Method and kit for multiplex amplification of nucleic acid

Abstract

The present invention relates to a multiplex amplification method to amplify a template nucleic acid of interest more efficiently, enabling the enrichment of target amplicons over the overlapping regions of the amplicons by removing amplicons corresponding to said overlapping regions based on their sizes. The invention also relates to kits and devices for carrying out the multiplexed amplification method of the invention and to a method for the sequencing of the target amplicons.

Description

[0001] METHOD AND KIT FOR MULTIPLEX AMPLIFICATION OF NUCLEIC ACID

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the field of nucleic acid amplification, and, more in particular, to a method and reagents for multiplex amplification of nucleic acids in a sample, to a kit for carrying out said method, and to a method for the sequencing of the nucleic acids.

[0004] BACKGROUND OF THE INVENTION

[0005] Nucleic acid amplification techniques, including PCR and LAMP methods, have been used in many fields of biology such as molecular biology and medicine, and they are commonly used for DNA profiling, food tests, environmental sanitation tests and animal and plant tests.

[0006] PCR amplification of long target nucleic acid regions of genes require multiple reactions to separately amplify the overlapping amplicons that tile the entire target sequences. When all primers are present in one reaction, the overlapping regions between the adjacent overlapping amplicons are preferentially amplified and dominate the reaction, resulting in the drop-out of the actual targeted amplicons and gaps in sequencing coverage.

[0007] A study by Schenk et al. (PLoS One, 2017 Jul 12;12(7):e0181062) describes the amplification of overlapping DNA amplicons in a single-tube multiplex PCR for targeted next generation sequencing of BRCA1 and BRCA2.

[0008] Given these challenges, there is still a need for methods for the amplification of nucleic acids based on multiplex amplification which enable the enrichment of the target amplicons over the amplicons which result from the amplification of the overlapping regions of the target nucleic acids.

[0009] SUMMARY OF THE INVENTION

[0010] Described herein is a multiplex amplification method to amplify a template nucleic acid of interest more efficiently, enabling the enrichment of target amplicons over the overlapping regions of the amplicons. It is also described herein a kit for carrying out said method, and a method for the sequencing of the target amplicons.

[0011] Thus, in a first aspect the present invention relates to a method for amplification of at least two amplicons from a template nucleic acid present in a sample comprising the steps of: (i) Performing a first multiplex amplification of at least two neighbouring regions from the template nucleic acid wherein the 3’ end part of the neighbouring upstream region overlaps with the 5’ end part of the neighbouring downstream region, wherein said amplification is carried out using a set of two primer pairs, and wherein each primer pair comprises a forward primer and a reverse primer specific for each of the regions, and wherein the amplification products comprise: a. Amplicons of a first type resulting from the amplification of the upstream region with the forward and reverse primers specific for said upstream region, b. Amplicons of a second type resulting from the amplification of the downstream region with the forward and reverse primers specific for said downstream region, and c. Amplicons of a third type resulting from the amplification of the template nucleic acid with the reverse primer of the primer pair specific for the upstream region and the forward primer of the primer pair specific for the downstream region,

[0012] (ii) removing the amplicons of the third type from the amplification mixture obtained in step (i) by a method which separates the amplicons of the first type and amplicons of the second type from the amplicons of the third type based on their sizes thereby obtaining an amplification mixture enriched in the amplicons of the first and of the second type, and

[0013] (iii) performing a second multiplex amplification of the mixture obtained in (ii) using a set of primer pairs specific for the amplicons of the first and of the second type.

[0014] In a second aspect, the invention relates to a kit for the amplification of at least two amplicons from a template nucleic, said kit comprising:

[0015] (I) a set of primer pairs, wherein each primer pair comprises a forward primer and a reverse primer specific for different regions within the template nucleic acid wherein the 3’ end neighbouring upstream region overlaps with the 5’ end part of the neighbouring downstream region, and

[0016] (ii) reagents adequate for the size-dependent separations of DNA molecules. In a third aspect, the invention relates to a method for the sequencing of a template nucleic acid, the method comprising generating amplicons from the template nucleic acid by a method according to the first aspect of the invention and sequencing the amplicons of the first and of the second type obtained in the third step of the first aspect of the invention.

[0017] BRIEF DESCRIPTION OF THE FIGURES

[0018] Figure 1 : Relative position on SERPINA1 gene of the 14 gene-specific PCR primer pairs for amplifying regions of interest in a single multiplex amplification reaction.

[0019] Figure 2: Relative position on SERPINC1 gene of the 13 gene-specific PCR primer pairs for amplifying regions of interest in a single multiplex amplification reaction- Design 1 .

[0020] Figure 3: Relative position on SERPINC1 gene of the 12 gene-specific PCR primer pairs for amplifying regions of interest in a single multiplex amplification reaction- Design 2.

[0021] Figure 4: Selective purification of amplicons differing in 47 bp in length with different ratios of SPRISelect beads / sample. a) shows the content of the first multiplex amplification in a Bioanalyzer 2100, where the high content of primers is shown, b) shows the content of the size-based purification of the second multiplex amplification using different beads / sample ratios in a Bioanalyzer 2100.

[0022] DETAILED DESCRIPTION OF THE INVENTION

[0023] Method for multiplex amplification of nucleic acids

[0024] The authors of the present invention have developed a method for multiplex amplification of nucleic acids in a sample. Said method avoids the amplification of undesired amplicons which correspond to the overlapping regions between adjacent overlapping amplicons, whose growth overtakes the reaction and hinders the amplification of the target amplicons.

[0025] Accordingly, in a first aspect, the invention relates to a method for amplification of at least two amplicons from a template nucleic acid present in a sample comprising the steps of:

[0026] (i) Performing a first multiplex amplification of at least two neighbouring regions from the template nucleic acid wherein the 3’ end part of the neighbouring upstream region overlaps with the 5’ end part of the neighbouring downstream region, wherein said amplification is carried out using a set of two primer pairs, and wherein each primer pair comprises a forward primer and a reverse primer specific for each of the regions, and wherein the amplification products comprise: a. Amplicons of a first type resulting from the amplification of the upstream region with the forward and reverse primers specific for said upstream region, b. Amplicons of a second type resulting from the amplification of the downstream region with the forward and reverse primers specific for said downstream region, and c. Amplicons of a third type resulting from the amplification of the template nucleic acid with the reverse primer of the primer pair specific for the upstream region and the forward primer of the primer pair specific for the downstream region,

[0027] (ii) removing the amplicons of the third type from the amplification mixture obtained in step (i) by a method which separates the amplicons of the first type and amplicons of the second type from the amplicons of the third type based on their sizes thereby obtaining an amplification mixture enriched in the amplicons of the first and of the second type, and

[0028] (iii) performing a second multiplex amplification of the mixture obtained in (ii) using a set of primer pairs specific for the amplicons of the first and of the second type.

[0029] As it is used herein, the term “amplification” refers to any known procedure for obtaining multiple copies of a target nucleic acid sequence or its complement or fragments thereof. The multiple copies may be referred to as amplicons or amplification products.

[0030] The term “amplicon”, as it is used herein, refers to an amplification product such as a nucleic acid that is amplified by a PCR reaction or other amplification reaction or method. Typically, the amplicon or amplification product refers to production of an amplified nucleic acid that contains less than the complete template nucleic acid or target nucleic acid or its complement, e.g., produced by using an amplification oligonucleotide that hybridizes to, and initiates polymerization from, an internal position of the target nucleic acid. Known amplification methods include both thermal and isothermal amplification methods. Replicase-mediated amplification, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand-displacement amplification (SDA), and transcription-mediated or transcription-associated amplification are non-limiting examples of nucleic acid amplification methods. In a particular embodiment, the amplification is an isothermal amplification. As used herein, "under isothermal conditions" means that the amplification reaction is conducted at a relatively constant temperature, thereby obviating the need for a thermal cycler. Exemplary, isothermal amplifications include, but is not limited to, Recombinase Polymerase Amplification (RPA), Loop Mediated Isothermal Amplification (LAMP), Helicase-dependent isothermal DNA amplification (HDA), Rolling Circle Amplification (RCA), Nucleic acid sequencebased amplification (NASBA), strand displacement amplification (SDA), nicking enzyme amplification reaction (NEAR), Polymerase Spiral Reaction (PSR), Hybridization Chain Reaction (HCR), Primer Exchange Reaction (PER), Signal Amplification by Exchange Reaction (SABER), transcription-based amplification system (TAS), Self-sustained sequence replication reaction (3 SR), Single primer isothermal amplification (SPIA), and cross-priming amplification (CPA).

[0031] The term “nucleic acid”, “nucleic acid molecule” “nucleotide sequence” or “polynucleotide” is used interchangeably in the present invention to refer to the polymeric form of the ribonucleoside phosphate ester (adenosine, guanosine, uridine or cytidine; "RNA molecules") or deoxy ribonucleosides (deoxiadenosine, deoxyguanosine, deoxythymidine or deoxycytidine; "DNA molecules") or any phosphoester analog thereof such as phosphorothioates and thioesters, in a single stranded or double stranded form. Thus, the term includes single stranded DNA or RNA molecules. It also includes double stranded molecules formed by DNA-DNA, DNA-RNA and RNA-RNA strands. The term "nucleic acid sequence" and, in particular, the DNA or RNA molecule, refers only to the primary or secondary structure of the molecule and does not limit any particular type of tertiary structure. Thus, this term encompasses double stranded DNA, as comprised in linear or circular DNA molecules, supercoiled DNA plasmids and chromosomes.

[0032] As used herein, the term "template nucleic acid" or “target nucleic acid” refers to a nucleic acid molecule that serves as a template in a nucleic acid amplification reaction. It may refer to a nucleic acid which is unknown (which may also be called an "unknown nucleic acid"), to a known nucleic acid (which may also be called a "target nucleic acid"), or a mixture thereof. It is a DNA and / or RNA. Any source of nucleic acid, in purified or non-purified form, can be utilized as the starting nucleic acid for amplification. The term "template nucleic acid" used in the present invention, therefore, is not only limited to any specific nucleic acid(s) (target nucleic acid(s)) to be assayed, but also includes nonspecific nucleic acid(s). In general, the term "template nucleic acid" or “template polynucleotide” refers to a nucleic acid molecule or polynucleotide in a starting population of nucleic acid molecules having a target sequence whose presence, amount, and / or nucleotide sequence, or changes in one or more of these, are desired to be determined. In general, the term "template sequence" refers to a nucleic acid sequence on a single strand of nucleic acid. The template sequence may be a portion of a gene, a regulatory sequence, genomic DNA, cDNA, RNA including mRNA, miRNA, rRNA, or others. The template sequence may be a target sequence from a sample or a secondary target such as a product of an amplification reaction. A template polynucleotide is not necessarily any single molecule or sequence. For example, a template polynucleotide may be any one of a plurality of target polynucleotides in a reaction, or all polynucleotides in a given reaction, depending on the reaction conditions. For example, in a nucleic acid amplification reaction with random primers, all polynucleotides in a reaction may be amplified. As a further example, a collection of targets may be simultaneously assayed using polynucleotide primers directed to a plurality of targets in a single reaction. As yet another example, all or a subset of polynucleotides in a sample may be modified by the addition of a primer-binding sequence (such as by the ligation of adapters containing the primer binding sequence), rendering each modified polynucleotide a target polynucleotide in a reaction with the corresponding primer polynucleotide(s).

[0033] The term “sample”, as it is used herein, relates to a material or mixture of materials containing one or more components of interest. The term "sample" includes "biological sample" which refers to a sample obtained from a biological subject, including a sample of biological tissue or fluid origin, obtained, reached, or collected in vivo or in situ. Suitable biological samples include a forensic sample, a synthetic sample or an environmental sample.

[0034] The nucleic acid may be obtained from different types of biological samples such as e.g. body fluids in general, whole blood, serum, plasma, red blood cells, white blood cells, buffy coat; swabs, including but not limited to buccal swabs, throat swabs, vaginal swabs, urethral swabs, cervical swabs, throat swabs, rectal swabs, lesion swabs, abscess swabs, nasopharyngeal swabs and anal swabs, urine, sputum, saliva, semen, lymphatic fluid, liquor, amniotic fluid, cerebrospinal fluid, peritoneal effusions, feces, pleural effusions, fluid from cysts, synovial fluid, vitreous humor; aqueous humor, bursa fluid, eye washes, eye aspirates, pulmonary lavage, lung aspirates, tissues, including but not limited to, liver, spleen, kidney, lung, intestine, brain, heart, muscle, pancreas, tumor tissue, biopsies and cell cultures, bacteria, microorganisms, viruses, plants, fungi including samples that derive from the foregoing or comprise the foregoing. Materials obtained from clinical or forensic settings or environmental samples such as soil that contain or are suspected to contain target nucleic acids can also be used as starting material. Furthermore, the skilled artisan will appreciate that extracts, or materials or portions thereof obtained from any of the above exemplary samples can also be used as source for the nucleic acids. Preferably, the source or sample from which the nucleic acids to be analysed are obtained is derived from a human, animal, plant, bacteria, virus, parasite or fungi. Preferably, the sample is selected from the group consisting of cells, tissue, bacteria, viruses and body fluids such as for example blood, blood products such as buffy coat, plasma and serum, urine, liquor, sputum, stool, CSF and sperm, epithelial swabs, vaginal swabs, cervix samples, biopsies, bone marrow samples and tissue samples, preferably organ tissue samples such as lung and liver or tumor tissue. The sample may be stabilized. Certain samples such as blood samples are usually stabilised upon collection, e.g. by contacting them with a stabilizer such as an anticoagulant in case of blood and samples derived from blood.

