WO2013113965A1 - Special polymerase chain reaction primers - Google Patents

Description

 SPECIAL PRIMERS FOR CHAIN REACTION OF THE

POLYMERASE

FIELD OF THE INVENTION

The present invention belongs to the field of molecular biology techniques, in particular to the field of polymerase chain reaction (PCR) technique. More specifically, it refers to primers specially designed for PCR for direct detection of amplified target sequences. This is achieved due to its special design and more specifically to the presence in the amplification products obtained, with the use of said primers, of single chain tails. These tails allow the capture of the amplification product to a functionalized substrate with capture probes and / or its simple detection with labeled reporter probes.

BACKGROUND OF THE INVENTION

The polymerase chain reaction (PCR) is a molecular biology technique to amplify one or more copies of a segment of DNA by several orders of magnitude, generating billions of copies of a particular DNA sequence.

Developed in 1983 by Kary Mullís, PCR is now a common and often indispensable technique, used in medical and biological research laboratories for a variety of applications. These include DNA cloning for sequencing, DNA-based phylogeny, or functional gene analysis; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic science and paternity tests) and the detection and diagnosis of infectious diseases.

The procedure is based on thermal cycles, which consist of repeated cycles of heating and cooling the reaction for DNA denaturation and enzymatic DNA processing. Primers (short DNA fragments), which contain sequences complementary to the target region, together with a DNA polymerase are key components to allow selective and repeated amplification. As the PCR progresses, the generated DNA itself will used as a template for replication, starting a chain reaction in which the DNA template is exponentially amplified. The PCR can be extensively modified to perform a wide variety of genetic manipulations. Almost all PCR applications employ a thermally stable DNA polymerase, such as Taq polymerase. This DNA polymerase enzymatically assembles a new DNA chain from building blocks of DNA, the nucleotides, using single-stranded DNA as a template and DNA primers, which are necessary for the initiation of DNA synthesis. The vast majority of PCR procedures use thermal cycles, that is, heating and alternating cooling of the sample of

PCR up to a series of defined temperature steps. These thermal cycle steps are necessary, first of all, to physically separate the two strands of a double DNA helix at an elevated temperature in a process called DNA denaturation. Then, at a lower temperature, by DNA polymerase each chain is used as a template in DNA synthesis to selectively amplify the target DNA. The selectivity of the PCR comes from the use of primers that are complementary to the region of DNA chosen for amplification under specific thermal cycle conditions.

Many variants of the PCR have been developed by making minor modifications to the standard PCR protocol, or by adjusting or varying different elements of the PCR such as, for example, the type of polymerase used.

In addition, different variants of the PCR result from the variation of the primers for amplification. For example, one of said known variants consists in the inclusion of tails at the 5 'end of the primer. Normally, the primers are completely complementary to the 3 'end of the target sequence.

However, polymerase is tolerant of fainting beyond the 3 'end. Tail primers include non-complementary sequences at their 5 'ends. A common procedure is to place restriction sites at the ends of these tails, facilitating their subsequent insertion into cloning vectors.

The presence of tails in the PCR amplification product can also be interesting since it allows direct capture by hybridization of the product to a substrate or surface that has complementary probes in it. It also allows the union of complementary probes marked for easy detection of the amplification product.

US 2004/0038194 describes a method for PCR amplification in which the primers contain a capture agent. The document mentions that, among others, the capture agent may be a sulfhydryl group, biotin, a cellulose binding domain or a specific nucleotide sequence. However, the document does not provide many indications on how a specific nucleotide sequence can be used as a capture agent. US 2003/0215926 provides a general idea of a method for obtaining double stranded DNA with single stranded tails comprising the use of hybrid primers formed by DNA at the 3 'end and by RNA at the 5' end. The 3 'DNA end is complementary to the target sequence and serves as a primer for amplification. Once the PCR has been carried out with these primers, a hybrid duplex of

RNA-DNA that is subsequently digested with RNase. This digestion results in a DNA duplex with a single chain tail at each end that can be detected immobilized with suitable probes. A similar approach is also described in US 2009/0203531. Hayashi G. et al. (Nucleic Acids Symposium Series, 2005, No. 49: 261-262;

ChemBio Chem, 2007, 8: 169-171; and Journal of Biotechnology (2008) 135: 157-160) follows a completely different approach that is based on the use of the special properties of L-DNA. Hayashi G. uses a D-DNA and L-DNA chimera as a primer. The primer comprises D-DNA at the 3 'end and L-DNA at the 5' end. During PCR, the polymerase stops its amplification at the boundary between D-DNA and L-DNA, leaving L-DNA in the form of single chains in the amplification product. The L-DNA can then be hybridized to complementary immobilized L-DNA probes.

The authors of the present invention provide specially designed primers, which contain different elements operatively linked that allow the generation of a double-stranded PCR amplification product with single-stranded tails at one or both ends of the duplex. BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Scheme depicting a possible design of the pair of primers according to the invention comprising both a fishing sequence and capture and reporter tails, respectively. Figure 2: Scheme of the proposed detection assay using specifically designed primer pairs and electrochemical and / or spectrophotometric detection.

