WO2013017702A1 - Méthode pour la détermination du génotype de séquences multiples - Google Patents

Méthode pour la détermination du génotype de séquences multiples Download PDF

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WO2013017702A1
WO2013017702A1 PCT/ES2011/070565 ES2011070565W WO2013017702A1 WO 2013017702 A1 WO2013017702 A1 WO 2013017702A1 ES 2011070565 W ES2011070565 W ES 2011070565W WO 2013017702 A1 WO2013017702 A1 WO 2013017702A1
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interest
amplification
reaction
sequencing
sequences
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PCT/ES2011/070565
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Spanish (es)
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Pablo CASTÁN GARCÍA
Ivana TONIC
Alistair Edward RITCHIE
Pedro Manuel Franco De Sarabia Rosado
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2B Blackbio S.L.
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Priority to PCT/ES2011/070565 priority Critical patent/WO2013017702A1/fr
Publication of WO2013017702A1 publication Critical patent/WO2013017702A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention describes a method for the determination of the genotype of multiple sequences of interest, more specifically, it describes a method for determining the genotype of multiple sequences simultaneously in a single reaction, and in particular, a method for the determination of the genotype of single nucleotide polymorphisms (SNPs) simultaneously in a single reaction.
  • SNPs single nucleotide polymorphisms
  • sequences which are the basis of the aforementioned syndromes, are very often separated long distances upstream / downstream of a particular genetic marker, therefore their parallel analysis is a requirement to be able to provide an accurate diagnosis.
  • a significant number of clinical uses occur when this kind of distant sequences (which often show different coupling patterns) are analyzed, including the determination of the consequences of complex diseases, uses in farmocogenetics, legal medicine, and epidemiological studies. In this sense, a significant number of genetic disorders are the result of the interaction between sequences distant from each other, contributing to the risk of developing a particular disease.
  • Single nucleotide polymorphisms represent the most abundant type of sequence variation in the human genome and can be useful for many diverse uses, including determining the genetic architecture of complex traits and diseases, applications in pharmacogenetics, legal medicine , and evolutionary studies. Many genetic disorders are the result of the interaction of more than one SNP, each contributing to the risk of developing a particular disease. In many cases these SNPs with which the risk of disease development are associated are very separated in the same gene, or even present in different genes. Traditionally the genotype of sequences and in particular SNPs is determined by microsequencing reactions.
  • the conventional method includes a PCR reaction to amplify a region of DNA that contains the sequence of interest and in particular the SNP of interest and to amplify a DNA sequence that flanks the sequence / SNP of interest and that can be used as a quality control. internal.
  • the microsequencing reaction is then carried out with which the sequence flanking the sequence / SNP of interest as well as the sequence of interest itself and in particular the SNP can be read and its genotype can be resolved.
  • Genotyping of multiple sequences and in particular of multiple SNPs is carried out by doing a PCR reaction and a sequencing reaction for each sequence and / or SNP of interest.
  • a sequencing reaction for example see Lin YS, Liu FG, Wang TY, Pan CT, Chang WT, Li WH (2010) "A simple method using Pyrosequencing TM to identify de novo SNPs in pooled DNA samples.”
  • Anal Biochem 363 (2): 275-287 The method requires multiple sequencing reactions that make it more expensive as well as lengthen the test.
  • large quantities of starting template DNA necessary to sequence the multiple regions of interest are required, thus making this process unfeasible for cases where the amount of starting template DNA sequencing is very low.
  • the present invention aims at determining the genotype of multiple sequences of interest, in particular, of multiple single nucleotide polymorphisms (SNPs) simultaneously in a single sequencing reaction.
  • SNPs single nucleotide polymorphisms
  • FIG. 1 shows a simple PCR reaction with a multiplex genotyping reaction.
  • FIG. 2 shows a multiplex PCR reaction with a multiplex genotyping reaction.
  • FIG. 3 shows the image of the agarose gel that results from the simple PCR, according to example 1.
  • FIG. 4 shows an overview of the overall quality of pyrosequencing reactions, according to example 1.
  • FIG. 5 shows the image of the pyrograms obtained from the simple and multiplex genotyping of SNP, according to example 1.
  • FIG. 6 shows the results of the genotype obtained for the template DNA, according to example 1.
  • FIG. 7 shows the image of the agarose gel that results from the simple PCR, according to example 2.
  • FIG. 8 shows a view of the quality of pyrosequencing reactions, according to example 2.
