B), - incubating, in the mixture A + B obtained, a reaction which has as substrate the products of the preceding reaction(s), the various operations being carried out without having to open the said tube.

Description

 Process for carrying out at least two successive reactions, the product of the first reaction serving as a substrate for the next reaction, avoiding the risk of contamination, in particular applicable to the DNA or RNA amplification techniques.

 The present invention relates to a method for performing at least two successive steps, including at least two successive reactions occurring in an aqueous medium, the product of the first reaction serving as a substrate for the next reaction, necessary for the execution of experience in molecular biology, and avoiding the risk of contamination.

The process according to the invention finds a particularly advantageous application in the technique of amplification of DNA sequences by PCR (Chain polymerase
Reaction).

 PCR is a technique very commonly used to allow the detection of very poorly represented sequences in a biological sample. One of the major drawbacks of this technique lies in the possibility of false positives due to contaminants. These are mostly made up of PCR amplification products previously made. If it is desired to prevent the amplification products from contaminating the subsequent reactions, it is necessary to physically separate the different stages of the reaction.

The protocol used to avoid PCR contamination is as follows
1) Extraction of DNA or RNAi in a room not containing amplified products (1 part room) use of a laminar flow and pipette host
Pasteur made of plastic for single use
2) Preparation of reagents (aliquots before PCR) and reaction mix in a room containing no other
DNA as synthetic oligonucleotides as primers (2nd part)
3) Mix of llADN to be tested with the mix in the 1st room and start of the PCR in the 1st room
4) Opening and exploitation of the results in a hybridization laboratory (3rd part)
5) Using microman positive displacement pipettes to deposit the DNA solution in the PCR buffer.

 It must be assumed in this system that the third piece can not be a source of contamination for others. This is rarely true in current basic research or medical biology structures.

Also, after five years of existence, the PCR remains a relatively little used technique in medical biology or in all the fields where it is useful to practice the analysis of a large number of samples.

 It has been proposed to modify the PCR technique, in order to improve the specificity of the amplified sequences. The proposed modification (Nested PCR) consists in performing two successive amplifications. For example, 1/50 of the first PCR is reamplified using primers in the internal position relative to the primers of the first PCR. This results in a sensitivity close to 100%.

 The specificity of the amplified sequence is total since four different hybridizations are necessary and this, on a small DNA sequence. The size of the amplified fragments is known and compared to molecular weight markers. Since amplification is visible from less than 5 initial targets, a simple reading of an agarose gel is sufficient. The use of a method of confirmation by molecular hybridization is, under these conditions, superfluous.

 One of the major drawbacks of this technique lies in the problem of contaminations, because it is necessary to open the tube in which the first amplification was performed to achieve the second amplification. The possibility of contaminating the second PCR with amplification products of the first PCR is therefore important. In fact, the use of this method remains limited.

The only possibility not to contaminate is to carry out the Nested PCR on a series of known dilutions, starting the second series of PCR with the lowest dilutions. Under these conditions, the sensitivity of the "Nested
PCR on low dilutions is such that amplification of less than 5 initial target sequences can be detected on agarose gel.

 The present invention aims, among other objectives, at remedying the disadvantages presented by the DNA amplification techniques or RNAs used hitherto, and in particular by the Nested PCR technique, by proposing a method making it possible, in particular, to carry out two amplifications. successive in the same tube and without having to open it after the first amplification.

 The present invention proposes, in its most general aspect, a method which makes it possible to carry out at least two successive steps, in particular at least two successive reactions preferably occurring in an aqueous medium, necessary for a molecular biology experiment, the product of the first reaction serves as a substrate for the next reaction and avoids the risk of contamination.

The process according to the invention is characterized in that it consists of
depositing in a tube, Eppendorf tube type, a volume of the first reaction medium (A),
covering the reaction medium (A) with an oil,
depositing the second reaction medium (B) directly on this oil,
to mix, after carrying out the reaction in the medium (A) and / or (B), the media (A) and (B),
and optionally, incubating in the A + B mixture obtained a reaction with the substrate, the products of the preceding reaction (s).

The tube in which the implementation of the process according to the invention is carried out may consist of an Eppendorf tube. The medium (A) which is deposited at the bottom of the tube is covered with a mineral oil (for example "Heavy
Mineral Oil "from Sigma) The medium (B) deposited directly on the oil, forms an aqueous drop which spreads and joins the walls of the tube.This drop remains suspended in the oil and is separated from (A) by an oil thickness Centrifugation, for example at 5000 g, after carrying out the reaction in the medium (A) or (B), makes it possible to lower this drop to the bottom of the tube and to mix (A) and ( B).