[0035] In further embodiments of the invention, the sample is selected from the group consisting of:

[0036] (i) a DNA containing biological sample;

[0037] (ii) a solution resulting from an DNA amplification procedure;

[0038] (iii) a solution resulting from a DNA size shearing procedure;

[0039] (iv) a solution resulting from a nucleotide sequencing reaction;

[0040] (v) a solution resulting from a restriction enzyme digestion comprising a mixture of nucleic acid molecule fragments;

[0041] (vi) an agarose solution containing nucleic acids; and

[0042] (vii) a DNA containing solution resulting from a DNA purification procedure.

[0043] In another embodiment, the biological sample is selected from the group consisting of cell lines, blood, serum, plasma, saliva, hair, urine, skin tissue, bone marrow, semen, rectal and vaginal fluid.

[0044] In a first step of the method according to the first aspect of the invention, a first multiplex amplification is carried out wherein at least two neighbouring regions from the template nucleic acid wherein the 3’ end part of the neighbouring upstream region overlaps with the 5’ end part of the neighbouring downstream region are amplified and wherein said amplification is carried out using a set of two primer pairs, and wherein each primer pair comprises a forward primer and a reverse primer specific for each of the regions, and wherein the amplification products comprise: a. Amplicons of a first type resulting from the amplification of the upstream region with the forward and reverse primers specific for said upstream region, b. Amplicons of a second type resulting from the amplification of the downstream region with the forward and reverse primers specific for said downstream region, and c. Amplicons of a third type resulting from the amplification of the template nucleic acid with the reverse primer of the primer pair specific for the upstream region and the forward primer of the primer pair specific for the downstream region.

[0045] In this invention, the method for amplification refers to at least the amplification of two amplicons from a template nucleic acid present in a sample. The method according to the invention generates amplicons of a first type, of a second type and of a third type. The amplification of the amplicons of the third type by the method according to the invention is not prevented with regards to the amplification of the amplicons of the first and of the second type. The method according to the invention may further generate amplicons of a fourth type. The amplicons of a fourth type result from the amplification of the template nucleic acid with primer combination that results in amplification products that contain more than one of the neighbouring regions. For example, amplicons of a fourth type may result from the amplification from the template nucleic acid with a forward primer which is the forward primer used for the amplification of the upstream neighbouring region and the reverse primer which is the reverse primer used for the amplification of the downstream neighbouring region.

[0046] In general, the amplification reaction is performed in a reaction mixture. By "reaction mixture", as used herein, is meant a composition including the relevant components to allow an amplification reaction to be performed. An exemplary reaction mixture can include, without limitation, a nucleic acid sample, primer pairs, and a suitable enzyme, such as a polymerase. A "polymerase" refers to an enzyme that performs template-directed synthesis of polynucleotides, e.g., DNA and / or RNA. The term encompasses both the full-length polypeptide and a domain that has polymerase activity. As used in the present description, the term “DNA polymerase” relates to an enzyme capable of catalyzing the polymerization of deoxynucleoside triphosphates. Generally, the enzyme initiates the synthesis in the 3' end of a primer hybridized with a template DNA sequence and proceeds towards the 5' end of the template DNA strand. DNA polymerases are well-known to those skilled in the art, including but not limited to DNA polymerases isolated or derived from Pyrococcus furiosus, Thermococcus litoralis, and Thermotoga maritime, or modified versions thereof. Additional examples of commercially available polymerase enzymes include, but are not limited to: Klenow fragment (New England Biolabs(R) Inc), Taq DNA polymerase (QIAGEN), 9 degrees NTMDNA polymerase (New England Biolabs(R) Inc.), Deep Vent™ DNA polymerase (New England Biolabs(R) Inc.), Manta DNA polymerase (Enzymatics(R)), Bst DNA polymerase (New England Biolabs(R) Inc.), and phi29 DNA polymerase (New England Biolabs(R) Inc.).

[0047] In an embodiment, the amplification in the first method according to the first aspect of the invention is performed by polymerase chain reaction (PCR).

[0048] One of ordinary skills in the art will appreciate that the reaction mixture may also include other components such as buffers, stabilisers, templates, nucleotides and the like and that these components may be dictated by the amplification reaction being performed.

[0049] It is to be understood that amplification parameters, such as nucleotide concentration, nucleic acid polymerases used for the amplification, buffer composition, number of amplification cycles, temperatures during the cycles, can be optimized as described herein or known in the art.

[0050] As used herein, a “multiplex amplification” refers to an amplification reaction in which different amplification reactions are carried out in the same amplification mixture, normally by the use of different primer pairs specific for each of the products that are simultaneously amplified.

[0051] As it is used herein, the term “primer” refers to a short strand of nucleic acid that is complementary to a sequence in another nucleic acid and serves as a starting point for DNA synthesis. A primer is selected to be "substantially" complementary to a strand of specific sequence of the template. A primer must be sufficiently complementary to hybridize with a template strand for primer elongation to occur. A primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being substantially complementary to the strand. Non- complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the template to hybridize and thereby form a template primer complex for synthesis of the extension product of the primer. The term "primer pair" as used herein refers to a pair of primers, consisting of one forward and one reverse primer (relative to the nucleic acid sequence to be amplified), which are necessary and sufficient for the amplification of a target nucleic acid sequence, for example, using PCR. As used herein, each primer pair comprises a forward primer and a reverse primer specific for each of the two neighbouring regions from the template nucleic acid.

[0052] In some embodiment, the forward primers and / or the reverse primers from the one or more of the primer pairs specific for the upstream and downstream regions used in step (i) comprise a target-specific region located in the 3’ end that hybridizes with the target nucleic acid region within the template nucleic acid and a custom region located in the 5’ end that does not hybridize with the target nucleic acid region. This results in that each of the amplicons obtained in step (i) contain 5’ and / or 3’ custom extensions which incorporate the sequence of the custom regions present in the forward or reverse primers. In yet another embodiment, the sequences of the custom regions within each of the forward and / or reverse primers used in step (i) are substantially identical.

[0053] In one embodiment of the method according to the first aspect of the invention, the forward primers and / or the reverse primers from the one or more of the primer pairs specific for the upstream and downstream regions used in the first step of the first aspect of the invention comprise a target-specific region located in the 3’ end that hybridizes with the target nucleic acid region within the template nucleic acid and a custom region located in the 5’ end that does not hybridize with the target nucleic acid region, thereby resulting in that each of the amplicons obtained in the first step of the first aspect of the invention contain 5’ and / or 3’ custom extensions which incorporate the sequence of the custom regions present in the forward or reverse primers.

[0054] As used herein, "hybridizes" refers to preferential hybridization under hybridization conditions where two nucleic acids, or portions thereof, that are substantially complementary, hybridize to each other and not to other nucleic acids that are not substantially complementary to either of the two nucleic acids. For example, specific hybridization includes the hybridization of a barcode region to a portion of a capture nucleic acid that is substantially complementary to the barcode or capture nucleic acid. In some embodiments nucleic acids, or portions thereof, that are configured to specifically hybridize are often about 80 percent or more, 81 percent or more, 82 percent or more, 83 percent or more, 84 percent or more, 85 percent or more, 86 percent or more, 87 percent or more, 88 percent or more, 89 percent or more, 90 percent or more, 91 percent or more, 92 percent or more, 93 percent or more, 94 percent or more, 95 percent or more, 96 percent or more, 97 percent or more, 98 percent or more, 99 percent or more or 100 percent complementary to each other over a contiguous portion of nucleic acid sequence. A specific hybridization discriminates over non-specific hybridization interactions (e.g., two nucleic acids that are not configured to specifically hybridize, e.g., two nucleic acids that are 80 percent or less, 70 percent or less, 60 percent or less or 50 percent or less complementary) by about 2-fold or more, often about 10-fold or more, and sometimes about 100-fold or more, 1000-fold or more, 10,000- fold or more, 100,000-fold or more, or 1 ,000,000-fold or more. Two nucleic acid strands that are hybridized to each other can form a duplex which includes a double stranded portion of nucleic acid.

[0055] A nucleic acid, or a portion thereof, "hybridizes" to another nucleic acid under conditions such that non-specific hybridization is minimal at a defined temperature in a physiological buffer (e.g., pH 6-9, 25-150 mM chloride salt). In some cases, a nucleic acid, or portion thereof, hybridizes to a conserved sequence shared among a group of target nucleic acids. In some cases, the target-specific region can hybridize with the target nucleic acid within the template nucleic acid and a custom region located in the 5’ end that does not hybridize with the target nucleic acid region. In some embodiments, the defined temperature at which specific hybridization occurs is room temperature. In some embodiments, the defined temperature at which specific hybridization occurs is higher than room temperature. In some embodiments, the defined temperature at which specific hybridization occurs is at least about 37, 40, 42, 45, 50, 55, 60, 65, 70, 75, or 80 degrees centigrade. In some embodiments, the defined temperature at which specific hybridization occurs is 37, 40, 42, 45, 50, 55, 60, 65, 70, 75, or 80 degrees centigrade.

[0056] In some embodiments, the custom region of the reverse primer of the primer pair specific for the upstream region and the custom region of the forward primer of the primer pair specific for the downstream region used in step (I) are not substantially complementary to each other.

[0057] The term “regions that are not substantially complementary to each other”, as used herein, refers to regions of the primer sequences which are non-complementary to each other (e.g., are at least 50 percent, at least 75 percent, at least 90 percent, or more non-complementary to each other) and the nucleic acids of each of the primer sequences, or portions thereof, do not hybridize with each other. In some embodiments, for a custom region of n nucleotides when aligned with the custom region of the other primer in the typical complementary manner (one region in the 5’ to 3’ direction and the other region in the 3’ to 5’ direction), the number of positions in the aligned structure containing complementary nucleotides is of n / 6 - x or less, wherein x is an integer which ranges from 0 to n / 6. In some embodiments, there are not at least two contiguous bases along any position of the custom region of a primer that are complementary to at least two contiguous bases along any position of the custom region of the other primer. In some embodiments, there are not at least two contiguous bases within the custom region of a primer that are complementary to at least two contiguous bases within the custom region of the other primer. In some embodiments, there are not at least two contiguous bases in any of the ends of the custom region of a primer that are complementary to at least two contiguous bases in any of the ends of the custom region of the other primer..

[0058] Primers containing regions that are not substantially complementary to each other, according to the invention, avoid any internal folding, or primer dimers due to primer-primer annealing.

[0059] In other embodiments, each strand of the amplicons of the third type is not susceptible to acquire a secondary structure upon annealing conditions, such as a hairpin structure

[0060] The term “not susceptible to acquire a secondary structure”, as used herein, when referred to each strand of the amplicons of the third type is used to indicate that each strand of the amplicons of the third type is not capable of annealing and acquiring a structure comprising a double stranded or higher order region and a region forming an unpaired loop. The formation of unpaired loop results in changes in the melting temperature (Tm) and / or annealing temperature (Ta). The presence of secondary structures, such as hairpin structures, as a result of the annealing of two primers can be excluded by determining the degree of the secondary structure after the primers have been allowed to anneal. The degree of a secondary structure can be quantified, for example, by quantitative polymerase chain reaction (qPCR) to evaluate the effect of the secondary structure, such as a hairpin, on the amplification efficiency of the qPCR. As an example, Fan H., et al., 2019 (J Biomol Struct Dyn. 37(11 ):2867-2874) discloses that Ct values of DNA amplification of an amplicon bearing a hairpin with a stem length of 6- , 9-, 15, and 20- bp were increased by 18%, 48%, 61 % and 77%, respectively, compared to an amplicon with no hairpin. These results demonstrate that the amplification efficiency of the amplicon decreased as the hairpin stem increased.

[0061] The degree of a secondary structure, such as a hairpin, in an amplicon can also be quantified, for example, by electrophoresis of the products after performing the qPCR. Fan H., et al., 2019 (J Biomol Struct Dyn. 37(11 ):2867-2874) discloses that, for the amplicon products bearing a hairpin with a stem length of 6-, 9-, 15, and 20- bp, the yields were 77%, 70%, 42%, and 11 %, respectively, compared to the amplicon with no hairpin, and that nonspecific products other than the targeted amplification product are formed.

[0062] A person skilled in the art would know how to design primer sequences which are non-complementary to each other, for example using softwares or algorithms such as those described by Untergasser A., et al., 2012 (Nucleic Acids Res. 2012 Jun 21 ;40(15):e115). These softwares and algorithms can analyze a sequence and predict potential secondary structures, such as hairpin structures, based on base pairing probabilities and thermodynamic stability.

[0063] Differential Scanning Calorimetry is also used to determine the thermodynamic properties of biomacromolecules, including polynucleotides, and specifically for the study of the hairpin or duplex structures (Methods in Cell Biology Vol. 84, 2008, 115-41 & Lah J., etal., J Nucleic Acids. 2011 :513910).

[0064] In one embodiment, the sequences of the custom regions within each of the forward primers used in step (I) are substantially identical. In one embodiment, the sequences of the custom regions within each of the reverse primers used in step (I) are substantially identical. In yet another embodiment, the sequences of the custom regions within each of the forward primers are substantially identical and the sequences of the custom regions within each of the reverse primers are substantially identical but different from the custom regions forming part of the forward primers.