  • FIG. 9 shows the image of the pyrograms of the simple and multiplex genotyping of SNP, according to example 2.
  • FIG. 10 shows the genotype results obtained for the template DNA, according to example 2.
  • FIG. 1 1 shows the image of the agarose gel that results from the simple PCR, according to example 3.
  • FIG. 12 shows the image of the agarose gel that results from multiplex PCR, according to example 3.
  • FIG. 13 shows an overview of the overall quality of pyrosequencing reactions, according to example 3.
  • FIG. 14 shows the genotype results obtained for the template DNA, according to example 3.
  • FIG. 15 shows the image of the agarose gel that results from the gelled PCR, according to example 4.
  • FIG. 16 shows an overview of the overall quality of pyrosequencing reactions, according to example 4.
  • sequences of interest includes, but is not limited to, genetic areas encoding or not (ie introns / exons), repetitions in tanden and satellites, 5 'UTR leader regions, high mutational intensity points ... and, in general, any DNA fragment whose modification leads to changes in the behavior of a particular organism in relation to the development of abnormalities.
  • target includes, but is not limited to, molecules, genes, or genomes, which contain a nucleic acid sequence or segment of a sequence that is interested in being characterized by identification, quantification or amplification.
  • Targets contemplated under this invention can be derived from any organism, including mammals and non-mammals, bacteria, viruses or fungi.
  • a retrovirus is an example of a target that can be identified or quantified using highly conserved sequences to make genomic mapping.
  • nucleic acid analyte may alternatively be used to identify a nucleic acid, a nucleic acid sequence, or a segment of a sequence of a nucleic acid within an organism, bacteria, or virus, which is subject to characterization.
  • gene includes, but is not limited to, a particular nucleic acid sequence within a DNA molecule that occupies a precise position on a chromosome and is capable of self-replication by encoding a specific polypeptide chain.
  • gene refers to a complete set of genes in the chromosomes of each cell of a specific organism.
  • nucleotide and nucleoside include, but are not limited to, nucleosides that contain not only the four nucleotide bases of natural DNA, ie, guanine purine bases (G ) and adenine (A) and pyrimidine cytosine (C) and thymine (T) bases, but also the uracil (U) RNA purine base, iso-G and iso-C non-natural nucleotide bases, universal bases , degenerate bases, and other modified nucleotides and nucleosides.
  • G guanine purine bases
  • A adenine
  • C pyrimidine cytosine
  • T thymine
  • U uracil
  • oligonucleotide includes, but is not limited to, polydeoxosiribonucleotides (containing 2-dexosi-D-ribose), polybibonucleotides (containing D-ribose), and any other type of polynucleotide that is a N-glycoside of a purine or pyrimidine base, and other polymers containing non-nucleotide characters (for example, any type of modified DNA or RNA chain from those used in different applications in molecular biology and medicine, where they can be used as a probe to investigate diseases, viral infections, identify genes, specific target regions within a chromosome ... etc.
  • modified oligonucleotides can be made of different nucleotide-derived base units, organized in various forms such as nucleic acids from proteins and polymers of synthetic nucleic acids with a specific sequence commercially available through Anti-Gene Development Group, Corvallis, Oregon, such as NEUGENE TM polymers) or non-standard junctions, provided that the polymers contain core-bases in a configuration that allows for matching and alignment of the bases, such as found in DNA and RNA.
  • oligonucleotides include double and single stranded DNA, as well as double and single stranded RNA and DNA: RNA hybrids, and also includes known types of modified oligonucleotides, such as, for example, oligonucleotides where one or more of the natural nucleotides are replaced by an analog; oligonucleotides that contain inter-nucleotide modifications such as, for example, those with non-anionic / non-cationic junctions (e.g., methyl phosphonates, phosphotriesters, phosphoromidates, carbamates, etc.), anionic junctions (e.g., phosphorothioates, phosphorodithioates, etc.) , and cationic junctions (eg amino-alkyl phosphoromidates, amino-alkyl phosphotriesters), which contain interaction domains such as, for example, proteins (including nucleases, toxins, antibodies, signal peptides,
  • amplification oligonucleotide includes, but is not limited to, an oligonucleotide that is complementary to the cDNA or RNA target molecule and provides the 3'-OH end as a substrate to which any DNA polymerase can add the nucleosides of a chain. increasing DNA in the 5'-3 'direction.
  • Simple includes, but is not limited to, a single test that is not carried out simultaneously with any other test.
  • Singleplex tests include individual tests that are carried out sequentially.