Figure 3: A) Gel electrophoresis of the amplification of a positive sample with the special designed primers in which 1, 2, 3 and 4 are the product of the amplification of a positive sample using the pair of primers L1 and L2 and the

Control NT is the product of the amplification in the absence of the sample (mold). B) Gel electrophoresis of the extracted sample and its comparison with the Molecular Weight Marker 200. The amplified product has a length of 240 bp that is consistent with the expected. Figure 4: Proof of concept of the detection of dsDNAs, dt (double stranded and double tail DNA) by ELONA assay Control: Response associated with nonspecific adsorption of the enzyme reporter probe; Product extracted from the PCR: Response obtained for the ADNds, dt extracted from the PCR product using the PureLink Quick Gel Extraction Kit .; Product not extracted from the PCR: Response obtained for the same PCR product before DNA extraction, dt. PCR sample diluted to 1 in 50.

Figure 5: Scheme of one of the possible approaches contemplated for cleaning the sample by removing the excess primer without reacting using magnetic beads.

Figure 6: Proof of concept of the feasibility of primer removal by fishing with magnetic beads. Control: Response associated with nonspecific adsorption of the enzymatically labeled reporter probe; PCR + magnets: Response obtained using unpurified PCR product after contact with non-functionalized streptavidin magnetic beads; PCR without fishing: Response obtained in the presence of excess primers; PCR + fishing: Response obtained using the product of PCR after "primer fishing" by fishing for magnetic streptavidin beads functionalized with the probe (fishing done for 60 minutes). In both experiments a concentration of 3330 μςΛτιΙ of magnetic beads was used. DNA was isolated from other PCR components using ethanol precipitation. The PCR product was diluted to 1 in 10 before fishing; The sample was further diluted to 1 in 6 before detection.

Figure 7: Evaluation of the components of the PCR regarding the efficiency of the "fishing of the primer". Control: Response associated with nonspecific adsorption of the enzymatically labeled reporter probe; Purified PCR: Response obtained in the PCR product after purification with ethanol and before "primer fishing". Purified "fishing": Response obtained in the PCR product after purification with ethanol and "fishing of the primer". Unpurified PCR: Response obtained in the PCR product without ethanol purification and before "primer fishing"; "Purified" fishing ": Response obtained in the PCR product without ethanol purification and" fishing "of the primer. The concentration of magnetic beads used in these experiments was 3330 μg / ml and "primer fishing" was carried out for 60 minutes. The PCR product was diluted to 1 in 10 before fishing; The sample was further diluted to 1 in 6 before detection. Figure 8: Proof of concept of the electrochemical detection of the actual product of ADNds, dt of the PCR. The PCR was diluted to 1 in 6 before "fishing" with modified magnetic beads (200 μg ml).

Figure 9: Test of the specificity of the capture sequences on the surface. Figure 10: Multiplex amplification test using modified primers and direct electrochemical detection.

DETAILED DESCRIPTION OF THE INVENTION

For the sake of clarity, some terms used in the context of the invention are defined below: The term "Primer" is the element capable of binding to one end of the sequence that is intended to be amplified, and which serves as a starting point for The polymerase begins the amplification reaction. A primer with special characteristics is the main object of the invention. In this sense, in the context of the invention, the primer is not only the DNA fragment complementary to the target sequence and which allows the start of the PCR, but in its broadest sense it also refers to the primer of the invention especially designed and comprising an operatively linked access sequence, optionally a fishing sequence, a PCR amplification terminator and a reporter capture queue. The primer of the invention has multiple functions due to its special design. "PCR" means polymerase chain reaction.

"Operatively linked" means that the different elements that make up the special primer of the invention are linked in the 3'-5 'direction in the specific sequence claimed, namely, access sequence / fishing sequence / terminator of PCR amplification / queues, in order to be operational and perform its function correctly.

"Target sequence" is the sequence that is intended to be amplified within the sample.

"Access sequence" is an element of the primer of the invention. It is the primer sequence capable of recognizing the target sequence and acting as such a primer for the PCR reaction. Preferably it is a DNA sequence.

"Oligonucleotide" is a nucleotide sequence that has a variable length.

"Nucleotides" are the structural units that form DNA and RNA. These structural units are formed by a nitrogen base such as adenine, guanine, cytosine, uracil or thymine, a five-carbon monosaccharide

(ribose or 2'-deoxyribose), and a phosphate group.

"Fishing sequence" is another element of the primer of the invention. It is a short chain of oligonucleotides of at least two nucleotides included within the primer of the invention. It is intended to remove the excess primer (if necessary) from the reaction medium by hybridization to a fishing probe complementary to the fishing sequence. It is preferably a DNA sequence

"Substrate" represents any type of surface on which a probe can be immobilized, such as a fishing probe or a capture probe. For example, in the case of a fishing probe the substrate can be magnetic beads or any other similar element such as cylinders, particles, cables or metallic, inorganic or polymeric surfaces of different sizes and geometries. In the case of a capture probe, the substrate can be an ELISA plate or the surface of an electrode or any other surface on which direct detection can be carried out. "PCR amplification terminator" is another element of the primer of the invention. Its function is to block the amplification reaction. The polymerase stops the reaction when it reaches the terminator. The terminator can be an organic terminator such as hydrocarbon chains of between 3 and 18 carbon atoms, a chain of hexaethylene glycol, triethylene glycol, polyethylene glycol, propyl, 1 ', 2'-dideoxyribose or bases or unnatural nucleic acids that are not recognized by the polymerase such as PNA, LNA, morpholino oligos or RNA.

"Capture or capture tail" is another element of the primer of the invention. It is operatively linked at 5 'to the PCR terminator and after the PCR reaction remains a single nucleic acid chain that is capable of hybridizing to an immobilized capture probe. It can be part of the direct or reverse primer within the pair of primers used in the PCR reaction.