[0065] The term “substantially identical” as used herein, describe a degree of similarity between nucleotide sequences. It refers to two or more sequences that have at least about 60 percent, at least about 70 percent, at least about 80 percent, about 90 percent to about 99 percent, about 95 percent to about 99 percent, and about 99 percent nucleotide identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm (using for example the alignment method of Needleman and Wunsch (J. Mol. Biol., 1970, 48: 443) as revised by Smith and Waterman (Adv. Appl. Math., 10 1981 , 2: 482)).

[0066] In another embodiment, the sequences of the custom regions within each of the forward and reverse primers of each primer set used in the first step of the first aspect of the invention are substantially identical and the set of primer pairs used in the third step of the first aspect of the invention consist of a single primer pair wherein the forward primer hybridizes with the 5’ custom extensions of all the amplicons of the first and of the second type and the reverse primer hybridizes with the 3’ custom extensions of all the amplicons of the first and of the second type. As used herein, the term "neighbouring upstream region" or “upstream region” and "neighbouring downstream region" or “downstream region” refers to regions within the same template nucleic acid that show a relative arrangement wherein the upstream region is located at a position which is 5’ with respect to the “downstream region”. According to the present invention, the neighbouring upstream and downstream region are overlapping regions, i.e. the 3’ end of the upstream region is located within the downstream region or, conversely, the 5’ end of the downstream region is located within the upstream region. In certain embodiments the upstream and downstream neighbouring regions show an overlap of at least 5 base pairs, at least 10 base pairs, at least 15 base pairs, at least 20 base pairs, at least 25 base pairs, at least 30 base pairs, at least 35 base pairs, at least 40 base pairs, at least 45 base pairs, at least 50 base pairs, at least 60 base pairs, at least 70 base pairs, at least 80 base pairs, at least 90 base pairs, at least 100 base pairs, at least 150 base pairs, at least 200 base pairs, at least 300 base pairs, at least 400 base pairs, at least 500 base pairs or more. In certain embodiments the upstream and downstream neighbouring regions show an overlap of 5 base pairs or less, 10 base pairs or less, 15 base pairs or less, 20 base pairs or less, 25 base pairs or less, 30 base pairs or less, 35 base pairs or less, 40 base pairs or less, 45 base pairs or less, 50 base pairs or less, 60 base pairs or less, 70 base pairs or less, 80 base pairs or less, 90 base pairs or less, 100 base pairs or less, 150 base pairs or less,

[0067] 200 base pairs or less, 300 base pairs or less, 400 base pairs or less or 500 base pairs or less.

[0068] In an embodiment, the amplicons of the first type resulting from the amplification of the upstream region with the forward and reverse primers specific for said upstream region, and the amplicons of a second type resulting from the amplification of the downstream region with the forward and reverse primers specific for said downstream region obtained in the first step of the first aspect of the invention have a length of 200 to 400 base pairs, of 210 to 390 base pairs, of 220 to 380 base pairs, of 230 to 370 base pairs, of 240 to 360 base pairs, of 250 to 350 base pairs, of 260 to 340 base pairs, of 270 to 330 base pairs, of 280 to 320 base pairs, of 290 to 310 base pairs, or of 300 base pairs. The amplicons of the first and of the second type may also have a length of 200 to 390 base pairs, of 200 to 380 base pairs, of 200 to 370 base pairs, of 200 to 360 base pairs, of 200 to 350 base pairs, of 200 to 340 base pairs, of 200 to 330 base pairs, of 200 to 320 base pairs, of 200 to 310 base pairs, of 200 to 300 base pairs, of 200 to 290 base pairs, of 200 to 280 base pairs, of 200 to 270 base pairs, of 200 to 260 base pairs, of 200 to 250 base pairs, of 200 to 240 base pairs, of 200 to 230 base pairs, of 200 to 220 base pairs, of 200 to 210 base pairs, or of 200 base pairs. The amplicons of the first and of the second type may also have a length of 210 to 400 base pairs, of 220 to 400 base pairs, of 230 to 400 base pairs, of 240 to 400 base pairs, of 250 to 400 base pairs, of 260 to 400 base pairs, of 270 to 400 base pairs, of 280 to 400 base pairs, of 290 to 400 base pairs, of 300 to 400 base pairs, of 310 to 400 base pairs, of 320 to 400 base pairs, of 330 to 400 base pairs, of 340 to 400 base pairs, of 350 to 400 base pairs, of 360 to 400 base pairs, of 370 to 400 base pairs, of 380 to 400 base pairs, of 390 to 400 base pairs, or of 400 base pairs.

[0069] In an embodiment, the amplicons of a third type resulting from the amplification of the template nucleic acid with the reverse primer of the primer pair specific for the upstream region and the forward primer of the primer pair specific for the downstream region obtained in the first step of the first aspect of the invention have a length of 100 to 190 base pairs, of 105 to 185 base pairs, of 110 to 180 base pairs, of 115 to 175 base pairs, of 120 to 170 base pairs, of 125 to 165 base pairs, of 130 to 160 base pairs, of 135 to 155 base pairs, of 140 to 150 base pairs, or of 145 base pairs. The amplicons of the third type may also have a length of 100 to 185 base pairs, of 100 to 180 base pairs, of 100 to 175 base pairs, of 100 to 170 base pairs, of 100 to 165 base pairs, of 100 to 160 base pairs, of 100 to 155 base pairs, of 100 to 150 base pairs, of 100 to 145 base pairs, of 100 to 140 base pairs, of 100 to 135 base pairs, of 100 to 130 base pairs, of 100 to 125 base pairs, of 100 to 120 base pairs, of 100 to 115 base pairs, of 100 to 110 base pairs, of 100 to 105 base pairs, or of 100 base pairs. The amplicons of the third type may also have a length of 110 to 190 base pairs, of 115 to 190 base pairs, of 120 to 190 base pairs, of 125 to 190 base pairs, of 130 to 190 base pairs, of 135 to 190 base pairs, of 140 to 190 base pairs, 145 to 190 base pairs, of 150 to 190 base pairs, of 155 to 190 base pairs, of 160 to 190 base pairs, of 165 to 190 base pairs, of 170 to 190 base pairs, of 175 to 190 base pairs, of 180 to 190 base pairs, of 185 to 190 base pairs, or of 190 base pairs.

[0070] In another embodiment, the difference between the length of the amplicons of the first and of the second type and the length of the amplicons of the third type may be of at least 50 base pairs, of at least 52 base pairs, of at least 54 base pairs, of at least 56 base pairs, of at least 58 base pairs, of at least 60 base pairs, of at least 62 base pairs, of at least 64 base pairs, of at least 66 base pairs, of at least 68 base pairs, of at least 70 base pairs, of at least 72 base pairs, of at least 74 base pairs, of at least 76 base pairs, of at least 78 base pairs, or of at least 80 base pairs or more. In yet another embodiment, the ratio of the lengths of the amplicons of the first type and of the second type and the length of the amplicons of the third type is of at least 1 .20: 1 , of at least 1 .25: 1 , of at least 1 .30: 1 , of at least 1 .35: 1 , of at least 1 .40: 1 , of at least

[0071] 1 .45: 1 , of at least 1 .50: 1 , of at least 1 .55: 1 , of at least 1 .60: 1 , of at least 1 .65: 1 , of at least

[0072] 1.70:1 , of at least 1.75:1 , of at least 1.80:1 , of at least 1.85:1 , of at least 1.90:1 , of at least

[0073] 1 .95: 1 , of at least 2.00: 1 , of at least 2.05: 1 , of at least 2.10: 1 , of at least 2.15: 1 , of at least

[0074] 2.20: 1 , of at least 2.25: 1 , of at least 2.30: 1 , of at least 2.35: 1 , of at least 2.40: 1 , of at least

[0075] 2.45: 1 , of at least 2.50: 1 , of at least 2.55: 1 , of at least 2.60: 1 , of at least 2.65: 1 , of at least

[0076] 2.70:1 , of at least 2.75:1 , of at least 2.80:1 , of at least 2.85:1 , of at least 2.90:1 , of at least

[0077] 2.95: 1 , of at least 3.00: 1 , of at least 3.05: 1 , of at least 3.10: 1 , of at least 3.15: 1 , of at least

[0078] 3.20:1 , of at least 3.25:1 , of at least 3.30: 1 , of at least 3.35: 1 , of at least 3.40: 1 , of at least

[0079] 3.45: 1 , of at least 3.50: 1 , of at least 3.55: 1 , of at least 3.60: 1 , of at least 3.65: 1 , of at least

[0080] 3.70:1 , of at least 3.75:1 , of at least 3.80:1 , of at least 3.85:1 , of at least 3.90:1 , of at least

[0081] 3.95: 1 , of at least 4.00: 1 , of at least 4.05: 1 , of at least 4.10: 1 , of at least 4.15: 1 , of at least

[0082] 4.20:1 , of at least 4.25:1 , of at least 4.30: 1 , of at least 4.35: 1 , of at least 4.40: 1 , of at least

[0083] 4.45: 1 , of at least 4.50: 1 , of at least 4.55: 1 , of at least 4.60: 1 , of at least 4.65: 1 , of at least

[0084] 4.70:1 , of at least 4.75:1 , of at least 4.80:1 , of at least 4.85:1 , of at least 4.90:1 , of at least

[0085] 2.45: 1 , or of at least 5.00: 1 .

[0086] In some embodiments, the amplicons of the first type and of the second type may have a length as defined above and may have a length difference with regards to the length of the amplicons of the third type of at least 50 base pairs, of at least 52 base pairs, of at least 54 base pairs, of at least 56 base pairs, of at least 58 base pairs, of at least 60 base pairs, of at least 62 base pairs, of at least 64 base pairs, of at least 66 base pairs, of at least 68 base pairs, of at least 70 base pairs, of at least 72 base pairs, of at least 74 base pairs, of at least 76 base pairs, of at least 78 base pairs, or of at least 80 base pairs or more.

[0087] In some embodiments, the amplicons of the third type may have a length as defined above and the amplicons of the first and of the second type may have a length difference with regards to the length of the amplicons of the third type of at least 50 base pairs, of at least 52 base pairs, of at least 54 base pairs, of at least 56 base pairs, of at least 58 base pairs, of at least 60 base pairs, of at least 62 base pairs, of at least 64 base pairs, of at least 66 base pairs, of at least 68 base pairs, of at least 70 base pairs, of at least 72 base pairs, of at least 74 base pairs, of at least 76 base pairs, of at least 78 base pairs, or of at least 80 base pairs or more. In some embodiments, the amplicons of the first and of the second type may have a length difference with regards to the length of the amplicons of the third type of at least 50 base pairs, of at least 52 base pairs, of at least 54 base pairs, of at least 56 base pairs, of at least 58 base pairs, of at least 60 base pairs, of at least 62 base pairs, of at least 64 base pairs, of at least 66 base pairs, of at least 68 base pairs, of at least 70 base pairs, of at least 72 base pairs, of at least 74 base pairs, of at least 76 base pairs, of at least 78 base pairs, or of at least 80 base pairs or more and the ratio of the lengths of the amplicons of the first and of the second type and the length of the amplicons of the third type may be as defined above.

[0088] In some embodiments, the multiplex amplification which is applied in step (i) of the method according to the invention results in the generation of a fourth type of amplicons as a result of the generation of amplification products resulting from combinations of a forward and a reverse primer different to those primer pairs resulting in the amplification of amplicons of the first, second and third type. For example, amplicons of a fourth type may result, without limitation, when the forward primer is the forward primer used for the amplification of the upstream neighbouring region and the reverse primer is the reverse primer used for the amplification of the downstream neighbouring region. In an embodiment, the amplicons of the fourth type have a length which is longer than the length of the amplicons of the first and of the second type.

[0089] In other embodiments, the multiplex amplification mixture which is applied in step (i) of the method according to the invention results in an amplification mixture which may contain DNA fragments other than the amplicons of the first, second, third or fourth type.

[0090] The term “DNA fragment” as used in herein is understood as meaning portions of the DNA of high molecular weight, which may derive, but is not limited to, from the original sample.

[0091] In some embodiments, the amplicons of a fourth type generated in step (i) of the method according to the first aspect of the invention and / or the DNA fragments present in the amplification mixture have a length which is longer than the length of the amplicons of the first and of the second type. In certain embodiments, the amplicons of a fourth type and / or the DNA fragments have a length of at least 401 base pairs, of at least 450 base pairs, of at least 500 base pairs, of at least 550 base pairs, of at least 600 base pairs, of at least 650 base pairs, of at least 700 base pairs, of at least 750 base pairs, of at least 800 base pairs, of at least 850 base pairs, of at least 900 base pairs, of at least 950 base pairs, of at least 1000 base pairs, of at least 1100 base pairs, of at least 1200 base pairs, of at least 1300 base pairs, of at least 1400 base pairs, of at least 1500 base pairs, of at least 1600 base pairs, of at least 1700 base pairs, of at least 1800 base pairs, of at least 1900 base pairs, of at least 2000 base pairs or more.

[0092] In a second step of the method according to the first aspect of the invention, the method for amplification comprises the removal of the amplicons of the third type from the amplification mixture obtained in the first step of the first aspect of the invention by a method which separates the amplicons of the first type and amplicons of the second type from the amplicons of the third type based on their sizes thereby obtaining an amplification mixture enriched in the amplicons of the first and of the second type.