  • multiplex assay includes, but is not limited to, multiple tests that are carried out simultaneously, in which the detection and analysis steps are generally carried out in parallel.
  • a multiplex assay will include the use of primers and probes, alone or in combination with other primers and additional probes for the identification, for example, of the influenza virus together with one or more additional viruses.
  • primers and probes for internal controls and assays may include for example oligonucleotides.
  • gelled amplification reagents includes, but is not limited to, stabilized amplification reagents that preserve their chemical and biochemical qualities.
  • amplification reagents include but are not limited to, reaction buffers, reaction enhancers, and enzymes involved in an enzymatic reaction, in this case the amplification of nucleic acids and the reactions associated with sequencing by synthesis, once all are included.
  • reaction buffers in this case multi-well tubes or plates, so that they are each dosed in the optimal reaction amounts, and do not interact or react with each other, immobilizing the biochemical reaction in which they intervene, being able to activate the enzymatic reaction at the will of the user, without having produced a significant decrease in their activity, having elapsed days, weeks, months or even years after mixing and stabilization.
  • the stabilization thus understood is achieved by the addition of a stabilizing mixture to a solution containing the reaction mixture, and the subsequent elimination of all or part of the water present in the resulting solution.
  • This removal of all or part of the water can be achieved by lyophilization processes, dried in a fluid bed, dried at room temperature and atmospheric pressure, dried at room temperature and low pressure, dried at high temperature and atmospheric pressure, and dried at high Temperature and low pressure.
  • the stabilization method preferably used is the stabilization by gelation, described in WO 02/072002, assigned to Biotools Biotechnological & Medical Laboratories, SA
  • the stabilizing mixture of the reaction mixture is preferably composed of trehalose, melezitosa , lysine or betaine and glycogen or raffinose, at different concentrations depending on the enzymatic reaction to stabilize. More preferably the gelation mixture is composed of trehalose, melezitose, glycogen and lysine.
  • stabilizers are preferably desiccation by vacuum at a temperature between 30 5 C and 40 5 C, depending on the enzymatic reaction to be stabilized. Specifically, in the present invention the moisture content is maintained between 10-30% water.
  • the present invention discloses a method for the determination of multiple sequences, in particular, single nucleotide polymorphisms (SNPs) simultaneously.
  • the method comprises the amplification of one or more target sequences present in a sample to produce an amplified product, and the genotyping of the amplified product with multiple sequencing oligonucleotides simultaneously.
  • the sample may be a DNA sample or an RNA sample and the target sequence is a sequence in the DNA sample or in the RNA sample comprising one or more sequences to be identified, in particular, SNP sites of interest and a adjacent sequence which can be used as internal quality control.
  • the amplification reaction may be a simple amplification reaction or a multiplex amplification reaction depending on the location of the sequence of interest, in particular the SNP sites in the sample.
  • the simple amplification reaction is used when two or more sequences of interest, in particular SNP sites of interest, are located at a distance not exceeding 1 kbp from each other.
  • a single reaction is designed with a pair of amplification oligonucleotides that covers all relevant sequences, in particular SNP sites in a single amplicon.
  • the multiplex amplification reaction is used when the sequences of interest, in particular the SNP sites of interest are located too far from each other to be included in a single amplicon, or due to the presence of a long intermediate sequence, or because the sequences of interest, particularly SNP sites are located in different genes or chromosomes
  • Multiplex amplification reactions are designed with a mixture of pairs of amplification oligonucleotides that share similar chemical compositions, so they can be used under the same concentrations of some reagents, same alignment temperatures and same extension times. Similar chemical compositions should be understood as nucleotide base units that need to be organized so that each unit has a complementary base to which it will bind.
  • the mixture of amplification oligonucleotide pairs comprises at least one pair of amplification oligonucleotides corresponding to each of the target sequences to be amplified.
  • the primers and amplification oligonucleotides used are complementary to the 3 '(three prime) ends of each parallel and anti-parallel strand of the strand of the target sequence.
  • the oligonucleotides align first to the sample and then the amplification reaction takes place.
  • One of the oligonucleotides of the amplification oligonucleotide pair is biotinylated so that the amplified product obtained is biotinylated.
  • the amplification reaction is the polymerase chain reaction (PCR), however, other amplification reactions such as, but not limited to, the ligase chain reaction (LCR), mutagenesis systems synthesis-directed (ARMS), specific allele PCR (ASPCR), degenerate PCR can be used for amplification of the target sequence without altering the scope or spirit of the present invention.