 The drop (B) is created automatically. The different oil-water interfaces are resistant to shock, handling and temperature changes (95 "-10" C).

As an indication, a tube having a volume (A) of 50 μl and a volume (B) of 150 μl can maintain at room temperature its appearance for several days. Aqueous drops can also be loaded with glycerol (used in enzymatic buffers).

 A tube prepared by the process according to the present invention can be frozen. The bubbles remain watertight by thawing. Several freeze-thaw operations do not modify, in any way, the device according to the invention.

 To ensure the tightness of the two compartments, a deposit blue diluted 1/100 in the upper compartment (B) was used.

 By way of example, the process according to the invention has been carried out on a 500 ml non-silicone Eppendorf tube. With such a tube, the volume of (B) for the upper bubble to be maintained must be at least 100 μl. This tube may however be modified, so that the minimum volume of the solution (B) is lowered to less than 50 1, as will be indicated below.

The invention will be better understood with the aid of the additional description which follows, given in a non-limiting manner, with reference to the accompanying drawings, in which:
FIG. 1a represents a tube prepared according to the present invention, before centrifugation, and FIG. 1b represents the same tube, after centrifugation,
FIG. 2 represents a tube according to the invention, modified so as to reduce the volume of the drop (B),
FIG. 3 illustrates, in the application of the process according to the invention to the production of a Nested PCR, the position of the primers.

There is shown schematically in Figure la a tube type Eppendorf tube, prepared by the process according to the invention, before centrifugation. The volume of the first reaction medium (droplet) was deposited at the bottom of the tube
A) and then covered the drop A with a mineral oil. This second reaction medium was deposited on this oil, which immediately forms an aqueous drop (drop B) which spreads and remains suspended in the oil. FIG. 1b shows the same tube after centrifugation. Drop B went down to the bottom of the tube and mixed with A.

In an exemplary embodiment, the process according to the invention was carried out on a non-silicone 500 micron Eppendorf microtube. The prepared tube had the following characteristics
drop A 50 til
drop B 100 pl
mineral oil 300 pl
dead space 50 1
total volume 500 p1.

With such a tube, the volume of the drop A is generally between 20 and 75 μl and that of gout
B between 100 and 200 p1.

 The good performance of the upper drop depends on the inside diameter of the tube. It is possible to reduce this diameter by introducing a ring into the tube, as shown in FIG. 2. Thus, if a ring is introduced into the tube that meets the above characteristics and if this ring decreases the internal diameter of the tube by 25%, a drop of 50 μm is then maintained indefinitely above the ring.

 The process according to the invention is particularly suitable for carrying out reactions having as substrate an amplification product.

 An example of implementation of the process according to the invention, applied to the production of a Nested PCR, will be given below.

Example
Two different Nested PCRs are made using the primers, cd / ab and cd '/ ab' (FIG. 3).

Equipment
Nested PCR is performed with primers amplifying DNA sequences of hepatitis B.

The starting DNA is that extracted from a reference serum of a patient in which the viral sequences can be detected by a filtration-hybridization (spot-test) method.

Method
The DNA to be tested is mixed with a PCR mix containing dNTPs (200 shots), a PCR buffer (MgCl2 = 1.5 mM), primers c and d and 2.5 units of TAQ polymerase enzyme. The total volume (DNA + mix) is 50 μl. The amount of primers c and d is 5 pmol. The reaction medium (A) is covered with mineral oil and an upper compartment (B) is created whose volume is 150 μl. The buffer and dNTP concentrations are identical to the previous ones. The amount of primers a and b is 80 pmol. This compartment, however, does not contain TAQ polymerase enzyme.

Realization of the PCR
After 25 cycles in a "thermal-cycler" during which the target was amplified by primers c and d in compartment (A), centrifugation at 5000 g for 5 min at 10 ° C caused the fusion of (A) and (B).

A factor of 4 dilution of the amount of DNA already amplified is obtained. The concentration ratio in internal primers / external primers in phase A + B is lower than that proposed in the various Nested PCR protocols already published (80/5 = 16). The other parameters of the PCR are otherwise equivalent. 35 cycles at 58 ° C are then programmed during which the internal fragment will be preferentially amplified due to the respective concentrations of the primers and the use of short elongation times (30 s).

 Results of the Nested PCR using the process according to the invention.

 The method using the process according to the invention, made on a series of 8 dilutions of 10 to 10, shows a band visible on gel until the 6th dilution (10-6) corresponding to about ten initial copies (well 2 at 8). There is no amplification in the next dilution (9) theoretically containing either a copy, or zero, or in the water control containing no DNA (10). the presence of PCR inhibitor is observed on the undiluted serum extraction.