[0093] In an embodiment, the separation of the amplicons of the first and of the second type from the amplicons of the third type based on the size in the second step of the first aspect of the invention is carried out by a method selected from the group consisting of Solid Phase Reversible Immobilization (SPRI), gel electrophoresis, ion-exchange chromatography, slalom chromatography, high-performance liquid chromatography, size-exclusion (gel-permeation) chromatography, density gradient centrifugation and microfluidics. In a preferred embodiment, the second step of the first aspect of the invention is carried out by SPRI.

[0094] The term "SPRI" as used herein is interpreted consistently with the understanding of one of ordinary skill in the related art, and generally refers to the patented technology of "Solid Phase Reversible Immobilization" (US 6,534,262 B2) wherein target nucleic acids are selectively precipitated under specific buffer conditions in the presence of a SPRI support, where said SPRI support is often carboxylated and paramagnetic. The precipitated target nucleic acids immobilize to said solid phase carrier and remain bound until removed by an elution buffer according to the operator's needs (US 6,534,262 B2 and DeAngelis, Margaret M. et al: Solid-Phase Reversible Immobilization for the Isolation of PCR Products. Nucleic Acids Res (1995), Vol. 23:22; 4742-4743, which is hereby incorporated by reference herein in its entirety for all purposes).

[0095] The term "carboxylated" as used herein is interpreted consistently with the understanding of one of ordinary skill in the related art, and generally refers to the modification of a material, such as a microparticle, by the addition of at least one carboxl group. A carboxyl group is either COOH or COO-.

[0096] The term "paramagnetic" as used herein is interpreted consistently with the understanding of one of ordinary skill in the related art, and generally refers to the characteristic of a material wherein said material's magnetism occurs only in the presence of an external, applied magnetic field and does not retain any of the magnetization once the external, applied magnetic field is removed. The term "SPRI support” as used herein generally refers to any type of solid phase carrier of any convenient size, of irregular or regular shape and which is fabricated from any number of known materials such as cellulose, cellulose derivatives, acrylic resins, glass, silica gels, polystyrene, gelatin, polyvinyl pyrrolidone, co-polymers of vinyl and acrylamide, polystyrene cross-linked with divinylbenzene or the like (as described, e.g., in Merrifield, Biochemistry 1964, 3, 1385-1390), polyacrylamides, latex gels, polystyrene, dextran, rubber, silicon, plastics, nitrocellulose, natural sponges, silica gels, control pore glass, metals, cross-linked dextrans (e.g., Sephadex™) agarose gel (Sepharose™), and other solid phase bead supports known to those of skill in the art although it will be appreciated that solid phase substrates may include a degree of porosity enabling penetration of fluids and / or biological molecule into the pores.

[0097] As described herein, the amplicons of the first and of the second type present in the amplification mixture of the first step of the first aspect of the invention can be isolated, on the basis of their molecular size, through the use of appropriate concentrations of a nucleic acid precipitating reagent, preferably a polyalkylene glycol, and a salt to result in the selective precipitation and facilitated adsorption of said amplicons to the functional group-coated surface of a suitable magnetically responsive solid phase carrier which functions as a bioaffinity adsorbent for said amplicons for isolation.

[0098] Thus, in one embodiment, the amplicons of the third type are removed from the amplification mixture obtained in the first step of the first aspect of the invention by means of the binding of the amplicons of the first and of the second type to a SPRI support which is capable of non-specifically binding to DNA in a size-dependent manner under conditions adequate for the binding of the amplicons of the first and of the second type to the SPRI support, followed by the separation of the SPRI support from the mixture and elution of the amplicons of the first and of the second type from the SPRI support, thereby obtaining an amplification mixture enriched in the amplicons of the first and of the second type.

[0099] In an embodiment, the SPRI support is a solid phase carrier.

[0100] In another embodiment, the solid phase carrier comprises a surface which is coated with a functional group that reversibly binds to DNA in a size-dependent manner.

[0101] In a further embodiment, the functional group of the surface coating of the solid phase carrier is selected from the group consisting of amine group-coating, carboxyl group-coating and encapsulated carboxyl group-coating. In a preferred embodiment, the functional group of the surface coating of the solid phase carrier is a carboxyl group- containing coat.

[0102] In yet another embodiment, the SPRI support is a magnetically responsive solid phase carrier.

[0103] According to the present invention, one embodiment of the method of the first aspect of the invention comprises selectively adsorbing (e.g., non-covalently binding) of the amplicons of the first and of the second type in a reversible manner to a solid phase carrier (e.g., paramagnetic microparticles) having a functional group coated (e.g., carboxyl-coated) surface by preparing a combination comprising a mixture of nucleic acid molecules, polyethylene glycol, salt, and solid phase carriers having a functional group-coated surface that reversibly binds nucleic acid molecules wherein the polyethylene glycol and salt are present in sufficient concentrations to selectively precipitate the target amplicons (e.g., the amplicons of the first and of the second type).

[0104] The amplification mixture is maintained under conditions appropriate for adsorption of the precipitated target nucleic acid molecules to the functional group- coated surfaces of the solid phase carriers, thereby producing solid phase carriers having the target amplification product bound thereto (e.g., the amplicons of the first and of the second type). Thus, in one embodiment the conditions allowing the binding of the amplicons of the first and of the second type to the SPRI support in the second step of the first aspect of the invention comprises adding a mixture of polyethylene glycol and a salt at concentrations adequate for the binding of the amplicons of the first and of the second type to the SPRI support.

[0105] As used herein the terms “selective” and “selectively” refer to the ability to isolate a particular DNA molecule, on the basis of molecular size (e.g., the amplicons of the first and of the second type), from an amplification mixture which includes or is a mixture of amplicons of different molecular size. The selective isolation of a particular type of amplicon is accomplished through the use of an appropriate precipitating reagent (e.g., polyalkylene glycol and / or a salt) to result in the precipitation and facilitated adsorption of a particular DNA molecule (e.g., characterized on the basis of size) to the surfaces of paramagnetic microparticles. As used herein, “facilitated adsorption” refers to a process whereby a precipitating reagent, (e.g., a polyalkyelene glycol) is used to promote the precipitation and subsequent adsorption of a DNA molecule (e.g. the amplicons of the first and of the second type), which were initially in mixture, onto the surface of a solid phase carrier. Suitable precipitating reagents include ethanol, isopropanol and polyalkylene glycols. Appropriate polyalkylene glycols include polyethylene glycol (PEG) and polypropylene glycol. Generally, PEG is used. Suitable PEG can be obtained from Sigma (Sigma Chemical Co., St. Louis, Mo., Molecular weight 8000, Dnase and Rnase fee, Catalog number 25322-68-3) The molecular weight of the polyethylene glycol (PEG) can range from about 6,000 to about 10,000, from about 6,000 to about 8,000, from about 7,000 to about 9,000, from about 8,000 to about 10,000. In a particular embodiment PEG with a molecular weight of about 8,000 is used. In general, the presence of PEG provides a hydrophobic solution which forces hydrophilic nucleic acid molecules out of soltution. The advantages of using PEG which is a nondenaturing water soluble polymer, rather than an organic precipitating reagent (e.g., ethanol, isoproponal or phenol), are attributed to its benign chemical properties. In an embodiment, the PEG-induced nucleic acid precipitates are adsorbed to the surfaces of magnetically responsive microparticles which can be physically manipulated to facilitate the isolation of essentially pure nucleic acid molecules from complex solutions comprising mixtures of nucleic acids.

[0106] Suitable salts which are useful for facilitating the adsorption of nucleic acid molecules targeted for isolation to the magnetically responsive microparticles include sodium chloride (NaCI), lithium chloride (LiCI), barium chloride (BaCh), potassium (KCI), calcium chloride (CaCh), magnesium chloride (MgCh) and cesium chloride (CsCI). In a preferred embodiment, sodium chloride is used. In general, the presence of salt functions to minimize the negative charge repulsion of the nucleic acid molecules. The wide range of salts suitable for use in the method indicates that many other salts can also be used and suitable levels can be empirically determined by one of ordinary skill in the art.

[0107] Thus, in another embodiment, the polyethylene glycol has an average molecular weight between about 6,000 and about 10,000 g / mol, and / or the salt is selected from the group consisting of a sodium salt, a magnesium salt, a calcium salt, a potassium salt, a lithium salt, a barium salt and a cesium salt.

[0108] In a further embodiment, in the second step of the first aspect of the invention, the polyethylene glycol is added until a concentration of about 3 to 50 % is reached, and / or the salt is added until a concentration of about 0.01 to 15 M is reached.

[0109] In another embodiment, the polyethylene glycol is added until a concentration of about 4 to 45 % is reached, 5 to 40 % is reached, 6 to 35 % is reached, 7 to 30 % is reached, 8 to 25 % is reached, 9 to 20 % is reached, 10 to 15 % is reached, or 12.5 % is reached. In another embodiment, the polyethylene glycol is added until a concentration of about 3 to 45 % is reached, 3 to 40 % is reached, 3 to 35 % is reached, 3 to 30 % is reached, 3 to 25 % is reached, 3 to 20 % is reached, 3 to 15 % is reached, 3 to 10 % is reached, 3 to 8 % is reached, 3 to 6 % is reached, or 3 to 4 % is reached. In another embodiment, the polyethylene glycol is added until a concentration of about 4 to 50 % is reached, 5 to 50 % is reached, 6 to 50 % is reached, 8 to 50 % is reached, 10 to 50 % is reached, 15 to 50 % is reached, 20 to 50 % is reached, 25 to 50 % is reached, 30 to 50 % is reached, 35 to 50 % is reached, 40 to 50 % is reached, 45 to 50 % is reached, or 50 % is reached.

[0110] In an embodiment, the salt is added until a concentration of about 0.05 to 12 M is reached, 0.10 to 10 M is reached, 0.20 to 8 M is reached, 0.40 to 6 M is reached, 0.60 to 6 M is reached, 0.80 to 4 M is reached, 1 .00 to 3 M is reached, 1 .20 to 2 M is reached, 1 .40 to 1 .80 M is reached, or 1 .50 M is reached. In another embodiment, the salt is added until a concentration of about 0.01 to 14 M is reached, 0.01 to 13 M is reached, 0.01 to 12 M is reached, 0.01 to 11 M is reached, 0.01 to 10 M is reached, 0.01 to 9 M is reached, 0.01 to 8 M is reached, 0.01 to 7 M is reached, 0.01 to 6 M is reached, 0.01 to 5 M is reached, 0.01 to 4 M is reached, 0.01 to 3 M is reached, 0.01 to 2 M is reached, 0.01 to 1 M is reached, 0.01 to 0.8 M is reached, 0.01 to 0.6 M is reached, 0.01 to 0.4 M is reached, 0.01 to 0.2 M is reached, 0.01 to 0.1 M is reached, 0.01 to 0.08 M is reached, 0.01 to 0.06 M is reached, 0.01 to 0.06 M is reached, 0.01 to 0.04 M is reached, 0.01 to 0.02 M is reached, or 0.015 M is reached. In another embodiment, the salt is added until a concentration of about 0.05 to 15 M is reached, 0.10 to 15 M is reached, 0.20 to 15 M is reached, 0.30 to 15 M is reached, 0.40 to 15 M is reached, 0.60 to 15 M is reached, 0.80 to 15 M is reached, 1 .00 to 15 M is reached, 1 .25 to 15 M is reached, 1 .50 to 15 M is reached, 2 to 15 M is reached, 4 to 15 M is reached, 6 to 15 M is reached, 8 to 15 M is reached, 10 to 15 M is reached, 12 to 15 M is reached, or 14.5 M is reached.

[0111] Isolation of the target amplification product (e.g. the amplicons of the first and of the second type) is accomplished by removing the target amplicons-coated carriers from the amplification mixture. The solid phase carriers (e.g., paramagnetic microparticles) can be recovered from the amplification mixture, for example, by applying a magnetic field to draw down the paramagnetic microparticles. More specifically, paramagnetic microparticles are preferably separated from solutions using magnetic means, such as applying a magnet field of at least 1000 Gauss. Other methods known to those skilled in the art can be used to remove the magnetic microparticles from the supernatant; for example, vacuum filtration or centrifugation can be used. Thus, in another embodiment the SPRI support is separated from the amplification mixture in the second step of the first aspect of the invention using a method selected from the group consisting of applying a magnetic field, applying vacuum filtration and centrifugation.

[0112] The remaining solution can then be removed; leaving paramagnetic microparticles having the target amplicons (e.g., the amplicons of the first and of the second type) adsorbed to their surface. Once separated from the mixture, the target amplicons adsorbed to the solid phase carrier can be recovered by contacting the microparticles with a suitable elution buffer. As a result, a solution comprising the target amplicons and paramagnetic microparticles is produced. Using appropriate means, for example, magnetic means, the microparticles are subsequently removed from the solution whereby the target amplicons are isolated, obtaining an amplification mixture enriched in the amplicons of the first and of the second type.

[0113] As used herein, a suitable elution buffer or elution solution can be water or any aqueous solution in which the salt concentration and polyalkylene concentration are below the concentrations required for binding of the amplicons onto the solid phase carrier (e.g., the magnetic microparticles). For example, useful buffers include, but are not limited to, TRIS-HCI, Tris-acetate, sucrose (20 %) and formamide (100 %) solutions. Elution of the amplicons from the solid phase carrier occurs quickly (e.g., in thirty seconds or less) when a suitable low ionic strength elution buffer is used. Once the bound amplicons have been eluted, the solid phase carrier is separated from the elution buffer.