  • PCR polymerase chain reaction
  • other amplification reactions such as, but not limited to, the ligase chain reaction (LCR), mutagenesis systems synthesis-directed (ARMS), specific allele PCR (ASPCR), degenerate PCR can be used for amplification of the target sequence without altering the scope or spirit of the present invention.
  • the amplification reaction is stabilized by the process and gelation reagents described in WO 02/072002.
  • the sequencing oligos are stabilized by the process and gelation reagents described in WO 02/072002.
  • the amplified product produced by a simple amplification reaction or a multiplex amplification reaction is a hybrid of at least two sequences of interest, in particular SNPs sites and their adjacent sequences.
  • This amplified product is then genotyped with a mixture of sequencing oligonucleotides. simultaneously in a single multiplex genotyping reaction.
  • the sequencing oligonucleotide mixture comprises at least one sequencing oligonucleotide for each of the sequences of interest, in particular SNP sites of interest.
  • the sequencing oligonucleotides bind a few bases upstream to the sequence of interest, in particular the SNP site to initiate the sequencing process.
  • genotyping of the amplified product is done by the pyrosequencing method, however, other genotyping methods such as, but not limited to, Sanger sequencing, mass sequencing systems by fragment stacking (MPSS), sequencing polarized, Illumination sequencing, SOLiD sequencing, SANGER microfluidic sequencing, hybridization sequencing or the like can also be used for genotyping the amplified product without altering the scope and spirit of the present invention.
  • MPSS fragment stacking
  • the genotyping reaction is carried out as follows: an oligonucleotide or hybrid sequencing primer with a single stranded amplified product or amplified amplicon or PCR amplicon serving as a template, and is incubated with the enzymes, DNA polymerase, ATP sulphorylase, luciferase and apyrase as well as with the substrates, 5 'phosphosulfate adenosine (APS), and luciferin.
  • an oligonucleotide or hybrid sequencing primer with a single stranded amplified product or amplified amplicon or PCR amplicon serving as a template, and is incubated with the enzymes, DNA polymerase, ATP sulphorylase, luciferase and apyrase as well as with the substrates, 5 'phosphosulfate adenosine (APS), and luciferin.
  • APS 5 'phosphosulfate adenosine
  • the first deoxyribonucleotide triphosphate (dNTP) is added to the reaction.
  • DNA polymerase catalyzes the incorporation of dNTPs into the DNA strand, if it is complementary to the base present in the template strand.
  • PPi pyrophosphate
  • ATP sulphorylase converts PPi to ATP in the presence of APS. This ATP conducts the luciferase-mediated conversion of luciferin to oxyluciferin that produces visible light in amounts proportional to the amount of ATP.
  • the light produced in the reaction catalyzed by luciferase is detected by a camera or a photomultiplier and is observed as a peak in the information of the raw data obtained (Pyrogram).
  • the height of each peak corresponds to the light signal that is proportional to the number of nucleotides incorporated during the genotyping reaction.
  • An enzyme that degrades nucleotides such as apyrase, degrades nucleotides that have not been incorporated and ATP. When degradation is complete, another nucleotide is added. This is a continuous process during the genotyping reaction. The addition of dNTPs is sequential.
  • dATP.S dexosiadenosine alpha-thio-triphosphate
  • dATP dexosiadenosine triphosphate
  • the sequencer such as, but not limited to, microsequencer used during the genotyping reaction is programmed with a target sequence that includes the sequences adjacent to each of the multiple sequences of interest, in particular SNPs sites. This allows the genotyping software to determine if the correct reference sequence is present.
  • FIG. 1 shows a simple PCR reaction with a multiplex genotyping reaction according to an exemplary embodiment of the present invention.
  • the DNA sample contains two SNP sites of interest (SNP1 and SNP2) that are located less than 1 kb from each other.
  • a target sequence of the DNA sample containing both SNP sites (SNP1 and SNP2) is amplified by polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • A01 and A01 ' are used, of which one of them, A01' is biotinylated.
  • the resulting PCR product is biotinylated and contains both SNP SNP1 and SNP2 sites.
  • two sequencing oligonucleotides S01 and S02 are aligned to the PCR product.
  • During genotyping reactions adjacent sequences and those that include both SNPs overlap.
  • FIG.2 shows a multiplex PCR with a multiplex genotyping reaction according to an exemplary embodiment of the present invention.
  • the DNA sample contains two SNP sites of interest (SNP1 and SNP2) that are located in two different genes.