 With the process according to the invention, the problem of contamination of Nested PCR is reduced to that of a PCR in a single step.

 An interpretation of the quantitative aspect of the signals can then be made. A net decrease of the signals is observed according to the increasing dilutions of the initial targets. A band with a size of 210 bp is visible in the low dilutions. It corresponds to an amplification of primer a and primer d. Indeed, 4 different products can theoretically be formed (c + d; a + b; c + b; a + d) in a Nested PCR. In this reaction, the occurrence of one or more bands depends on the initial amount of target and the elongation times programmed. The probability of formation of these "parasitic" amplification products is even greater than the initial amount of target is important.

 Composition with a conventional single-stage PCR using primers c and d.

PCR was run for 35 cycles (30 sec at 94 ° C, 1 min at 58 ° C, 1 min at 74 ° C) using primers c and d in a volume of 50 1 and with 1 U of TAQ Pol Cetus. on a 5 μl aliquot of the dilutions 0, -1 and -2 used previously, a sensitivity gain is obtained for the 4
Nested PCR of 104 versus PCR in a single step.

 Results of the conventional Nested PCR.

 Nested PCR was performed on the same material with different primers (c, a, b 'and d'), but using the conventional procedure. Between the two PCRs, the tubes of the first PCR were opened in descending order of dilution (from 0 to 8). It is noted that, despite the use of the anti-contamination protocol and positive displacement pipettes, the second PCR was contaminated by the first.

 We find the presence of a minority band at 340 corresponding to the amplification a-d 'for the same low dilutions (0, -1, -2 and -3).

 The activity of TAO polymerase Cetus after 25 amplification cycles (time at 94 ° C = 17 min 30).

In order to estimate the activity of the TAQ polymerase at the beginning of the second amplification, a Nested
PCR, using the method according to the invention and cd '/ ab' primers on already tested dilutions of a target DNA. This DNA can be amplified only by the internal primers. Before the first PCR, the concentration of the enzyme is 2.5 U / 50 μl. After centrifugation, the amplification of the internal fragment can then be obtained from the internal primers. The performance of PCR performed under these conditions is equivalent to that obtained with "fresh enzyme" and under normal conditions with visible band up to dilution 10 in both cases.

 Demonstration of the mutual impermeability of the two buffers A and B.

 For the process according to the invention can be usefully implemented, it is essential that the two compartments are mutually sealed.

 To ensure the tightness of the two compartments (A) and (B), it can not be appealing to the use of dyes is a technique too imprecise. We proceeded as follows. A positive control diluent for the HIV amplification was placed in (B) and (A) a PCR buffer comprising the pol3 and TAQ polymerase primers. After PCR and 24 hours of autoradiography, there is no pol3 amplification in the tube.

 The process according to the invention can receive many other applications, in particular in the field of PCR or other techniques derived therefrom. As an indication, the following table summarizes some of the possible applications of the process according to the invention.

Among these applications, mention may be made more particularly
PCR and one-step probe hybridization.

 The mix is a PCR containing the TAQ pol enzyme, the primers, and the DNA to be analyzed. After PCR, the bubble A containing a hybridization buffer and a specific probe of the amplification product is mixed by centrifugation with the PCR made in B. The different hybridization times adapted to the probe used are carried out in a "fhermal- cycler "can accept the tube in its entirety.

The hybridization product can then be deposited on a gel or be filtered in the case of a spot.

 It is also possible to envisage the use of a spectroscopically modifying oligonucleotide in case of hybridization. Such an oligonucleotide could be used either as internal primers of a Nested PCR or as hybridization probe. By using a system that analyzes the hybridization through the tube, PCR is performed without opening the tubes, thereby allowing to solve the problem of contaminations.

 Direct PCR analysis on biological sample.

Techniques for the rapid extraction of DNA or serum or cellular RNA using "lysis buffers" have been proposed. These make it possible to avoid a conventional phenol-chloroform extraction and to treat the sample so that it can be directly amplifiable. These techniques use a proteolytic enzyme and a detergent compatible with PCR (NP40,
Triton). In place of the detergent, physical procedures (heating, sonication) can also be used. Buffer A is a cell or serum lysis buffer using Proteinase K. The buffer is a PCR mix with 2.5 U of the TAQ enzyme and primers. After action of sonication of the tube and Proteinase K for 1 hour, inactivation of Proteinase K for 10 min at 95 ° C, centrifugation is done followed by a PCR adapted to the primers used.