"Reporting queue" is an element of the primer of the invention analogous to the capture tail. Like the capture tail, it is operatively linked to the PCR terminator and after the PCR reaction remains a single nucleic acid chain that is capable of hybridizing to a reporter probe. It can be part of the direct or reverse primer within the pair of primers used in the PCR reaction.

"Amplification product" is the product obtained in the PCR reaction using the primer or primer pair of the invention. It consists of a double-stranded DNA duplex with a single chain tail at one end (amplification product of ADNds, st) in case only a single one is used primer of the invention in the PCR reaction, or of a double-stranded DNA duplex with a single-stranded tail at both ends (dsDNA amplification product, dt) in the case where two primers of the invention are used . "Nucleic acid" includes both natural nucleic acids, such as DNA or

RNA, such as artificial nucleic acids such as PNA, GNA, LNA, BNA, TNA or Morpholino nucleic acids.

"Direct primer" is the primer that extends in the PCR from the start codon to the stop codon of the template DNA. "Reverse primer" is the primer that extends from the stop codon to the start codon of the template DNA.

"Fishing probe" is a nucleic acid probe designed to be complementary to the fishing sequence of the primer of the invention. It is envisioned that it hybridizes to the fishing sequence in order to separate the unreacted excess primer from the amplification product.

"Capture or capture probe" is a nucleic acid probe designed to be complementary to the capture tail of the primer of the invention. It is intended to hybridize to the capture tail in order to immobilize (capture) the amplification product to a substrate. "Reporter probe" is a nucleic acid probe designed to be complementary to the reporter tail of the primer of the invention. It is planned to hybridize to the queue. The reporting probe is labeled with any means that provides a direct or indirectly detectable signal through an intermediate reaction. The possible mapping of the reporter probe includes, but is not limited to, redox electrochemical molecules, enzymes capable of producing a detectable reaction, fluorophores, molecules capable of producing electrochemiluminescence, color changes, chemiluminescence or fluorescence, liposomes, metal particles / cylinders , inorganic particles / cylinders, polymer particles / cylinders, magnetic particles / cylinders, quantum dots, dendrimers, radioactive markers.

A first aspect of the invention relates to a primer for amplification. by PCR comprising operatively linked in the 3'-5 'direction: a. An access sequence consisting of a 10-35 nucleotide oligonucleotide capable of specifically hybridizing to the 3 'end of the sequence to be amplified (target sequence), b. Optionally, a fishing sequence for excess primer removal after the PCR reaction consisting of at least 2 nucleotides, the sequence that is designed to hybridize to a probe (fishing probe), optionally immobilized to a substrate and which does not interact with the target sequence, c. A terminator of PCR amplification, d. A capture or reporter queue for hybridization of the resulting amplification product to an optionally immobilized probe on a substrate or a labeled probe, respectively (capture probe and reporter probe, respectively), consisting of a nucleic acid with a sequence with a length of at least two bases designed for hybridization to said optionally immobilized probe on a substrate or said labeled probe.

The primer of the invention is specially designed such that, once used in a PCR amplification of a target sequence, a DNA duplex is obtained as an amplification product comprising a single chain tail (in the case where use in the reaction a single primer according to the invention) or two single chain tails at each end of the duplex (in the case where a pair of primers comprising two primers according to the invention is used). The presence of these tails allows the capture of the PCR product on a substrate such as an ELISA plate, a biochip or an electrode functionalized with homologous sequences (reverse and complementary) to one of the tails (capture tail), and / or the marking of said DNA duplex by hybridization of a labeled homologous probe (reverse and complementary) to the second tail (reporter tail). The presence of the capture tails has the advantage that it allows direct capture by hybridization of the amplification product without the need for any modification and / or functionalization. Similarly, the reporting queue allows detection Direct amplification product without complicated sample processing.

The primer of the invention comprises, operatively linked in the 3'-5 'direction, an access sequence, a fishing sequence, a PCR amplification terminator and a capture / reporter tail depending on whether the tail is intended for be captured by hybridization to a substrate or if it will be used to report the resulting product.

The access sequence is the part of the primer capable of recognizing and hybridizing to the 3 'end of the sequence that is intended to be amplified, that is, the target sequence. The access sequence is the element where polymerase begins amplification using the target sequence as a template. It is the active part of the primer as such. The access sequence is a DNA oligonucleotide of 10 to 35 nucleotides.

The fishing sequence is a predetermined sequence of at least 2 nucleotides, and preferably of not less than 15 nucleotides, which has the function of allowing the removal of the unreacted primer from the reaction medium after PCR amplification has taken place. . The presence of excess primer in the amplification product may interfere with the result of the detection step as demonstrated in the examples (see Figure 4). Thus, it may be desirable to remove it before carrying out the detection. Since the fishing sequence is also amplified in the PCR reaction, it is easy to remove non-amplified primers (i.e., excess primers) and separate them from the amplification product in the reaction medium by hybridization to a fishing probe that it can be immobilized on a substrate such as, for example, magnetic beads. Any other means commonly used by the person skilled in the art can be used for the fishing stage. The fishing sequence is an optional sequence within the primers of the invention, but in preferred embodiments the fishing sequence is present. In a particular embodiment, the fishing sequence is a DNA sequence.