[0114] Optionally, impurities (e.g., DNA impurities) can be removed by washing the solid phase support (e.g., the paramagnetic microparticles) with the target amplicons bound thereto (e.g., by contacting the microparticles with a suitable wash buffer solution) before separating the microparticle-bound nucleic acid from the solid phase magnetically responsive microparticle. The composition of the wash buffer of wash solution is chosen to ensure that impurities either bound directly to the solid phase carrier, or associated with the adsorbed DNA are dissolved. The pH and solute composition and concentration of the wash buffer or wash solution can be varied according to the types of impurities which are expected to be present. For example, ethanol exemplifies a preferred wash buffer useful to remove excess PEG and salt. The magnetic microparticles with bound DNA can also be washed with more than one wash buffer solution. The paramagnetic microparticles can be washed as often as required (e.g., three to five times) to remove the desired impurities. However, the number of washings is preferably limited to in order to minimize loss of yield of the bound DNA. A suitable wash buffer solution has several characteristics. First, the wash buffer solution must have a sufficiently high salt concentration (a sufficiently high ionic strength) that the nucleic acid bound to the magnetic microparticles does not elute off of the microparticles, but remains bound. A suitable salt concentrations is greater than about 0.1 M and is preferably about 0.5M. Second, the buffer solution is chosen so that impurities that are bound to the DNA or microparticles are dissolved. The pH and solute composition and concentration of the buffer solution can be varied according to the types of impurities which are expected to be present. Suitable wash solutions include the following: 0.5x5 SSC; 100 mM ammonium sulfate, 400 mM Tris pH 9, 25 mM MgCh and 1 percent bovine serum albumin (BSA); and 0.5M NaCI. A preferred wash buffer solution comprises 25 mM Tris acetate (pH 7.8), 100 mM potassium acetate (KOAc), 10 mM magnesium acetate (Mg2OAc), and 1 mM dithiothreital (DTT).

[0115] Suitable magnetically responsive paramagnetic microparticles have sufficient surface area to permit efficient binding and are further characterized by having surfaces which are capable of reversibly binding nucleic acid molecules. Suitable solid phase carriers include, but are not limited to, other particles, fibers, beads and or supports which have an affinity for DNA and which can embody a variety of shapes, that are either regular or irregular in form, provided that the shape maximizes the surface area of the solid phase, and embodies a carrier which is amenable to microscale manipulations. Generally, paramagnetic microparticles are used.

[0116] As used herein, the term “paramagnetic microparticles” refers to microparticles which respond to an external magnetic field (e.g., a plastic tube or a microtiter plate holder with an embedded rare earth (e.g., neodymium) magnet but which demagnetize when the field is removed. Thus, the paramagnetic microparticles are efficiently separated from a solution using a magnet, but can be easily resuspended without magnetically induced aggregation occurring. Preferred paramagnetic microparticles comprise a magnetite rich core encapsulated by a pure polymer shell. Suitable paramagnetic microparticles comprise about 20-35 % magnetite / encapsulation ratio. For example, magnetic particles comprising a magnetite / encapsidation ration of about 23 %, 25 %, 28 %, 30 %, 32 % or 34 % are suitable for use in the present invention. Magnetic particles comprising less than about a 20 % ratio are only weakly attracted to the magnets used to accomplish magnetic separations. The use of encapsulated paramagnetic microparticles, having no exposed iron, or FesC on their surfaces, eliminates the possibility of iron interfering with polymerase function in certain downstream manipulations of the isolated DNA. However the larger the magnetite core the higher the chance of encapsulation leakage (e.g., release of iron oxides). Suitable paramagnetic microparticles for use in the instant invention can be obtained for example from Bangs Laboratories Inc., Fishers, Ind. (e.g., estapor(R) carboxylate-modified encapsulated magnetic microspheres).

[0117] Suitable paramagnetic microparticles should be of a size that their separation from solution, for example by magnetic means or by filtration, is not difficult. In addition, preferred paramagnetic microparticles should not be so large that their surface area is minimized or that they are not suitable for microscale manipulation. Suitable sizes range from about 0.1 pm mean diameter to about 100 pm mean diameter. A preferred size is about 1 .0 pm mean diameter.

[0118] As used herein, the term “functional group-coated surface” or “functional group of the surface ” refers to a surface of the solid phase carrier which is coated with moieties which reversibly bind nucleic acid (e.g., DNA, RNA or polyamide nucleic acids (PNA)). One example is a surface which is coated with moieties which each have a free functional group which is bound to the amino group of the amino silane or the microparticle; as a result, the surfaces of the microparticles are coated with the functional group containing moieties. The functional group acts as a bioaffinity adsorbent for polyalkylene glycol precipitated DNA. In one embodiment, the functional group is a carboxylic acid. A suitable moiety with a free carboxylic acid functional group is a succinic acid moiety in which one of the carboxylic acid groups is bonded to the amine of amino silanes through an amide bond and the second carboxylic acid is unbonded, resulting in a free carboxylic acid group attached or tethered to the surface of the paramagnetic microparticle. Suitable solid phase carriers having a functional group coated surface that reversibly binds nucleic acid molecules are for example, magnetically responsive solid phase carriers having a functional group-coated surface, such as, but not limited to, amino-coated, carboxyl-coated and encapsulated carboxyl group-coated paramagnetic microparticles.

[0119] In a yet another embodiment, in those cases in which (a) amplicons of a fourth type are generated during the first step of the method of the first aspect of the invention and / or (b) DNA fragments are present in the amplification mixture obtained in the first step of the method according to the first aspect of the invention; (a) and (b) having a length which is longer than the length of the amplicons of the first and of the second type, then the method of the first aspect of the invention further comprises an additional step prior to the second step of the first aspect of the invention, wherein the amplicons of the fourth type and / or the DNA fragments are removed from the amplification mixture obtained in the first step of the method of the first aspect of the invention by means of the binding of said amplicons of the fourth type and / or of said DNA fragments to a SPRI support which is capable of non-specifically binding to DNA in a size-dependent manner under conditions adequate for the binding of said amplicons of the fourth type and / or of said DNA fragments to the SPRI support, followed by the separation of the SPRI support from the mixture, thereby obtaining an amplification mixture free of the amplicons of the fourth type and / or of the DNA fragments.

[0120] The amplification mixture is maintained under conditions appropriate for adsorption of the precipitated nucleic acid molecules to the functional group-coated surfaces of the solid phase carriers, thereby producing solid phase carriers having the amplification product bound thereto (e.g. the amplicons of the fourth type). Thus, in one embodiment the conditions allowing the binding of the amplicons of the fourth type and / or of the DNA fragments to the SPRI support comprises adding a mixture of polyethylene glycol and a salt at concentrations adequate for the binding of the amplicons of the fourth type to the SPRI support.

[0121] In a further embodiment, the conditions allowing the binding of the amplicons of the fourth type and / or of the DNA fragments to the SPRI support requires adding polyethylene glycol until a concentration of about 2 to 30 % is reached, and / or adding the salt until a concentration of about 0.005 to 8 M is reached.

[0122] In another embodiment, the polyethylene glycol is added until a concentration of about 2.5 to 25 % is reached, 3 to 20 % is reached, 3.5 to 15 % is reached, 4 to 10 % is reached, 6 to 8 % is reached, or 7 % is reached. In another embodiment, the polyethylene glycol is added until a concentration of about 2 to 25 % is reached, 2 to 20 % is reached,

[0123] 2 to 15 % is reached, 2 to 10 % is reached, 2 to 5 % is reached, or 2 to 4 % is reached. In another embodiment, the polyethylene glycol is added until a concentration of about 2.5 to 30 % is reached, 3 to 30 % is reached, 3.5 to 30 % is reached, 4 to 30 % is reached, 6 to 30 % is reached, 8 to 30 % is reached, 10 to 30 % is reached, 15 to 30 % is reached, 20 to 30 % is reached, 25 to 30 % is reached, or 30 % is reached.

[0124] In an embodiment, the salt is added until a concentration of about 0.01 to 7 M is reached, 0.05 to 6 M is reached, 0.10 to 5 M is reached, 0.20 to 4 M is reached, 0.40 to

[0125] 3 M is reached, 0.60 to 2 M is reached, 0.80 to 1.00 M is reached, or 0.90 M is reached. In another embodiment, the salt is added until a concentration of about 0.005 to 7 M is reached, 0.005 to 6 M is reached, 0.005 to 5 M is reached, 0.005 to 4 M is reached, 0.005 to 3 M is reached, 0.005 to 2.5 M is reached, 0.005 to 2 M is reached, 0.005 to 1 .5 M is reached, 0.005 to 1 M is reached, 0.005 to 0.5 M is reached, 0.005 to 0.1 M is reached, 0.005 to 0.05 M is reached, 0.005 to 0.01 M is reached, or 0.005 M is reached. In another embodiment, the salt is added until a concentration of about 0.01 to 8 M is reached, 0.05 to 8 M is reached, 0.10 to 8 M is reached, 0.15 to 8 M is reached, 0.20 to 8 M is reached, 0.25 to 8 M is reached, 0.30 to 8 M is reached, 0.40 to 8 M is reached, 0.50 to 8 M is reached, 1.00 to 8 M is reached, 1.50 to 8 M is reached, 2.00 to 8 M is reached, 2.50 to 8 M is reached, 3 to 8 M is reached, 4 to 8 M is reached, 5 to 8 M is reached, 6 to 8 M is reached, or 7 M is reached.

[0126] In a third step of the method according to the first aspect of the invention, a second multiplex amplification of the mixture obtained in the second step of the first aspect of the invention is carried out using a set of primer pairs specific for the amplicons of the first and of the second type. This step is carried out essentially as described in the amplification method according to step (i) using primer pairs specific for each of the amplicons generated in said step (i) and which have been recovered during step (ii).

[0127] In one embodiment, the sets of primer pairs specific for each of the amplicons of the first and of the second type used in step (iii) comprise forward primers and reverse primers comprising at least a region located in the 3’ end that hybridizes with the custom extensions located in each of the amplicons of the first and of the second type.

[0128] In yet another embodiment, the sequences of the custom regions within each of the forward and reverse primers of each primer set used in step (i) are substantially identical and the set of primer pairs used in step (iii) consist of a single primer pair wherein the forward primer hybridizes with the 5’ custom extensions of all the amplicons of the first and of the second type and the reverse primer hybridizes with the 3’ custom extensions of all the amplicons of the first and of the second type.

[0129] In an embodiment, the amplification in the step (iii) of the method according to the first aspect of the invention is performed by polymerase chain reaction (PCR).

[0130] Kit of the invention and uses thereof

[0131] In a second aspect, the invention relates to a kit for the amplification of at least two amplicons from a template nucleic, said kit comprising:

[0132] (I) a set of primer pairs, wherein each primer pair comprises a forward primer and a reverse primer specific for different regions within the template nucleic acid wherein the 3’ end neighbouring upstream region overlaps with the 5’ end part of the neighbouring downstream region, and

[0133] (ii) reagents adequate for the size-dependent separations of DNA molecules.

[0134] As it is used herein, the term “kit” refers to a product containing the different reagents necessary for carrying out the uses and methods of the invention which is packed so as to allow their transport and storage. Materials suitable for packing the components of the kit include crystal, plastic (e.g., polyethylene, polypropylene, polycarbonate), bottles, vials, paper or envelopes.

[0135] All the terms and embodiments described in any of the previous aspect of the invention are equally applicable to the second aspect of the invention.

[0136] Also disclosed herein are kits that can be used for the amplification of at least two amplicons from a template nucleic acid. The kit can include a disclosed device, and one or more packages, receptacles, labels, or instructions for use. The kit may include at least one buffer. The kit may also include other components to facilitate using the device and methods thereof. Examples of such components include, but are not limited to, one or more additional reagents, such as one or more dilution buffers; one or more reconstitution solutions; one or more wash buffers; one or more storage buffers, one or more control reagents, (one or more additional component(s), such as a sample collection device (e.g., a syringe, cotton swab, tongue depressor, and the like), and the like. Components (e.g., reagents, components, etc.) may also be provided in a form that is usable in a particular assay, or in a form that requires addition of one or more other components before use (e.g. in concentrate or lyophilized form). Suitable buffers include, but are not limited to, phosphate buffered saline, sodium carbonate buffer, sodium bicarbonate buffer, borate buffer, Tris buffer, MOPS buffer, HEPES buffer, and combinations thereof. The choice of buffers and reagents will depend on the particular application, e.g., setting of the assay (point-of-care, research, clinical), analyte(s) to be assayed, the detection moiety used, etc. Such components may be provided individually or in combination(s) and may be provided in any suitable container such as a vial, a bottle, box, or a tube.

[0137] The kit may also include a packaging configured to contain the device and other components. The packaging may be a sealed packaging, such as a sterile sealed packaging. By "sterile" it is meant that there are substantially no microbes (such as fungi, bacteria, viruses, spore forms, etc.). In some embodiments, the packaging may be configured to be sealed, e.g., a water vapor-resistant packaging, optionally under an airtight and / or vacuum seal.