 Realization of RT - PCR in a single tube.

Using a reverse transcription (RT) and TAQ Polymerase compatible A buffer, RT-PCR is performed in one of the two RNA-containing buffers. Solution B comprises internal primers to those used in A. A Nested PCR is then prepared after centrifugation. In this way, a RT-Nested is made
PCR in a single tube.

 Realization of two simultaneous PCRs.

 Internal control of contamination.

 Standard PCR is made in buffer B with the DNA to be analyzed. In buffer A, a PCR is made with the mix used for the PCR carried out in B with enzyme, but without DNA and with primers internal to the previous ones. Centrifugation is done after PCR. Thus, if a small amplification appears, it reflects a contamination occurred in the mix or the tube. This contamination is either the entire target, the amplification product used in the diagnosis, or the small amplification product used as a control. This control, if it is positive, reflects a contamination. This process is equivalent to a tube test.

 PCR binding.

 Various methods for synthesizing an amplification product from two other amplification products have been described. Both initial amplification products have one of their common terminal sequences and can themselves serve as primers for PCR. These techniques are used to make constructs that are then subcloned and used for mutagenesis experiments or to obtain longer fragments for sequencing.

 In situ PCR.

 This is a newly developed technique, published in July 1990 in "Amplification and detection of lentiviral DNA internal cells, Haase RT et al., Proc.

Nat. Acad. Sci., July 1990, No. 87; 13; As a first step, cells are treated with formaldehyde, thus rendering them permeable to the different PCR reagents, an in situ PCR reaction is then performed using 2 pairs of specific target primers as shown. This choice of primers allows the PCR ligation of the different strands whose elongation is incomplete under the intracellular conditions (protein-DNA interaction) .The cells are then analyzed by in situ hybridization. this in situ PCR is estimated at 12% per cycle, a method 100 times more sensitive than in situ hybridization.

 A Nested PCR using the method according to the invention would allow to add a very important signal gain to the PCR in situ and thereby reduce the total number of cycles.

 Decontamination processes.

 Since the amplification product is by definition a source of contamination, two possibilities can be retained for eliminating these products. The first is to try to specifically destroy the amplification products possibly present in the tube before starting the PCR reaction. This approach is based on the biochemical differences between the native target and the amplification products: size, 5 'hydroxylated protruding ends or llbluntl', special nucleotides incorporated in the PCR reaction. The second is to make the amplification products immeasurable by a chemical treatment, after PCR, while being able to analyze the result of the reaction.

 In both cases, whether the decontamination is carried out by specific destruction of the amplification products before PCR, or by inactivation of the amplification products after PCR, the process according to the present invention will advantageously be used.

 The present invention also extends to kits or kits for diagnostic or research purposes, which make use of the method according to the invention.

 This allows for "integrated" kits in which two successive stages can be performed in the same tube (see Table).

The process according to the invention, in particular applied to DNA amplification techniques or RNA, has many advantages. I1 allows7 in particular
to shorten the operating times necessary for the PCR exploitation of a biological sample, by reducing the manipulations,
- to lower the cost of the analyzes, by reducing the number of tubes necessary for the execution of these analyzes,
- to adjust, when applied to the Nested PCR, the problem of contamination due to amplification products.
BOARD