  • Two target sequences of the DNA sample, each containing one of the SNP sites of interest, are amplified by the use of two pairs of amplification oligonucleotides, wherein the first pair of amplification oligonucleotides (A01 and A01 ') It is for the first SNP site of interest (SNP1) and the second couple of amplification oligonucleotides (A02 and A02 ') is for the second SNP site of interest.
  • the amplification oligonucleotides A01 'and A02' are biotinylated.
  • the resulting PCR products are biotinylated and contain an SNP site.
  • two sequencing oligonucleotides S01 and S02 are aligned to the PCR products. During genotyping reactions adjacent sequences and those that include both SNPs overlap
  • the method according to one of the exemplary embodiments of the present invention uses a single amplification reaction and a single genotyping reaction reducing the work and costs related to the preparation of the sample, numerous tests, need to have a large amount of Starting DNA and large number of sequencing reactions. In addition, the method is technically less complicated and also reduces the overall cost.
  • kits consist of: i) all the necessary reagents in the optimal amounts and concentrations for the generation of a simple or multiplex amplification reaction of the targets of interest ( precisely amplification oligonucleotides, DNA polymerase, dexosinucleotides and reaction buffer) all pre-mixed and stabilized by means of the gelation process and,
  • the CDKN2a gene contains two SNPs, Rs10757283 and Rs1081 1661, which are associated with an increased risk of suffering from type II diabetes mellitus. These SNPs are linked in equilibrium and are separated by 77 bases.
  • the following amplification oligonucleotides are designed: (i) a 23-base unmodified forward oligonucleotide and (ii) a 20-base reverse-biotinylated oligonucleotide.
  • the forward oligonucleotide has a theoretical alignment temperature of 60.8 5 C and the reverse oligonucleotide has an alignment temperature of 62.4 5 C.
  • the optimal reaction alignment temperature was determined experimentally at 55 5 C.
  • the two SNPs are read by the sequencing oligonucleotide 1 of 17 bases, which hybridizes 3 bases upstream of Rs1081 1661 and has an alignment temperature of 50.0 5 C, and sequencing oligonucleotide 2 of 19 bases, which hybrid adjacent to Rs10757283 and has an alignment temperature of 53.7 5 C.
  • the sequencing oligonucleotide 1 reads the sequence SEQ ID NO: 1 (TTCYCATGAC) with the dispensing order SEQ ID NO: 2 (GTCTCGATGA), and the sequencing oligonucleotide 2 reads the sequence SEQ ID NO: 3 ( YTGATATTCT) with the dispensing order SEQ ID NO: 4 (GCTCGATAT).
  • the sequences are read in the order of dispensing SEQ ID NO: 5 (GCTAGCTCGATGA).
  • a reaction mixture is prepared with the amplification oligonucleotides of CDKN2a.
  • DNA polymerase enzyme manufactured by Biotools Biotechnological & Medical Laboratories SA, 5 ⁇ of a reaction that is marketed together with the enzyme, between 0.1 ⁇ and 0.3 ⁇ of a 10mM solution containing the four dexosiribonucleotides that form the dexosiribonucleic acid chain (dATP, dTTP, dGTP, dCTP) and between 0.2 ⁇ and 0.5 ⁇ of a solution 10 ⁇ of the pair of amplification oligonucleotides described in the materials section to generate a 246 bp amplicon containing the two SNPs of interest (Rs10757283 and Rs1081 1661).
  • FIG. 3 shows the results of the agarose gel. 30ng of template DNA was loaded onto the agarose gel together with a control without template DNA (NTC), where 10 ⁇ of PCR product was mixed with 2 ⁇ of loading buffer and ran on a 2% agarose gel in TBE 0.5x to 100V for 30 minutes.
  • NTC control without template DNA
  • FIG. 4 shows the overall quality of pyrosequencing reactions.
  • Wells A1 and A2 correspond to the single-stranded PCR product with oligo sequencing 1.
  • Wells A3 and A4 correspond to the single-stranded PCR product with sequencing oligo 2.
  • Wells A5 and A6 correspond to the single-stranded PCR product with oligos sequencing 1 and 2 at equimolar concentrations. The blue circles represent the high quality of the sequence obtained.
  • FIG. 5 represents the pyrograms of SNP simple and multiplex genotyping.
  • the first pyrogram represents the resulting peaks when the sequencing oligo 1 is added to the template DNA.
  • the second pyrogram represents the resulting peaks when oligo sequencing 2 is added to the template DNA.