[0138] Following construction of the device, it can be optionally dried, e.g., by mild desiccation, blow drying, lyophilization, or exposure to ambient air at ambient temperature, for a time sufficient for the article to be dry or at least macroscopically dry. Once the device is dry or at least macroscopically dry, it may be sealed in a container (e.g., such as an impermeable or semipermeable polymeric container) in which it can be stored and shipped to a user. Once sealed in a container, the device may have, in some embodiments, a shelf life of at least 2 to 4 months, or up to 6 months or more, when stored at a temperature of 25 degrees centigrade (e.g., without loss of more than 20 percent, 30 percent or 50 percent of binding activity).

[0139] In addition to the above-mentioned components, the kit according to the present invention can further include instructions for using the components of the kit to practice the disclosed method. The instructions for practicing the methods are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or sub-packaging) etc. In some embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g., CD-ROM, diskette, flash drive, etc. In some other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g., via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.

[0140] In some embodiments, the kit includes instructions that correlate the number, intensity, or both with the size of the amplicons in the sample. In some embodiments, the kit includes a reference card that correlates the number and intensity with the size of the amplicons in the sample. In some embodiments, the kit further includes a smart device (e.g., tablet, smart phone, etc.).

[0141] In another embodiment, the set of primer pairs of the kit comprise forward primers and / or reverse primers from the one or more of the primer pairs specific for the upstream and downstream regions used in the first step of the first aspect of the invention and said forward primers and / or reverse primers comprise a target-specific region located in the 3’ end that hybridizes with the target nucleic acid region within the template nucleic acid and a custom region located in the 5’ end that does not hybridize with the target nucleic acid region.

[0142] In another embodiment, the set of primer pairs of the kit further comprise sequences of the custom regions within each of the forward primers of each set of primer pairs used in the first step of the first aspect of the invention which are substantially identical and / or sequences of the custom regions within each of the reverse primers of each set of primer pairs used in the first step of the first aspect of the invention which are substantially identical. In another embodiment, the kit further comprises a set of primer pairs designed for the amplification of amplicons of the first and of the second type used in the third step of the first aspect of the invention containing in their 5’ and 3’ ends sequences resulting from the 5’ and 3' custom extensions present in the set of primer pairs.

[0143] In one embodiment, the reagents of the kit adequate for the size-dependent separations of DNA molecules is a SPRI support. In yet another embodiment, the SPRI support is a solid phase carrier.

[0144] The kit may also comprise an aqueous solution of paramagnetic microparticles having a functional group-coated surface capable of reversibly binding nucleic acid molecules.

[0145] Thus, in a further embodiment, the solid phase carrier of the kit comprises a surface which is coated with a functional group that reversibly binds to DNA in a sizedependent manner. In another embodiment, the functional group of the surface coating of the solid phase carrier of the kit is selected from the group consisting of amine group- coating, carboxyl group-coating and encapsulated carboxyl g roup-coati ng. In a preferred embodiment, the functional group of the surface coating of the solid phase carrier is a carboxyl group-containing coat.

[0146] In addition the kit may comprise either at least one preformulated binding buffer comprising polyalkylene glycol and salt, at concentrations which are empirically determined to be appropriate to reversibly bind nucleic acid molecules characterized by a particular molecular size range onto the functional group-coated surfaces of the paramagnetic microparticles, or reagents for the formulation of a binding buffer.

[0147] Thus, in another embodiment, the kit further comprises a mixture of polyethylene glycol and a salt. In a further embodiment, the polyethylene glycol has an average molecular weight between about 6,000 and about 10,000 g / mol, and / or wherein the salt is selected from the group consisting of a sodium salt, a magnesium salt, a calcium salt, a potassium salt, a lithium salt, a barium salt and a cesium salt.

[0148] Additionally, the kit may comprise at least one preformulated high ionic strength buffer suitable for use as a wash buffer, or reagents for preparing such buffer, and a preformulated elution buffer or reagents for its preparation.

[0149] In an embodiment, the kit comprises one or more of the following components:

[0150] (i) a suitable wash solution, wherein said wash solution is capable of removing impurities bound to the SPRI support but does not affect the binding of the DNA to the SPRI support, (ii) a suitable elution solution, wherein the elution solution is capable of disrupting the binding of the DNA to the SPRI support,

[0151] (iii) an enzyme for nucleic acid sequence amplification, wherein said enzyme is a DNA polymerase, and

[0152] (iv) a dNTP mixture.

[0153] Suitable wash buffers or wash solutions capable of removing impurities bound to the SPRI support not affecting the binding of the DNA to the SPRI support have been descried in the first aspect of the invention and apply equally to the second aspect of the invention. The term “impurities” or “contaminants” refers to molecules including proteins, other cellular components or undesired nucleic acids present in the amplification mixture, either bound directly to the SPRI support or associated with the adsorbed DNA are dissolved.

[0154] Suitable elution buffers or elution solutions capable of disrupting the binding of the DNA to the SPRI support have been descried in the first aspect of the invention and apply equally to the second aspect of the invention.

[0155] In a preferred embodiment, the wash solution is a high ionic strength buffer and / or the elution solution is a low ionic strength buffer.

[0156] The term “DNA polymerase” and “dNTPs” have been descried in the first aspect of the invention and apply equally to the second aspect of the invention.

[0157] In another embodiment, the kit further comprises a magnetic plate holder for applying a magnetic field, a vacuum filtration system for applying vacuum filtration, or a centrifuge for performing the centrifugation.

[0158] The magnetic plate holder designed to optimize the features of the magnetic field are known to be crucial to the efficiency of automated processing. The design of the magnetic plate holder is instrumental in producing a magnetic field having the requisite uniformity and field strength to maximize the efficiency achievable with automatic processing. Field strength of up to and over 1600 Gauss can be achieved with the use of N35 rare earth magnets configured with alternating North and South polar spaced 9 mm apart. Since the magnetically responsive microparticles are paramagnetic they will attract to either pole.

[0159] Method for the sequencing of nucleic acids

[0160] In a third aspect, the invention relates to a method for the sequencing of a template nucleic acid, the method comprising generating amplicons from the template nucleic acid by a method according to the first aspect of the invention and sequencing the amplicons of the first and of the second type obtained in the third step of the first aspect of the invention.

[0161] All the terms and embodiments described in any of the previous aspect of the invention are equally applicable to the second aspect of the invention.

[0162] The term “sequencing” or “DNA sequencing” is commonly applied to several methods and technologies that are used for determining the order of the nucleotide bases adenine, guanine, cytosine, and thymine in a molecule of DNA. As of today, a number of different sequencing techniques exist, that are commonly subsummized under first generation sequencing, second generation sequencing, and third generation, or single molecule sequencing (SMS), all of which are known in the art.

[0163] Any DNA sequencing method known in the art can be used in the methods provided herein. Non-limiting examples of DNA sequencing methods useful in the methods provided herein include long-read molecule sequencing, for example, as described in Liu, Y. et al. (2020). Pan-genome of wild and cultivated soybeans. Cell, 182(1 ), 162-176, Next Generation Sequencing (NGS) technologies, for example, as described in Egan, A.N, et al. (2012) American Journal of Botany 99(2): 175-185; genotyping by sequencing (GBS) methods, for example, as described in Elshire, R.J., et al. (2011 ) PLoS ONE 6(5):el9379; Molecular Inversion Probe (MIP) genotyping, as described, for example, in Hardenbol, P., et al. (2003) Nature Biotechnology 21 (6):673- 678; or high throughput genotyping by whole-genome resequencing, as described, for example in Huang, X et al., (2009) Genome Research 19: 1068-1076.

[0164] In another embodiment, the sequencing comprises short read-length sequencing reactions.

[0165] The term “short read-length sequencing” refers to sequencing of read lengths less than 600 base pairs, particularly less than 500 base pairs.

[0166] In another embodiment, the method for sequencing of the third aspect of the invention comprises generating the sequence of the template nucleic acid from the sequences of the amplicons of the first and the second type obtained in the third step of the first aspect of the invention.

[0167] ***

[0168] The invention is described below by means of the following examples which are to be construed as merely illustrative and not limitative of the scope of the invention.

[0169] EXAMPLES Example 1 : SERPINA1 gene variants identification by next generation sequencing (NGS) using a unique first multiplex amplification reaction for amplicon library preparation.

[0170] 1- Material and methods

[0171] 1.1 . First multiplex amplification of a template nucleic acid

[0172] 14 gene-specific PCR primer pairs were designed using Primer3 v4.1.0 (http: / / primer3.ut.ee) to amplify 7 exonic regions of SERPINA1 gene containing variants associated with Alpha-1 antitrypsin deficiency (A1ATD). SERPINA1 is a gene consisting of 20,946 base pairs under the NCBI accession number NG_008290.1 (entry of 2 June 2024). These gene-specific primers contain a region targeting SERPINA1 in the 3’ region of the primer and the tag sequence used in Illumina's Nextera XT Index kits in the 5’ region of the primer (SEQ ID NO: 1 : 5’-

[0173] TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3’ for the forward primers and SEQ ID NO: 2: 5’- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3’ for the reverse primers). The length of the gene-specific primers ranged from 51 to 57 base pairs (bp). 7 forward gene-specific PCR primer from a primer pair were located between 1 bp -12 bp upstream to the reverse gene-specific PCR primer from a different primer pair such that a short amplicon partially overlapping the amplicons generated by each primer pair could obtained in a single multiplex amplification reaction.

[0174] Amplification reactions were prepared using 0.35 ng-1000 ng of DNA, 0.2 pM or 0.3 pM of each primer and 2x QIAGEN Multiplex PCR Master Mix (Cat. No. i ID: 206145). Cycling was performed as follows: one cycle of 95 °C for 15 minutes, 23 cycles of 95 °C for 30 seconds and 58 °C for 1 minute and 72 °C for 1 minute, and one cycle of 72 °C for 7 minutes.

[0175] 7.2- Purification of the amplicons of the first and of the second type from the amplification mixture

[0176] PCR products were purified with Agencourt SPRISelect Reagent beads in two steps using ratios beads / sample of 0.2 and 0.4 (Beckman Coulter product No: B23318).

[0177] 7.3- Second multiplex amplification of the amplicons of the first and of the second type of the amplification mixture Purified products were amplified through Nextera XT Index kits (Illumina Cat. ID: FC-131-2001 and FC-131-2004) using 2x QIAGEN Multiplex PCR Master Mix (Cat. No. I ID: 206145. Cycling was performed as follows: one cycle of 95 °C for 15 minutes, 14 cycles of 95 °C for 30 seconds and 55 °C for 1 minute and 72 °C for 1 minute, and one cycle of 72 °C for 10 minutes. The indexed libraries were subsequently normalized with SequalPrep™ Normalization Kit (Thermo Fisher, Cat. No.: A1051001 ) and pooled. The pool was quantified using the Qubit High Sensitivity dsDNA Assay (Thermo Fisher, Cat. No.: Q32854). Following the manufacturer's instructions, the pooled libraries were sequenced on an Illumina MiSeq (MiSeq v2 kit or MiSeq Nano v2 kit, Illumina Cat. ID: MS-103-1003) using a 2 x 250 bp paired-end sequencing protocol. Reads were aligned to Complete Human Chromosome 14 (hg38 chr14) and variants called through a dedicated CLC Genomics Workbench v22 pipeline from QIAGEN

[0178] 2- Results

[0179] 2.1- Set of primer pairs for SERPINA 1 amplification and amplicons generated

[0180] The relative position of the 14 gen-specific primer pairs over SERPINA1 gene and the amplicons generated (first, second and third type, according to the invention) in the first multiplex amplification reaction are illustrated in Figure 1.

[0181] Each primer pair consists of a forward primer (F) and a reverse primer (R) that amplify a region of interest ranging from 200 bp to 247 bp, such that the amplicons generated range from 267 to 314 bp. The details of the primer pair sets are shown in table 1.

[0182] Table 1. Primer Pairs for amplifying region of interest within SERPIN A1 gene and amplicons of interest. Each primer pair consists of a forward primer (Fn) and a reverse (Rn) primer suitable for generating an amplicon of interest. Some forward primers (Fn+1 ) and reverse primers (Rn) (both in bold) are suitable for generating a short amplicon that partially overlaps with some of the amplicons of interest.

[0183] For some primer pairs that target neighboring regions, the F primer of one of the primer pairs and the R primer of the different primer pair are suitable for generating a short amplicon that partially overlaps with the amplicons of interest generated by their corresponding primer pairs. The details of these primers and the short amplicons generated is shown in Table 2. Table 2. Primers Fn+1 and Rn that are suitable for generating a short amplicon that partially overlaps with some of the amplicons of interest. Distance between the 3' end of those primers.

[0184] Then, during the first multiplex amplification reaction not only the 14 amplicons of interest (first type and second type) are generated but also 7 additional short overlapping amplicons (third type). The details of the first, second and third type of amplicons generated during the first multiplex amplification reaction are shown in table 3.

[0185] Table 3. Amplicons generated with the primer pair sets in the first single multiplex amplification reaction. 2.2- Samples tested

[0186] A panel of 73 samples covering more than 99.9% of the A1AT mutations frequency in the general population from different biological sources (Dried Blood Spot (DBS), Saliva, Cell Lines and Synthetic Controls) and different DNA extraction methods (QIAamp DNA from QIAgen, Lysis and Neutralization from Sigma, Extracta DNA Prep for PCR from QuantaBio and Kapa Express Extract from Kapa Biosystems) were sequenced by a NGS assay using a unique multiplex amplification reaction for the amplicon library preparation.