The PCR terminator is an element capable of stopping polymerase activity. The terminator also acts as a spacer between part of the primer that is subjected to amplification and its tail. In the context of the Any invention capable of stopping the PCR reaction can be used, such as, for example, hexaethylene glycol, triethylene glycol, propyl, 1 ', 2'-dideoxyribose or bases or unnatural nucleic acids that are not recognized by polymerase, such such as PNA, LNA, Morfolino oligos or RNA. In a preferred embodiment, the PCR terminator comprises hydrocarbon chains of 3 to 18 carbon atoms (C ₃ -Ci ₈ ).

The final part of the primer of the invention is the tail that, after amplification, remains a single chain at the end of the DNA duplex resulting from the PCR reaction. The queue can be a capture queue or an information queue depending on whether it is destined for immobilization.

(capture) of the PCR amplification product to a substrate such as an ELISA plate, a biochip or an electrode or if it is intended to be attached to a homologous reporter probe for detection. The capture and reporter tails can be formed from any nucleic acid whether they are naturally occurring nucleic acids such as DNA or RNA or artificial nucleic acids such as PNA, GNA, LNA, BNA, TNA or Morpholino nucleic acids.

The primer of the invention may be designed as a direct or reverse primer. It can be used in a PCR reaction as a single primer to be used in combination with a normal primer or it can be used as a pair of primers comprising two primers of the invention.

This is, in fact, the following aspect of the invention, namely, a pair of primers for PCR amplification consisting of a direct primer and a reverse primer wherein one or both are primers according to the invention.

In a particular embodiment of the invention, the direct primer is a primer according to the present invention and the reverse primer is a normal primer.

In another particular embodiment, the reverse primer is a primer according to the invention and the direct primer is a normal primer.

In another additional and preferred particular embodiment, both the direct and reverse primer for PCR amplification are primers according to the invention. In this embodiment, the direct primer comprises a capture tail and the reverse primer comprises a reporter queue, or vice versa, the direct primer comprises a reporter queue and the reverse primer It comprises a capture queue.

Another aspect of the invention is the use of the primer or pair of primers according to the invention. The primer and / or primer pair of the invention are used for PCR amplification reactions. In a particular embodiment, after the use of the primers in the PCR, the excess of unreacted primers is removed and separated from the amplification product by hybridization to a fishing probe, optionally immobilized to a substrate such as magnetic beads, which comprises a sequence capable of hybridizing specifically to the fishing sequence. A further aspect of the invention is a PCR amplification product that can be obtained by using at least one primer according to the invention. The PCR amplification product comprises a DNA duplex with a single chain tail corresponding to the capture or reporter tail present in the primer according to the invention. Another related aspect of the invention is an amplification product of the

PCR that can be obtained by using a pair of primers according to the invention. The preferred embodiment of the invention relates to the PCR amplification product obtained by using a pair of primers composed of two primers according to the invention, this product comprising a DNA duplex with two single stranded tails, one at each end of the duplex, corresponding to a capture and reporting queue.

A further aspect of the invention is a group of two or more primer pairs according to the invention for a multiplex PCR. In a particular embodiment, in the group of primer pairs, the capture tails of each of the primer pairs are different from each other and are designed to be individual for each access sequence, and the reporting queues of each of the primer pairs are designed to be common with each other or individual for each access sequence.

A final aspect of the invention relates to a method for detecting the presence of a target sequence in a biological sample comprising: a) preparing capture probes capable of hybridizing to the tail of capture of the primer according to the invention or of the pair of primers according to the invention, and / or reporter probes capable of hybridizing to the primer tail of the primer according to the invention or of the pair of primers according to the invention, b) performing a PCR amplification with the less a primer according to the invention or with a pair of primers according to the invention to obtain an amplification product, c) optionally removing the excess primer that remains after the PCR amplification of step b) by hybridization to a fishing probe , optionally immobilized to a substrate, comprising a sequence capable of hybridizing specifically to the fishing sequence, d) allowing hybridization between the capture probes and / or the reporting probes of step a) with the capture tails and / or the reporting tails of the amplification product, e) detecting the presence of hybridization between the capture probes and / or the reporting probes of step a) with the c capture waves and / or the reporting tails of the amplification product, where the presence of said hybridization implies the presence of the target sequence in the sample and non-hybridization implies the absence of said target sequence in the sample.

The process of the invention can be implemented as a continuous amplification and detection process in a system comprising unit operations of PCR amplification, separation / removal of excess primers by hybridization of fishing sequences with immobilized fishing probes and detection by hybridizing capture tails to sensors / biochips / substrates functionalized with capture probes and reporting queues to marked report probes.

In a particular embodiment, the detection method of the invention comprises capture probes immobilized to a substrate that includes microtiter plates, glass / polymer / silicon substrates, biochips or electrodes. In another particular embodiment, the detection method of the invention comprises a reporter probe with a marker that includes redox electrochemical molecules, enzymes capable of producing a detectable reaction, fluorophores, molecules capable of producing electrochemiluminescence, chemiluminescence or fluorescence, liposomes, particles. metallic, inorganic particles, quantum dots, dendrimers or radioactive markers.

In the process of the invention, the nature of the capture and reporter probes may be that of a natural or synthetic nucleic acid such as DNA, RNA, PNA, GNA, LNA, BNA, TNA or Morpholino nucleic acids and mixtures of these acids. nucleic

In the process of the invention, hybridization detection is carried out by any means suitable for detection, such as, for example, electrochemical, optical, gravimetric, thermometric, magnetic, micromechanical, electrophoresis and affinity chromatography methods, among others. .