  • the third pyrogram represents the resulting peaks when the oligos of sequencing 1 and 2 are added at equimolar concentrations. The peaks determine the incorporation of each new additional dexosinucleotide.
  • FIG. 6 shows the genotypic results obtained for the template DNA
  • Wells A1 and A2 correspond to the single stranded PCR product with the sequencing oligo 1.
  • Wells A3 and A4 correspond to the single-stranded PCR product with the sequencing oligo 2.
  • Wells A5 and A6 correspond to the single-stranded PCR product with the sequencing oligos 1 and 2 at equimolar concentrations. Consequently, any person skilled in the art understands that in wells from A1 to A4 where only one SNP was genotyped and the result of a single genotype is shown. The multiplex reactions shown in wells A5 to A6 give two results, of course.
  • the image described in FIG. 3 shows that the amount of PCR product produced is approximately equivalent as long as 30ng of template DNA is added to the reaction. Slight differences appear due to errors in the pipetting, during the assembly of the PCR, or when the gel is loaded.
  • the control without template DNA shows some signals that occur as a result of a residual signal from the PCR product loaded into the adjacent well in the gel or due to aerosol formation during PCR assembly. In any case, the difference between PCR reactions and controls without template DNA is evident. Since the amount of PCR products is very similar, any difference in genotyping results is due to simple versus multiple genotyping methods. The quality of the PCR products remains similar in all cases and is not variable, which is evident to one skilled in the art. The results illustrated in FIG.
  • the image illustrated in FIG. 5 describes the resulting pyrograms obtained.
  • Well-defined peaks appear at the expected positions, coinciding with the reference sequence,
  • (a) the first pyrogram was obtained from the genotyping reaction where only oligo sequencing 1 was used.
  • (b) the second pyrogram was obtained from the genotyping reaction where only the sequencing oligo 2 was used.
  • (c) the third pyrogram was obtained from the genotyping reaction where the sequencing oligos 1 and 2 were used
  • the resulting pyrogram of the multiplex genotyping corresponds to the sequences obtained with the sequencing oligo 1 superimposed with the sequence obtained with the sequencing oligo. 2.
  • the two polymorphic bases present a great approximation with the results of simple reactions.
  • the results illustrated in FIG. 6 describe the genotypes obtained by multiple sequencing according to the genotypes obtained by simple sequencing.
  • the genotype of SNP 1 is shown in wells A1, A2, A5 and A6 with position 1, and is T / T in all cases.
  • the genotype of SNP 2 is shown in wells A2 and A3 with position 1 and in wells A5 and A6 with position 2. Therefore, the result of the same genotypes from both single genotyping and multiple genotyping is shown. . Therefore, it is apparent to any person skilled in the art that it is possible to obtain genotype data related to multiple SNPs using multiple sequencing oligos with the same single stranded template DNA.
  • the TCF7a gene contains two SNPs, Rs12255372 and Rs7903146, which are associated with damage to sulfonylurea metabolism. These SNPs are linked in equilibrium and are separated by more than 50,000 bases of coding and non-coding sequence.
  • the following amplification oligonucleotides are designed to generate a 278 bp amplicon and a 246 bp amplicon.
  • the 278 bp amplicon uses (i) an 18-base unmodified forward oligonucleotide and (ii) a 21-base reverse-biotinylated oligonucleotide.
  • the forward oligonucleotide has a theoretical alignment temperature of 56.9 5 C and the reverse oligonucleotide has an alignment temperature of 57.9 5 C.
  • the optimal reaction alignment temperature was determined experimentally at 55 5 C.
  • the 246 bp amplicon uses (i ) a 20-base unmodified forward oligonucleotide and (ii) a 20-base reverse-biotinylated oligonucleotide.
  • the forward oligonucleotide has a theoretical alignment temperature of 54.9 5 C and the reverse oligonucleotide has an alignment temperature of 54.9 5 C.
  • the optimal reaction alignment temperature was determined experimentally at 55 5 C.
  • the two SNPs are read by the 16 base sequencing oligonucleotide 1, which hybridizes from the third base prior to Rs1081 1661 and has a alignment temperature of 53.3 5 C, and the 19 base sequencing oligonucleotide 2, which hybridizes from first base prior to Rs10757283 and has an alignment temperature of 48.1 5 C.