[0187] DNA samples from DBS and saliva were not quantified and diluted after extraction. DNA from cell lines were diluted at 0.4 ng / pL and two serial 1 :500 dilutions were performed for Synthetic Controls.

[0188] Table 4 summaries the number of samples included in experiment per its source and method of extraction used. Table 5 shows all genotypes included (Synthetic Controls correspond to the last 4 entries of the table). Table 4. Sample types and DNA extraction methods used in the experiment.

[0189] Sample type DNA Extraction Method # Samples

[0190] Saliva Sigma 12

[0191] Saliva QuantaBio 10

[0192] Saliva QIAgen 21

[0193] Saliva Kapa Biosystems 10

[0194] Dried Blood Spot (DBS) Sigma 14

[0195] Cell lines (Coriell) Manufacturer’s CoA 2

[0196] Synthetic Controls Oligonucleotide synthesis inserted in plasmids 4 This sample panel covers more than 99.9% of the A1AT mutations frequency in the general population.

[0197] Table 5. Genotypes, variant description and number of samples in experiment.

[0198]

[0199] SNVs and small InDeis, as different zygosity (heterozygous, compound heterozygous and homozygous) are included. 2.3- Amplicon library preparation of SERPIN A1 gene regions of interest

[0200] Each DNA from the 73 samples from were added to a reaction well plate and amplified in a first multiplex amplification reaction using the 14 gene-specific primer pairs described above in the amplification conditions described in Material and Methods section.

[0201] After amplification, SPRISelect Reagent beads were added to each reaction well in a ratio 0.2 beads / sample. After 2 minutes incubation of the beads and the sample, a magnet was used to collect SPRISelect Reagent beads on the walls of each the reaction wells. Supernatant was transferred to a new reaction well plate.

[0202] SPRISelect Reagent beads were added to each reaction well in a ratio 0.4 beads / sample. After 1 -minute incubation, a magnet was used to collect SPRISelect Reagent beads on the walls of each the reaction wells. Supernant was discarded. The SPRISelect Reagent beads on each reaction well were washed with 200 microL of Ethanol 80% and the supernatant discarded. 20 microL of ultrapure water was added to each reaction well to elute the amplicons bound to the beads and the elution liquid transferred to a new reaction well plate.

[0203] Nextera XT Index kits were added to each reaction well and amplification was performed following conditions described in Material and Methods section to generate indexed amplicon libraries that were subsequently normalized with SequalPrep™ Normalization Kit and pooled. After quantification, the pooled libraries were sequenced on an Illumina MiSeq and variants identified as described in Material and Methods section.

[0204] 2.4- Variant identification

[0205] The method demonstrates very high accuracy for all kind of mutations, type of sample and DNA extraction methods. All samples showed a 100% coverage of the regions of interest with a depth of coverage well over 60X (60 reads for each position to be detected). All variants were correctly detected in all samples and no unexpected variants were detected in the regions of interest. The overall call rate was therefore 100%. A panel of 2221 bp was analysed for each of the samples except for the 4 synthetic controls where a reduced panel of 1024 bp was analyzed. In total, 157.345 positions were analysed. Table 6 summarizes final percentage of agreement for this experiment. Table 6. Summary result of the experiment Number of false negative (#FN), Number of false positive (#FP), Number of true negative (#TN), Number of true positive (#TP), positive percentage of agreement (PPA%), negative percentage of agreement (NPA%), percentage of overall agreement (OA%) and TPPV - (Technical Positive Predictive Value). PPA = TP / (TP+ FN), NPA = TN / (TN + FP), TPPV = TP / (TP+FP), OA=(TP+TN) / # positions

[0206] Example 2: SERPINC1 gene variants identification by next generation sequencing (NGS) using a unique first multiplex amplification reaction for amplicon library preparation. Design 1.

[0207] 1- Material and methods

[0208] 1.1- First multiplex amplification of a template nucleic acid

[0209] 13 gene-specific PCR primer pairs were designed using Primer3 v4.1.0 (http: / / primer3.ut.ee) for amplifying 7 exonic regions of the SERPINC1 gene containing variants associated with hereditary antithrombin deficiency (hATD). SERPINC1 is a gene consisting of 20,575 base pairs under the NCBI accession number NG_012462.1 (entry of 17 March 2024). These gene-specific primers contain a region targeting SERPINC1 in the 3’ region of the primer and the tag sequence used in Illumina's Nextera XT Index kits in the 5’ region of the primer as in example 1. The length of the gene-specific primers ranged from 50 to 62 bp. 6 forward gene-specific PCR primers from a primer pair were located between 29 bp - 79 bp upstream to the reverse gene-specific PCR primer from a different primer pair such that a short amplicon partially overlapping the amplicon amplicons generated by each primer pair could be obtained in a single multiplex amplification reaction.

[0210] Amplification reactions were prepared using 50 ng of DNA, 0.1 pM to 0.3 pM of each primer and 2x QIAGEN Multiplex PCR Master Mix (Cat. No. i ID: 206145). Cycling was performed as follows: one cycle of 95 °C for 15 minutes, 23 cycles of 95 °C for 30 seconds and 62 °C for 1 minute and 72 °C for 1 minute, and one cycle of 72 °C for 7 minutes.

[0211] 7.2- Purification of the amplicons of the first and of the second type from the amplification mixture

[0212] PCR products were purified with Agencourt SPRISelect Reagent beads in one step using ratios beads / sample of 0.4 (Beckman Coulter product No: B23318). 1.3- Second multiplex amplification of the amplicons of the first and of the second type of the amplification mixture

[0213] Purified products were amplified as in example 1 . 2- Results

[0214] 2.1- Set of primer pairs for SERPINC1 amplification and amplicons generated

[0215] The relative position of the 13 gene-specific primer pairs over SERPINCI gene and the amplicons generated (first, second and third type, according to the invention) in the first multiplex amplification reaction are illustrated in Figure 2. Each primer pair consists of a forward primer (F) and a reverse primer (R) that amplify a region of interest ranging from 172 bp to 201 bp, such that the amplicons generated range from 288 bp to 307 bp. The details of the primer pair sets are shown in table 7.

[0216] Table 7. Primer Pairs for amplifying region of interest within SERPINC1 gene and amplicons of interest. Each primer pair consists of a forward primer (Fn) and a reverse (Rn) primer suitable for generating an amplicon of interest. Some forward primers (Fn+1 ) and reverse primers (Rn) (both in bold) are suitable for generating a short amplicon that partially overlaps with some of the amplicons of interest.

[0217] For some primer pair sets that targets contiguous regions, the F primer of one of the primer sets and the R primer of the different primer set are suitable for generating a short amplicon that partially overlaps with the amplicons of interest generated by their corresponding primer pair sets. The details of these primers and the short amplicons generated is shown in Table 8. Table 8. Primers Fn+1 and Rn that are suitable for generating a short amplicon that partially overlaps with some of the amplicons of interest. Distance between the 3' end of those primers.

[0218] Then, during the multiplex amplification reaction not only the 13 amplicons of interest (first type and second type) are generated but also 6 additional short overlapping amplicons (third type). The details of the first, second and third type of amplicons generated during the first multiplex amplification reaction are shown in Table 9.

[0219] Table 9. Amplicons generated with the primer pair sets in the first single multiplex amplification reaction. 2.2- Samples tested

[0220] A panel of 37 samples (Saliva samples collected in buccal swabs or whole blood samples collected in EDTA) were sequenced by a NGS assay using a unique multiplex amplification reaction for the amplicon library preparation. These samples contained several variants and SNPs in the SERPINC1 gene that had been previously confirmed by Sanger sequencing. Table 10 shows all genotypes included.

[0221] DNAs from blood and saliva were extracted using QIAamp Blood DNA Mini Kit (QIAGEN) following manufacturer’s instructions with two minor modifications: starting quantity material was 400 pl and the elution volume was set to 50 pl. Extracted DNAs were stored at - 20 °C. Table 5. Genotypes and number of samples in experiment.

[0222] 2.3- Amplicon Library preparation of SERPINC1 gene region of interest Each DNA isolated from biological samples were added to a reaction well plate and amplified in a first multiplex amplification reaction using the 13 gene-specific primer pairs described above in the amplification conditions described in the Material and Methods section.

[0223] After amplification, SPRISelect Reagent beads were added to each reaction well in a ratio 0.4 beads / sample. After 1 -minute incubation of the beads, a magnet was used to collect SPRISelect Reagent beads on the walls of each of the reaction wells. Supernatant was discarded. Then, the SPRISelect Reagent beads on each reaction well were washed with 180 pl of Ethanol 85% and the supernatant discarded. 25 pl of ultrapure water was added to each reaction well to elute the amplicons bound to the beads and the elution liquid transferred to a new reaction well plate.

[0224] Nextera XT Index kits were added to each reaction well and amplification was performed following conditions described in the Material and Methods section to generate indexed amplicon libraries that were subsequently normalized with SequalPrep™ Normalization Kit and pooled. After quantification, the pooled libraries were sequenced on an Illumina MiSeq as in Example 1.

[0225] 2.4- Variant identification

[0226] All 37 samples showed a 100% coverage of the regions of interest with a depth of coverage well over 100X (100 reads for each position to be detected). All variants were correctly detected in all samples and no unexpected variants were detected in the regions of interest. The overall call rate was therefore 100%.

[0227] Table 11. Overall Call Rate of the verification study performed with a set of samples from volunteer donors.

[0228] Example 3: SERPINC1 gene variants identification by next generation sequencing (NGS) using a unique first multiplex amplification reaction for amplicon library preparation. Design 2.

[0229] 1- Material and methods

[0230] 1.1- First multiplex amplification of a template nucleic acid

[0231] 12 gene-specific PCR primer pairs were designed using Primer3 v4.1.0 (http: / / primer3.ut.ee) for amplifying 7 exonic regions of the SERPINC1 gene containing variants associated with hereditary antithrombin deficiency (hATD). SERPINC1 is a gene consisting of 20,575 base pairs under the NCBI accession number NG_012462.1 (entry of 17 March 2024). These gene-specific primers contain a region targeting SERPINC1 in the 3’ region of the primer and the tag sequence used in Illumina's Nextera XT Index kits in the 5’ region of the primer as in example 1. The length of the gene-specific primers ranged from 51 to 62 bp. 5 forward gene-specific PCR primers from a primer pair were located between 0 bp - 6 bp upstream to the reverse gene-specific PCR primer from a different primer pair such that a short amplicon partially overlapping the amplicon amplicons generated by each primer pair could be obtained in a single multiplex amplification reaction.

[0232] Amplification reactions were prepared using 50 ng of DNA, 0.1 pM to 0.3 pM of each primer and 2x QIAGEN Multiplex PCR Master Mix (Cat. No. I ID: 206145). Cycling was performed as follows: one cycle of 95 °C for 15 minutes, 23 cycles of 95 °C for 30 seconds and 62 °C for 1 minute and 72 °C for 1 minute, and one cycle of 72 °C for 7 minutes.

[0233] 1.2- Purification of the amplicons of the first and of the second type from the amplification mixture

[0234] PCR products were purified with Agencourt SPRISelect Reagent beads in one step using ratios beads / sample of 0.4 (Beckman Coulter product No: B23317).

[0235] 1.3- Second multiplex amplification of the amplicons of the first and of the second type of the amplification mixture

[0236] Purified products were amplified as in example 1 .

[0237] 2- Results

[0238] 2.1- Set of primer pairs for SERPINC1 amplification and amplicons generated

[0239] The relative position of the 12 gene-specific primer pairs over SERPINCI gene and the amplicons generated (first, second and third type, according to the invention) in the first multiplex amplification reaction are illustrated in Figure 3.

[0240] Each primer pair consists of a forward primer (F) and a reverse primer (R) that amplify a region of interest ranging from 172 bp to 209 bp, such that the amplicons generated range from 288 bp to 317 bp. The details of the primer pair sets are shown in table 12. Table 12. Primer Pairs for amplifying region of interest within SERPINC1 gene and amplicons of interest. Each primer pair consists of a forward primer (Fn) and a reverse (Rn) primer suitable for generating an amplicon of interest. Some forward primers (Fn+1 ) and reverse primers (Rn) (both in bold) are suitable for generating a short amplicon that partially overlaps with some of the amplicons of interest.

[0241] For some primer pair sets that targets contiguous regions, the F primer of one of the primer sets and the R primer of the different primer set are suitable for generating a short amplicon that partially overlaps with the amplicons of interest generated by their corresponding primer pair sets. The details of these primers and the short amplicons generated is shown in Table 13. Table 13. Primers Fn+1 and Rn that are suitable for generating a short amplicon that partially overlaps with some of the amplicons of interest. Distance between the 3' end of those primers.

[0242] Then, during the multiplex amplification reaction not only the 12 amplicons of interest (first type and second type) are generated but also 5 additional short overlapping amplicons (third type). The details of the first, second and third type of amplicons generated during the first multiplex amplification reaction are shown in Table 14.

[0243] Table 14. Amplicons generated with the primer pair sets in the first single multiplex amplification reaction. 2.2- Variant identification

[0244] The same panel of sample used in Example 2 were used for the preparation of the corresponding Amplicon Library of SERPINC1 gene region of interest. As in experiment 2, all 37 samples showed a 100% coverage of the regions of interest and all variants were correctly detected in all samples and no unexpected variants were detected in the regions of interest.