A particular embodiment of the process of the invention comprises a multiplex PCR with a group of primers or primer pairs according to the invention. The following examples are intended to illustrate the invention, but are not intended to limit it.

EXAMPLES

1. The objectives:

The objective of this work is the development of a pair of primers designed specifically that will allow the generation of ADNds that carry, at each end, a tail of ADNss (ADNds, dt) as shown in Figure 1.

These tails will allow the capture / detection of ADNds, dt according to the desired application. In this proof of concept work the ADNss tails will be used to capture the ADNds product, dt on ELISA plates and / or electrodes, followed by the introduction of an informative marker (in this specific case a DNA sequence complementary to that of the queues of ADNss attached to a enzyme marker) and its detection; The complete test can be summarized as seen in Figure 2. The fact that ELISA and electrochemical detections have been used in this proof of concept work does not limit the applicability of the proposed amplification procedure to these detection approaches, as will be evident. For an expert person. This could be coupled to any type of detection platform that involves the capture of the DNA amplicon on the surface by hybridization to a probe immobilized on a surface and, eventually, the marking of the amplicon as for example; resonance detection of surface plasmons, detection based on fluorescence, detection based on electrochemiluminescence or luminescence, DNA biochips, etc.

2. Experimental results

2. 1 Amplification: Description of the pair of primers

In the amplification stage the following pair of primers has been used:

Direct primer (L1): 5'-surface capture tail / C8 / Fishing sequence (18 bases in length) -Catch (dtq-3-1) (21 bases in length) 3 ':

5 ^' -GTTTTCCCAGTCACGAC-spacer C8-CAGGAAACAGCTATGACC-CGT GCGTCTCGTGAGCAGAAG-3 ^' (SEQ ID NO 1)

Special reverse primer (L2): 5'-Reporting queue / C8 / Primer dtq-3-2 (19 bases in length) -3 ':

5 ^' -TGTAAA ACGACGGCCAGT-spacer C8-TGCAAGGTCGTGCGGAGCT-3 ^' (SEQ ID NO 2) In which:

- the surface capture tail and the reporter tail are the sequences for the two DNA tails used to facilitate the capture on the surface of the dsDNA, dt and the capture of the reporter marker (in this specific example, an enzyme) respectively. - C8 is an internal modification that contains eight groups (CH ₂ ) that has the function of stopping the polymerase reaction.

- The fishing sequence is the sequence used to capture the direct unreacted primers. The absence of a similar sequence in the reverse primer is due to the fact that, for the specific application proposed in this proof of concept work, the reverse primer did not generate any interference.

The pair of primers used in this proof of concept work was designed to amplify the DQB1 ^* 02:01 - ^* 02:06 alleles (list of alleles based on publication 3.3.0 of the IMGT sequence database / HLA) of the MHC region of chromosome 6 involved in the predisposition to celiac disease.

Amplification protocol

The PCR was carried out using a commercial thermocycler (iCycler, Biorad) using a 3 temperature protocol and a temperature rise of 1 ^qC / second:

- 96 ^< 2 min

- 10 x 96 ^< 15 seconds, 65 ° C 60 seconds

- 20 x 96 ^< 15 seconds, 61 ° C 50 seconds, 72 ^< C 30 seconds

- - \ 0 ° C 10 minutes.

The amplification of a positive sample (containing the DQB1 ^* 02:01 - ^* 02:06), using the pair of primers designed specifically, was carried out according to the PCR protocol mentioned above and using the following mixture composition of the PCR - Primer -L1 400 nM

- Primer -L2 400 nM

- "Ready PCR" (from Innotrain) 30 μΙ

- Axi Taq (from Innotrain) 0.8 μΙ - Sample 25-100 ng / well

- ddH ₂ 0 to 100 μΙ

The effectiveness of the amplification was checked using gel electrophoresis. The gel electrophoresis of Figure 3A clearly indicated that the presence of the PCR terminator and the different tails did not prevent amplification from occurring. In addition, the fact that, in the case of the NT control, no amplification was recorded, also demonstrated that the new set of primers maintained its specificity.

The expected size of the PCR was 240 bp; In order to confirm the identity of the amplicon, it was extracted from the electrophoresis gel using the "PureLink Quick Gel Extraction Kit" (Invitrogens). The gel depicted in Figure 3B clearly shows that the sizes of the amplicons extracted were consistent with those expected.

2.2 Use of ADNds, dt in the ELONA test

In order to confirm the presence of the two tails in the amplification product obtained and to simulate the expected detection approach (Figure 2) some PCR products were used before and after extraction (as in section 2.2) to Perform an "Enzyme-linked oligonucleotide assay" (ELONA).

Preparation of ELONA plates:

Maleimide modified ELISA plates were modified with a short chain of 17 base oligonucleotides (capture probe) complementary and inverse to the surface capture tail and modified at its 3 'end with a TEG-SH group that allows its immobilization on the ELONA plates.

The reporting sequence for the final sandwich assay consisted of a complementary and inverse 18 base oligonucleotide short chain (reporter probe) of the marker capture tail and modified at its 3 'end with a horseradish peroxidase enzyme (HRP) to allow its colorimetric detection.

Functionalization of the plate and ELONA test: one . Immobilization of the DNA probe: the capture probe was immobilized at 37 ° C for 2 hours in a 250 nM solution in 100 mM Na ₂ HP0 ₄ , 150 mM NaCI and 10 mM EDTA at pH 7.2.