  • the sequencing oligonucleotide 1 reads the sequence SEQ ID NO: 9 (AATKACCATA) with the dispensing order SEQ ID NO: 10 (GATGACAT), and the sequencing oligonucleotide 2 reads the sequence SEQ ID NO: 1 1 (AYTATATAATTTAATTGCCGTATGAGG) with the dispensing order SEQ ID NO: 12 (GACTGATAT).
  • the sequences are read in the order of dispensing SEQ ID NO: 13 (GACTGCATACA).
  • a reaction mixture is prepared with the amplification oligonucleotides of TCF7L2.
  • DNA polymerase enzyme manufactured by Biotools Biotechnological & Medical Laboratories SA, 5 ⁇ of reaction buffer that is sold together with the enzyme, between 0.1 ⁇ and 0.3 ⁇ of a 10mM solution containing the four deoxyribonucleotides that make up the deoxyribonucleic acid chain (dATP, dTTP, dGTP, dCTP) and between 0.2 ⁇ and 0.5 ⁇ of a 10 ⁇ solution of the pair (four in total) of amplification oligonucleotides described in the materials section for generate a 278 bp amplicon and a 246 bp amplicon in a single multiplex amplification reaction.
  • Each amplicon contains SNP Rs12255372 and SNP Rs7903146 respectively.
  • FIG. 7 Shows the results of the agarose gel. 30ng 2% agarose gel was loaded onto the template DNA together with a control without template DNA (NTC). Each PCR replica should have approximately equivalent amounts of the PCR products. The theoretical sizes of the PCR products are 278pb and 246pb.
  • FIG. 8 Shows the overall quality of pyrosequencing reactions.
  • Wells B1 and B2 contain two single chain PCR products only with oligo 1 sequencing.
  • Wells B3 and B4 contain two single chain PCR products only with oligo 2 sequencing.
  • Wells B5 and B6 contain two single chain PCR products with oligo 1 and 2 sequencing at equimolar concentrations. The blue circles represent the high quality of the sequence obtained.
  • FIG. 9 represents the pyrograms of SNP simple and multiplex genotyping. The first pyrogram represents the resulting peaks when oligo 1 sequencing is added to the template DNA. The second pyrogram represents the resulting peaks when oligo 2 sequencing is added to the template DNA. The third pyrogram represents the resulting peaks when sequencing oligos 1 and 2 are added at equimolar concentrations to the template DNA. The peaks determine the incorporation of each new additional dexosinucleotide.
  • FIG. 10 shows the genotypic results obtained for the template DNA
  • Wells B1 and B2 contain two single chain PCR products only with oligo 1 sequencing.
  • Wells B3 and B4 contain two single chain PCR products only with oligo 2 sequencing.
  • Wells B5 and B6 contain two single chain PCR products with oligo 1 and 2 sequencing at equimolar concentrations. Consequently, any person skilled in the art understands that in wells B1 to B4 a single SNP was genotyped since only one genotype is shown. The multiplex sequencing reactions shown in wells B5 and B6 clearly yield two results.
  • the image described in FIG. 7 shows that the amount of PCR product produced is approximately equivalent as long as 30ng of template DNA is added to the reaction. Slight differences appear due to errors in the pipetting, during the assembly of the PCR, or when the gel is loaded.
  • the control without template DNA can give signal drawn from the PCR product loaded into the adjacent well in the gel or by aerosol formation during PCR assembly. The signal strength of the PCR product may be such that it is difficult to distinguish between the 278 bp PCR product and the 246 bp PCR product, however this seems to be uniform for both reactions in all wells. In any case, the difference between the PCR reaction and the control without template DNA is evident.
  • any difference in genotyping results is due to simple versus multiple genotyping methods.
  • the quality of the PCR products remains similar in all cases and is not variable, which is evident to one skilled in the art.
  • the results illustrated in FIG. 8 show high quality data in genotyping reactions as expected. The blue circles appear to confirm the high quality. A failure in the reaction is indicated by a red circle and means low quality. The inconsistency between the reference sequence and the results obtained or a poor signal ratio appears yellow. All reactions in this case pass the quality test regardless of the number of sequencing oligos that were added.
  • the image illustrated in FIG. 9 describes the pyrograms obtained. Well defined peaks appear in the expected positions, coinciding with the reference sequence, (a) the first pyrogram was obtained from the genotyping reaction using only oligo 1 sequencing. (b) the second pyrogram was obtained from the genotyping reaction using only oligo 2 sequencing. (c) the third pyrogram was obtained from the genotyping reaction using oligos 1 and 2 sequencing. The pyrogram resulting from multiple genotyping corresponds to the sequences obtained with the sequencing oligo 1 superimposed with the sequence obtained with the sequencing oligo 2. The two polymorphic bases present a great approximation with the results of simple reactions.