[0245] Summary of the main features of the different designs used in Examples 1 , 2 and

[0246] 3 are shown in Table 15.

[0247] Table 15: Summary of the main features of the different designs used in Examples 1 , 2 and 3.

[0248] Example 4: Minimal difference in length to allow purification of amplicons based on their size using SPRISelect Reagent beads.

[0249] In order to confirm the minimal difference in length between two amplicons to be separated based on their size, a DNA library consisting of two amplicons, one having 314 bp and the other one having 267 bp (47 bp length difference), was prepared using two primer pairs containing the tag sequences used in Illumina's Nextera XT Index kits in the 5’ region of the primers as in the experiments above.

[0250] After amplification, the amplicons were purified using SPRISelect Reagent beads at different ratios beads / sample of 0.1 , 0.2, 0.3 and 0.4 (Beckman Coulter product No: B23318) following the conditions described in previous experiments. Eluates containing the amplicons bound to the beads were transferred to a new reaction well plate where a second amplification reaction with Nextera XT Index kits was performed following conditions described in previous experiments. Amplicons were further quantified in the Bioanalyzer instrument (Agilent). Figure 4a shows the content of the first multiplex amplification, where the high content of primers is shown. Figure 4b shows the content of the size-based purification with different ratios beads / sample of the second multiplex amplification. It can be seen that both amplicons of 267 and 314 bp have been purified from the primers and that both amplicons are purified together at all ratios of beads / sample. Note that Nextera XT index kits add a total of 69 bp to each amplicon so the final length of the amplicons is 336 and 383 bp.

Claims

CLAIMS1. A method for amplification of at least two amplicons from a template nucleic acid present in a sample comprising the steps of:(I) Performing a first multiplex amplification of at least two neighbouring regions from the template nucleic acid wherein the 3’ end part of the neighbouring upstream region overlaps with the 5’ end part of the neighbouring downstream region, wherein said amplification is carried out using a set of two primer pairs, and wherein each primer pair comprises a forward primer and a reverse primer specific for each of the regions, and wherein the amplification products comprise: a. Amplicons of a first type resulting from the amplification of the upstream region with the forward and reverse primers specific for said upstream region, b. Amplicons of a second type resulting from the amplification of the downstream region with the forward and reverse primers specific for said downstream region, and c. Amplicons of a third type resulting from the amplification of the template nucleic acid with the reverse primer of the primer pair specific for the upstream region and the forward primer of the primer pair specific for the downstream region,(ii) removing the amplicons of the third type from the amplification mixture obtained in step (i) by a method which separates the amplicons of the first type and amplicons of the second type from the amplicons of the third type based on their sizes thereby obtaining an amplification mixture enriched in the amplicons of the first and of the second type, and(iii) performing a second multiplex amplification of the mixture obtained in (ii) using a set of primer pairs specific for the amplicons of the first and of the second type.

2. The method according to claim 1 , wherein the forward primers and / or the reverse primers from the one or more of the primer pairs specific for the upstream and downstream regions used in step (i) comprise a target-specific region located in the 3’ end that hybridizes with the target nucleic acid region within the template nucleic acid and a custom region located in the 5’ end that does not hybridize with the targetnucleic acid region, thereby resulting in that each of the amplicons obtained in step (i) contain 5’ and / or 3’ custom extensions which incorporate the sequence of the custom regions present in the forward or reverse primers.

3. The method according to claim 2, wherein the custom region of the reverse primer of the primer pair specific for the upstream region and the custom region of the forward primer of the primer pair specific for the downstream region used in step (i) are not substantially complementary to each other.

4. The method according to any one of claims 1 to 3, wherein each strand of the amplicons of the third type is not susceptible to acquire a secondary structure upon annealing conditions, such as a hairpin structure.

5. The method according to any one of claims 2 to 4, wherein the sequences of the custom regions within each of the forward primers used in step (I) are substantially identical and / or wherein the sequences of the custom regions within each of the reverse primers used in step (I) are substantially identical.

6. The method according to any one of claims 1 to 5, wherein the set of primer pairs specific for each of the amplicons within the amplicons of the first and of the second type used in step (ill) comprise forward primers and reverse primers comprising at least a region located in the 3’ end that hybridizes with the custom extensions located in each of the amplicons of the first and of the second type.

7. The method according to any one of claims 2 to 6, wherein the sequences of the custom regions within each of the forward and reverse primers of each primer set used in step (I) are substantially identical and wherein the set of primer pairs used in step (ill) consist of a single primer pair wherein the forward primer hybridizes with the 5’ custom extensions of all the amplicons of the first and of the second type and the reverse primer hybridizes with the 3’ custom extensions of all the amplicons of the first and of the second type.

8. The method according to any one of claims 1 to 7, wherein the amplicons of the first and of the second type obtained in step (I) have a length of 200 to 400 base pairs.

9. The method according to any one of claims 1 to 8, wherein the amplicons of the third type have a length of 100 to 190 base pairs.

10. The method according to any one of claims 1 to 9, wherein the length of the amplicons of the first and of the second type is of at least 50 base pairs longer than the length of the amplicons of the third type.

11. The method according to any one of claims 1 to 10, wherein the ratio of the lengths of the amplicons of the first and of the second type and the length of the amplicons of the third type is of at least 1.2:1.

12. The method according to any one of claims 1 to 11 , wherein the amplification in steps (I) or (iii) is performed by polymerase chain reaction (PCR).

13. The method according to any one of claims 1 to 12, wherein the sample is selected from the group consisting of:(i) a DNA containing biological sample;(ii) a solution resulting from an DNA amplification procedure;(iii) a solution resulting from a DNA size shearing procedure;(iv) a solution resulting from a nucleotide sequencing reaction;(v) a solution resulting from a restriction enzyme digestion comprising a mixture of nucleic acid molecule fragments;(vi) an agarose solution containing nucleic acids; and(vii) a DNA containing solution resulting from a DNA purification procedure.

14. The method according to claim 13, wherein the biological sample is selected from the group consisting of cell lines, blood, serum, plasma, saliva, hair, urine, skin tissue, bone marrow, semen, rectal and vaginal fluid.

15. The method according to any one of claims 1 to 14, wherein the separation of the amplicons of the first and of the second type from the amplicons of the third type based on the size in step (II) is carried out by a method selected from the group consisting of Solid Phase Reversible Immobilization (SPRI), gel electrophoresis, ionexchange chromatography, slalom chromatography, high-performance liquid chromatography, size-exclusion (gel-permeation) chromatography, density gradient centrifugation and microfluidics.

16. The method according to claim 15, wherein step (ii) is carried out by SPRI and wherein the amplicons of the third type are removed from the amplification mixture obtained in (i) by means of the binding of the amplicons of the first and of the second type to a SPRI support which is capable of non-specifically binding to DNA in a sizedependent manner under conditions adequate for the binding of the amplicons of the first and of the second type to the SPRI support, followed by the separation of the SPRI support from the mixture and elution of the amplicons of the first and of the second type from the SPRI support, thereby obtaining an amplification mixture enriched in the amplicons of the first and of the second type.

17. The method according to claim 16, wherein the SPRI support is a solid phase carrier.

18. The method according to any one of claims 16 or 17, wherein the solid phase carrier comprises a surface which is coated with a functional group that reversibly binds to DNA in a size-dependent manner.

19. The method according to claim 18, wherein the functional group of the surface coating of the solid phase carrier is selected from the group consisting of amine group-coating, carboxyl group-coating and encapsulated carboxyl g roup-coati ng.

20. The method according to claim 19, wherein the functional group of the surface coating of the solid phase carrier is a carboxyl group-containing coat.21 . The method according to any one of claims 16 to 20, wherein the SPRI support is a magnetically responsive solid phase carrier.

22. The method according to any one of claims 16 to 21 , wherein the SPRI support is separated from the amplification mixture in step (ii) using a method selected from the group consisting of applying a magnetic field, applying vacuum filtration and centrifugation.

23. The method according to any one of claims 16 to 22, wherein the conditions allowing the binding of the amplicons of the first and of the second type to the SPRI support in step (ii) comprises adding a mixture of polyethylene glycol and a salt atconcentrations adequate for the binding of the amplicons of the of the first and of the second type to the SPRI support.

24. The method according to claim 23, wherein the polyethylene glycol has an average molecular weight between about 6,000 and about 10,000 g / mol, and / or wherein the salt is selected from the group consisting of a sodium salt, a magnesium salt, a calcium salt, a potassium salt, a lithium salt, a barium salt and a cesium salt.

25. The method according to any one of claims 23 or 24, wherein the polyethylene glycol is added until a concentration of about 3 to 50 % is reached, and / or wherein the salt is added until a concentration of about 0.01 to 15 M is reached.

26. The method according to any one of claims 1 to 25, further comprising an additional step prior to step (II), wherein a) amplicons of a fourth type generated in step (I) and having a length which is longer than the length of the amplicons of the first and of the second type, and / or b) DNA fragments present in the amplification mixture having a length which is longer than the length of the amplicons of the first and of the second type are removed from the amplification mixture obtained in step (I) by means of the binding of said amplicons of the fourth type and / or of said DNA fragments to a SPRI support which is capable of non-specifically binding to DNA in a sizedependent manner under conditions adequate for the binding of said amplicons of the fourth type and / or of said DNA fragments to the SPRI support, followed by the separation of the SPRI support from the mixture, thereby obtaining an amplification mixture free of the amplicons of the fourth type and / or of the DNA fragments.

27. The method according to claim 26, wherein the conditions allowing the binding of the amplicons of the fourth type and / or of the DNA fragments to the SPRI support requires adding polyethylene glycol until a concentration of about 2 to 30 % is reached, and / or adding the salt until a concentration of about 0.005 to 8 M is reached.

28. A kit for the amplification of at least two amplicons from a template nucleic using the method according to any one of claims 1 to 27, said kit comprising:(i) a set of primer pairs, wherein each primer pair comprises a forward primer and a reverse primer specific for different regions within the template nucleic acid wherein the 3’ end neighbouring upstream region overlaps with the 5’ end part of the neighbouring downstream region, and(ii) reagents adequate for the size-dependent separations of DNA molecules.

29. The kit according to claim 28, wherein the reagents adequate for the size-dependent separations of DNA molecules is a SPRI support.

30. The kit according to any one of claims 28 or 29, wherein the forward primers and / or the reverse primers from the one or more of the primer pairs specific for the upstream and downstream regions used in step (i) comprise a target-specific region located in the 3’ end that hybridizes with the target nucleic acid region within the template nucleic acid and a custom region located in the 5’ end that does not hybridize with the target nucleic acid region.

31. The kit according to claim 30, wherein the sequences of the custom regions within each of the forward primers of each set of primer pairs used in step (i) are substantially identical and / or wherein the sequences of the custom regions within each of the reverse primers of each set of primer pairs used in step (i) are substantially identical.

32. The kit according to any one of claims 28 to 31 , further comprising a set of primer pairs designed for the amplification of amplicons of the first and of the second type used in step (iii) containing in their 5’ and 3’ ends sequences resulting from the 5' and 3' custom extensions present in the set of primer pairs.

33. The kit according to any one of claims 28 to 32, wherein the SPRI support is a solid phase carrier.

34. The kit according to claim 33, wherein the solid phase carrier comprises a surface which is coated with a functional group that reversibly binds to DNA in a sizedependent manner.

35. The kit according to claim 34, wherein the functional group of the surface coating of the solid phase carrier is selected from the group consisting of amine g roup-coati ng, carboxyl group-coating and encapsulated carboxyl group-coating.

36. The kit according to claim 35, wherein the functional group of the surface coating of the solid phase carrier is a carboxyl group-containing coat.

37. The kit according to any one of claims 28 to 36, wherein the kit further comprises a magnetic plate holder for applying a magnetic field, a vacuum filtration system for applying vacuum filtration, or a centrifuge for performing the centrifugation.

38. The kit according to any one of claims 28 to 37, wherein said kit further comprises a mixture of polyethylene glycol and a salt.

39. The kit according to claim 38, wherein the polyethylene glycol has an average molecular weight between about 6,000 and about 10,000 g / mol, and / or wherein the salt is selected from the group consisting of a sodium salt, a magnesium salt, a calcium salt, a potassium salt, a lithium salt, a barium salt and a cesium salt.

40. The kit according to any one of claims 28 to 39, wherein said kit further comprises one or more of the following components:(I) a suitable wash solution, wherein said wash solution is capable of removing impurities bound to the SPRI support but does not affect the binding of the DNA to the SPRI support,(II) a suitable elution solution, wherein the elution solution is capable of disrupting the binding of the DNA to the SPRI support,(ill) an enzyme for nucleic acid sequence amplification, wherein said enzyme is a DNA polymerase, and(iv) a dNTP mixture.41 . The kit according to claim 40, wherein the wash solution is a high ionic strength buffer and / or wherein the elution solution is a low ionic strength buffer.

42. A method for the sequencing of a template nucleic acid, the method comprising generating amplicons from the template nucleic acid by a method according to any of claims 1 to 27 and sequencing the amplicons of the first and of the second type obtained in step (ill).

43. The method according to claim 42, wherein the sequencing comprises short readlength sequencing reactions.

44. The method according to any one of claims 42 or 43, comprising generating the sequence of the template nucleic acid from the sequences of the amplicons of the first and the second type obtained in step (ill).

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