<tb><SEP> Solution <SEP> A <SEP> Solution <SEP> B <SEP> Purpose
<tb><SEP> Hybridization Buffer <SEP>
<tb><SEP> with <SEP> probe <SEP> buffer <SEP> PCR <SEP> hybridize <SEP> the <SEP> product <SEP> PCR
<tb><SEP> molecular <SEP> without <SEP> open <SEP> the <SEP> tube
<tb><SEP> specific
<tb><SEP> Extraction Buffer <SEP> Avoid <SEP> Extrac <SEP> Step
<tb><SEP> (type <SEP> proteinase <SEP> tion <SEP> of <SEP> DNA <SEP> and <SEP> so
<tb><SEP> K <SEP> +) <SEP> + <SEP> sonication <SEP> buffer <SEP> PCR <SEP> of <SEP> contaminations
<tb><SEP> Inactivation <SEP> PK <SEP> Before
<tb><SEP> PCR <SEP> by <SEP> the <SEP> Heat
<tb><SEP> reverse
<tb><SEP> Transcription (<SEP> RT) <SEP> Buffer <SEP> PCR <SEP> RT <SEP> PCR
<tb> of <SEP> RNA <SEP> + <SEP> primers <SEP> + <SEP> primers <SEP> (PCR <SEP> done <SEP> on <SEP> a <SEP> cDNA)
<tb><SEP> (random <SEP> priming <SEP> or
<tb><SEP> specific <SEP> primers
<tb><SEP>5'aval)<SEP> + <SEP> Taq
<tb><SEP> RT <SEP> then <SEP> PCR <SEP> PCR <SEP> nested
<tb><SEP> (common <SEP> buffer) <SEP>(<SEP> + <SEP> primers <SEP> RT-Nested <SEP> PCR <SEP> on <SEP> ARN
internal <tb><SEP>
<tb><SEP> ligation <SEP> to <SEP> 10 <SEP>"C<SEP> with <SEP> PCR <SEP> using <SEP> a <SEP> primer
<tb><SEP><SEP> specific <SEP> linkers <SEP> and <SEP> the <SEP> strand <SEP> LM-PCR
<tb><SEP> antisense <SEP> of <SEP> linker <SEP> (Ligated <SEP> Mediated <SEP> PCR)
<tb><SEP> PCR <SEP> standard <SEP> controls <SEP> PCR <SEP> for <SEP><SEP> simultaneous presence <SEP> 2 <SEP> PCR <SEP><SEP> on
<SEQ ID>SEP>SEP> Exogenous <SEP><SEP> DNA <SEP> SEP <SEP> DNA <SEP> Amount
<tb> (type <SEP> HEA) <SEP> on <SEP> DNA <SEP> tested <SEP> internal <SEP> control
<tb><SEP> tested <SEP> for amplification
<tb><SEP> Buffer <SEP><SEP> processes from <SEP> deconta
<tb><SEP> PCR <SEP> no <SEP> contaminated <SEP> mination <SEP> from <SEP> 1 '<SEP> extrac- <SEP> PCR <SEP> from <SEP> DNA <SEP> decontaminated
<tb><SEP> tion <SEP> to <SEP> analyze <SEP> + <SEP> DNA <SEP> mined
<tb><SEP> PCR <SEP><SEP> PCR <SEP>b'-c<SEP> PCR <SEP> ac
<tb><SEP> (b '<SEP> complementary <SEP> of <SEP> b)
<Tb>

Claims (6)

CLAIMS.  1. A method for performing at least two successive steps, including at least two successive reactions occurring in an aqueous medium, required for a molecular biology experiment, the product of the first reaction serving as a substrate for the next reaction7 in the same tube, Eppendorf tube type, characterized in that it consists  depositing in the tube a volume of the first reaction medium (A)  to cover the medium (A) with an oil,  depositing the second reaction medium (B) directly on an oil,  - mixing, after carrying out the reaction in the medium (A) and / or (B) the media (A) and (B),  incubating in the A + B mixture a reaction with the substrate, the products of the preceding reaction (s),  the various operations being performed, without having to open said tube.  2. Method according to claim 1, characterized in that the first reaction medium is deposited at the bottom of the tube in the form of a drop (A), in that the drop (A) is covered with mineral oil, in that said second reaction medium (B) is deposited on said oil, the latter forming an aqueous drop (B) separated from (A) by an oil thickness, and in that, after reaction in (A) and / or (B), the drop (A) is lowered to the bottom of the tube, in particular by centrifugation, thus effecting the mixing of (A) and (B).  3. Method according to claim 1 or 2, applied to the realization of a Nested PCR, characterized in that the medium (A) comprises the DNA to be tested, nucleoside triphosphates, a PCR buffer, a polymerization enzyme and a pair of external primers, in that the medium (B) comprises a PCR buffer, nucleoside triphosphates and a pair of internal primers7 in that, after a first amplification carried out in (A), the media are mixed ( A) and (B) and that a second amplification is performed on the A + B mix.  4. Method according to claim 1, applied to the realization in a single step of a PCR and a hybridization by a probe, characterized in that the medium (B) comprises the DNA to be tested, a pair of primers and a polymerization enzyme while (A) comprises a hybridization buffer and a probe specific for the amplification product, in that, after carrying out the PCR in (B), (A) and (B) are mixed together and in that the results of the hybridization are analyzed.  5. Process according to claim 1, applied to a PCR carried out directly on a biological sample, characterized in that the medium (A) consists of a cell or serum lysis buffer using a proteolytic enzyme, in that the medium (B) is a PCR mix containing a polymerization enzyme and a pair of primers and in that, after inactivation of the enzyme, (A) and (B) are mixed and PCR is performed.  6. Tube, Eppendorf tube type, characterized in that it is obtained according to the method defined in one of claims 1 and 2.