2. The wells were washed 3 times with ultra pure water to remove excess probes.

3. Blocking: 10 mM mercaptohexanol (MCH) in water was added to each well and incubated at 37 ° C for 1 hour.

4. The wells were washed 3 times with ultra pure water.

5. Hybridization: The dsDNA targets, dt (PCR sample diluted to 1 in 50) in 10 mM Tris + 1 M NaCl (pH 7.4) were incubated for 1 hour at 37 ° C.

6. The wells were washed 3 times with PBS Tween buffer (Sigma Aldrich) to remove excess target DNA.

7. Sandwich hybridization: The enzyme-labeled reporter sequence (reporter probe) was added to each well and incubated for 1 hour at 37 ° C.

8. The wells were washed 3 times with PBS Tween buffer to remove excess enzyme labeled reporter probe.

9. 100 µΙ of TMB ELISA substrate was added to each well and allowed to react for 15 minutes.

10. Next, 50 μΙ of 1 M H ₂ S0 ₄ was added to each well to stop the enzymatic reaction.

eleven . The signal was measured by reading the absorbance of the solution in each well at 420 nm using a commercial plate reader.

The results obtained for this group of experiments are presented in Figure 4. From Figure 4 it is clear that the amplification product obtained (ADNds, dt) using the proposed pair of primers contains the two tails of ADNss that allow its specific capture on the surface and the marking that facilitates its colorimetric detection (ELONA test) . The specificity of the Detection is confirmed by the intensity recorded for the control experiment (approximately 10 times lower than the specific response). In the specific example used as a preliminary concept test, the high concentration of primers used, together with the low amplification efficiency, produced a PCR product with a large number of unreacted primers that appear to compete with the tail during the test, making it necessary to eliminate these primers in excess before the detection of ADNds, dt.

However, in a more efficient amplification (see section 2.5) there was no need to remove unreacted primers. 2.3 Elimination of unreacted primers (primer fishing) using magnetic beads (optimal)

As an example of a scenario in which it is necessary to eliminate the excess of unreacted primers, an amplification was carried out, according to a non-optimized protocol and in the presence of a high concentration of primers. Subsequently, a protocol for the elimination of excess primers present in the PCR product was designed and carried out by capturing the excess of unreacted primers by hybridization to probes (fishing probes) immobilized on magnetic beads modified with streptavidin . In order to achieve this, a fishing sequence was incorporated into the primers; the capture probes immobilized on the magnetic beads were short-chain oligonucleotides (18 bases) inverse and complementary to the L1 fishing sequence segment. It is important to indicate that in ADNds, dt, this sequence is part of the sequence that is part of the duplex, and therefore is not viable for hybridization to probes immobilized on streptavidin coated magnetic beads. We refer to this elimination of excess unreacted primers as "primer fishing".

In Figure 5, the proposed approach for purification of the PCR product by primer fishing is depicted. The pearls used in this work were the Myone Dynabeads (Invitrogen), which were functionalized with the fishing sequence according to the following protocol:

one . Preparation of the pearls: 50 μΙ of pearls were washed (starting concentration: 10 mg / ml) three times with ultra pure water.

2. Pearl modification: 100 μΙ of biotin modified fishing probe (1 μΜ in 10 mM Tris pH 7.4 + 1 M NaCI + 1 mM EDTA) was added to the magnetic beads and incubated for 30 minutes with stirring soft.

3. The magnetic beads were collected by a magnetic field and washed 3 times with 10 mM Tris pH 7.4 + 1 M NaCl + 1 mM EDTA.

The removal of excess unreacted primers from the PCR product was carried out according to the following protocol:

one . Fishing: The oligonucleotide modified beads were mixed with 150 μΙ of PCR product (approximately 100 nM) and incubated for 1 hour with gentle agitation.

2. PCR product collection: Pearls with excess of unreacted primers captured were separated using a magnet and the supernatant PCR product was collected and used for analysis.

In this group of experiments, a second control experiment was implemented, together with the evaluation of the nonspecific adsorption of the enzyme-labeled reporter sequence, which consisted in the repetition of the fishing protocol but with the use of unmodified magnetic beads.

The effect of primer fishing on the ELONA detection of the ADNds PCR product, dt, was evaluated according to the ELONA assay protocol described above (Figure 6). As can be seen in Figure 6, "primer fishing" effectively reduced interference from unreacted primers, which resulted in a significant improvement in the detection of dsDNAs, dt (PCR + fishing vs. PCR without fishing).

In order to evaluate the effectiveness of the "fishing protocol" described above in cleaning the actual PCR product, the "fishing of primers" was carried out on untreated aliquots and ethanol precipitates of the same PCR product and ELONA results were compared. As can be seen in Figure 7, the response obtained with the PCR product and the ethanol precipitated samples were very similar, demonstrating that the components of the PCR product, for example, Taq, dNTPS, etc. they do not interfere with "primer fishing" using a probe functionalized with streptavidin beads.

2.4- Proof of concept using electrochemical detection

In order to demonstrate the flexibility of the proposed amplification protocol with different detection strategies, direct detection of the dsDNAs was carried out, using an electrochemical process instead of optical transduction. An array of electrodes was cleaned and functionalized with thiolated DNA probes. The probes immobilized on the surface and the reporting probes were analogous to those previously described for the ELONA experiments.

The dsDNA (fish using 200 μg / ml of oligonucleotide modified magnetic beads) was diluted to 1 in 6 with PBS Tween, contacted with electrodes and allowed to hybridize for 90 minutes, and then washed with PBS Tween . Subsequently, the electrodes were contacted with a 10 nM solution of the enzyme labeled reporter sequence and allowed to hybridize for 1 hour, and washed again.

Finally, the TMB substrate was added and allowed to react for approximately 10 seconds, and the response was measured using a rapid pulse ammeter (Figure 8), and as the dsDNA can be observed, dt was easily detected using electrochemical transduction.

2.5- Multiplex amplification using modified primers and detection

Four different primer pairs were designed (three pairs for the identification of the desired alleles - the DQ region of chromosome 6 and a fourth pair for internal control) according to the sequence specific primer (CES) approach.

Pair of primers 3b. (specific for DQB1 ^* 02:01, ^* 02:02, ^* 02:04, ^* 02:05, ^* 02:06) 5'-GTTTTCCCAGTCACGAC-espac \ ador C3-CGTGCGTCTCGTGAGCAGAAG-3 ' Direct (dtq-3-1) (SEQ ID NO 3)

5'-TGTAAAACGACGGCCAGT-spacer C3- GGCTGTTCCAGTACTCGGCGG-3 'Inverse (dtq-4-2) (SEQ ID NO 4)

Specific probe 3b. 5'-GTCGTGACTGGGAAAAC-TEG-SH 3 '(SEQ ID NO 5)

Pair of primers 4y. (specific for: DQB1 ^* 02:01, ^* 02:02, ^* 02:04, ^* 02:05, ^* 02:06, ^* 03:02, ^* 03:07, ^* 03:08, ^* 03: 1 1 , ^* 03:18, ^* 03:32, ^* 06:29)

5'-AGCGGATAACAATTTCACACAGGA-espac \ ador C3- AACGGGACGGAGCGCG TGCG TCT-3 'Direct (dtq-4-3) (SEQ ID NO 6) 5'-TGTAAAACGACGGCCAGT-spacer C3-

GGCTGTTCCAGTACTCGGCGG-3 'Inverse (dtq-4-2) (SEQ ID NO 7)

Specific probe 4y.

5'-TCCTGTGTGAAATTGTTATCCGCT-TEG-SH 3 '(SEQ ID NO 8)

Pair of 5z primers: (specific for: DQA1 ^* 05:01, ^* 05:05, ^* 05:08, ^* 05:09) 5'-TGTAAAACGACGGCCAGT-spacer C3-CACTGGGTCAGCCCAACAT-3 '

Direct (qa-5-3) (SEQ ID NO 9)

5'-GTGTCCGACTTATGCCC-espac \ ador C3- TTGCAGTCATAACTCTCCTCAGC-3 'Inverse (qa-5-4) (SEQ ID NO 10)

Specific probe 5z: 5 'GGGCATAAGTCGGACAC-TEG-SH 3' (SEQ ID NO 1 1)

Pair of primers HGH. (internal control)

5'-TGTAAAACGACGGCCAGT-spacer C3-GGGAGAGGCAGCGACCTGTA- 3 'Direct (HGH-F2) (SEQ ID NO 12)

5'-AGGCAGAA TCGACTCACCGCTA-espac \ ador C3- GGAGAGCAAGAGGCCAGCAC-3 'Inverse (HGH-R2) (SEQ ID NO 13)

HGH specific probe. 5 'TAGCGGTGAGTCGATTCTGCC-TEG-SH 3' (SEQ ID NO 14) Generic marking element:

5 'ACTGGCCGTCGTTTTACA - HRP 3' (SEQ ID NO 15) Where:

The generic tide tail is the tail of ADNss used for hybridization to the reporter sequence labeled with enzyme and common to all primer pairs.

The sequences, specific for each amplicon, for the capture on the surface of the products of ADNds, dt are italicized.

In this evaluation, the amplification of the different samples was carried out according to an optimized thermocycling protocol.

- Initial denaturation: 1 minute at 96 ° C

35 cycles

- Denaturation: 15 seconds at 96 ° C

- Hybridization + Elongation: 50 seconds at 61 ° C.

The concentrations of the primers and other components of the amplification mixture are listed in the following table.

Oligo Final concc in PCR ^* dtq-3-1 1000 nM dtq-4-2 1000 nM dtq-4-3 300 nM qa-5-3 400 nM qa-5-4 400 nM

HGH-F2 150 nM

HGH-R2 150 nM Volume / concentration component

amplification used ddH ₂ 0 Add up to 10 μΙ

3x3 "ready PCR" 3 μΙ

 (Buffer for

 INNO-TRAIN PCR)

DNA mold 25-100 ng / well

AxiTag (Taq- 0.08 μΙ

 polymerase

 INNO-TRAIN)

Electrochemical detection was performed in a similar way to the methodology presented in the previous section, where the short working oligonucleotides, complementary and inverse to the four different capture tails, were immobilized on the electrode surface of an electrode array.

First, a demonstration of the specificity of the capture tails on a surface designed with the electrochemical detection of the dsDNA products was carried out, amplifying a totally positive sample with 4 different primer pairs in a uniplex format ( Figure 9).

Finally, the possibility of obtaining and detecting the different dsDNA products, necessary in the detection format proposed in a multiplex format with the amplification and typing of a series of real samples and the comparison of typing results with those obtained using the conventional typing technique (Figure 10).

The following table shows the results of electrochemical typing using the proposed approach and its comparison with the results obtained in the conventional typing approach.

Sample TYPICAL ELECTROCHEMICAL TYPES OF REFERENCE

 3b 4y 5z HGH 3b 4y 5z HGH

8087 +/- + + + + + + +

8092 ++

FRCBS 25 + +

 FRCBS 12 + + + + + +

FRCBS 20 + + + + + + + +

FRCBS31 + +

Claims

one . - The primer for PCR amplification which comprises operatively linked in the 3'-5 'direction: a. An access sequence consisting of an oligonucleotide of 10 to  Nucleotides capable of specifically hybridizing to the 3 'end of the sequence to be amplified, b. Optionally, a fishing sequence for excess primer removal after the PCR reaction consisting of at least 2 nucleotides, the sequence that is designed to hybridize to a probe, optionally immobilized to a substrate and that does not interact with the sequence Diana, c. A terminator of PCR amplification, d. A capture or reporter queue for hybridization of the resulting amplification product to an optionally immobilized probe on a substrate or a labeled probe respectively, consisting of a nucleic acid with a sequence with a length of at least two bases designed for hybridization to said probe optionally immobilized on a substrate or said labeled probe. 2. - The primer according to claim 1, wherein the access sequence and the fishing sequence are two DNA sequences. 3. - The primer according to any of the preceding claims, wherein the fishing sequence has not less than 15 nucleotides. 4. - The primer according to any of the preceding claims, wherein the PCR amplification terminator can be an inorganic terminator such as hexaethylene glycol, triethylene glycol, propyl, 1 ', 2'-dideoxyribose or bases or non-natural nucleic acids that do not they are recognized by polymerase such as PNA, LNA, morpholino oligos or RNA. 5. - The primer according to any of the preceding claims, wherein the capture or reporter tail comprises a nucleic acid selected from DNA, PNA, Morfolino or LNA oligos and mixtures of these nucleic acids. 6. - The primer according to any of the preceding claims, wherein the primer is a direct or reverse primer. 7. - Pair of primers for PCR amplification consisting of a direct primer and a reverse primer, in which one or both are according to claims 1 to 6. 8. - Pair of primers according to claim 7, wherein the direct primer comprises a capture queue and the reverse primer comprises a reporter queue or wherein the direct primer comprises a reporter queue and the reverse primer comprises a capture queue . 9. - Use of a primer according to any of claims 1 to 6 or of a pair of primers according to any of claims 7 to 8 to carry out a PCR amplification. 10. - Use according to claim 9, wherein, after the PCR reaction, the excess primers are removed and separated from the amplification product by hybridization to a fishing probe, optionally immobilized to a substrate, comprising a sequence capable of hybridizing specifically to the fishing sequence. eleven . - PCR product that can be obtained by using at least one primer according to claim 1 consisting of a DNA duplex with a single chain tail corresponding to the capture or reporter tail present in the primer according to claim 1 . 12. - PCR product that can be obtained by using a pair of primers according to claim 8, comprising a DNA duplex with two single-stranded tails at each end of the duplex, corresponding to the capture tail and / or primer pair informer according to claim 8. 13. - A group of two or more primer pairs according to any of claims 7-8 for a multiplex PCR, in which the capture tails of each of the primer pairs are different from each other and are designed to be individual for each access sequence, and in which the queues of each of the primer pairs are designed to be common to each other or individual for each access sequence. 14. A method for detecting the presence of a target sequence in a biological sample comprising: a) preparing capture probes capable of hybridizing to the capture tail of the primer according to any one of claims 1 to 6 or of the pair of primers according to any of claims 8 to 9, and / or report probes capable of hybridizing to the primer queue according to any one of claims 1 to 6 or of the pair of primers according to any of claims 8 to 9, b) perform amplification by PCR with at least one primer according to any one of claims 1 to 6 or with a pair of primers according to any one of claims 7 to 8 to obtain an amplification product according to claims 1 or 12 respectively, c) optionally eliminating the primers in excess remaining after PCR amplification of stage b) by means of enzymatic digestion or hybridization to an optional fishing probe mind immobilized to a substrate, comprising a sequence capable of hybridizing specifically to the fishing sequence, d) allowing hybridization between the capture probes and / or the reporting probes of step a) with the capture tails and / or the reporting tails of the amplification product, e) detecting the presence of hybridization between the capture probes and / or the reporting probes of step a) with the capture tails and / or the reporting tails of the amplification product, where the presence of said hybridization implies the presence of the specific sequence in the sample and non-hybridization implies the absence of said problem sequence in the sample. 15. A method according to claim 14, wherein the capture probes are immobilized to a substrate. 16. - A method according to any of claims 14 and 15, wherein the reporter probe comprises a marker that includes redox electrochemical molecules, enzymes capable of producing a detectable reaction, fluorophors, molecules capable of producing electrochemiluminescence, color changes, chemiluminescence or fluorescence , liposomes, metal particles / cylinders, inorganic particles / cylinders, polymer particles / cylinders, magnetic particles / cylinders, quantum dots, dendrimers, radioactive markers. 17. - A method according to claims 14 and 16, wherein the capture probe and the reporter probe can be a nucleic acid selected from DNA, PNA, morpholino oligos, LNA or combinations thereof. 18. - A method according to any of claims 14 to 17, wherein the detection of hybridization is carried out by means of electrochemical, optical, gravimetric, thermometric, magnetic and micromechanical procedures. 19. - A method according to any of claims 14 to 18 wherein the PCR amplification is a multiplex PCR and is carried out with a group of primers according to claim 13.