  • the results illustrated in FIG. 10 describe the genotypes obtained by multiple sequencing according to the genotypes obtained by simple sequencing.
  • the genotype of SNP 1 is shown in wells B1, B2, B5 and B6 with position 1, and is G / G in all cases.
  • the genotype of SNP 2 is shown in wells B2 and B3 with position 1 and in wells B5 and B6 with position 2, and is T / C in each case.
  • TCF7L2 MULTIPLEX GELIFIED PCR, MULTIPLE GENOTIPATE WITH GELIFIED SEQUENCING OLIGONUCLEOTIDES Introduction:
  • This example reproduces Example 2 using the gelation process applied to multiplex PCR reactions and sequencing oligos used for multiple genotyping.
  • a reaction mixture is prepared with the amplification oligonucleotides of TCF7L2.
  • This reaction mixture is composed of DNA polymerase enzyme at a final concentration of 6 U, manufactured by Biotools Biotechnological & Medical Laboratories SA, reaction buffer that is marketed together with the enzyme at a final concentration of 1 X, the four dexosiribonucleotides that form the dexosiribonucleic acid chain (dATP, dTTP, dGTP, dCTP) at a final concentration between 200 ⁇ and 250 ⁇ each and a solution at a final concentration between 75 nM and 150 nM each of: i) the two pairs (four oligos in total) of amplification oligos described in the materials section to generate at the same time in a multiplex amplification reaction a 278bp amplicon and a 246bp amplicon.
  • Each amplicon contains SNP Rs12255372 and SNP Rs7903146 respectively, ii) the pair of amplification oligonucleotides individually for the simple PCR reactions corresponding to the amplification of SNP Rs12255372 and SNP Rs7903146 independently.
  • the reaction mixtures (both simple and multiplex) are stabilized by the gelation process.
  • a gelation mixture composed of trehalose, melezitose, glycogen and lysine is added to the reaction mixtures described above which contain all the reagents necessary to carry out the PCR reactions.
  • the gelation process is completed after drying under vacuum conditions and at a temperature below 40 5 C, resulting in a stabilized reaction mixture containing between 10% and 30% water.
  • the two sequencing oligos were stabilized by the gelation process following the same protocol described above for the PCR reaction mixture.
  • the gelled oligos are re-hydrated with 85 ⁇ of Milli-Q water. Prepare a 96 Q96 plate with 20 ⁇ hybridization buffer, and with the following combinations of sequencing oligos:
  • FIG. 1 1 shows the results of the agarose gel. 10 ⁇ of the simple PCR products were loaded on the 2% agarose gel. Each replica of the PCR reaction must contain approximately equivalent amounts of PCR product. The expected product sizes are 278bp and 246bp.
  • FIG. 12 shows the results of the agarose gel. 10 ⁇ of the products of the multiplex PCR reaction were loaded on the 2% agarose gel. Each replica of the PCR reaction must contain approximately equivalent amounts of PCR product. The expected product sizes are 278bp and 246bp, although with a 2% agarose gel it is not possible to distinguish between these two bands individually.
  • FIG. 13 shows the overall quality of pyrosequencing reactions.
  • Wells C1, C2 and C3 correspond to the two single-stranded PCR products obtained only with oligo sequencing 1.
  • Wells C4, C5 and C6 correspond to the two single-stranded PCR products obtained only with the sequencing oligo 2.
  • Wells C7, C8 and C9 correspond to the two single-stranded PCR products obtained with a mixing at equimolar concentration of the oligos of sequencing 1 and 2. The blue circles represent the high quality of the sequence obtained.
  • FIG. 14 shows the genotypic results obtained for the template DNA
  • Pocilios C1, C2 and C3 correspond to the two single-stranded PCR products obtained with only the oligo of sequencing 1.
  • C4, C5 and C6 wells correspond to the two single stranded PCR products obtained with only sequencing oligo 2.
  • C7, C8 and C9 wells correspond to the two single stranded PCR products obtained with a mixing at equimolar concentrations of the oligos of sequencing 1 and 2. Consequently, any person skilled in the art understands that in the wells of C1 to C6 where only one SNP was genotyped, the result of a single genotype is shown. The multiple genotyping reactions shown in wells C7, C8 and C9 give two results, of course.
  • FIG. 1 and FIG. 12 show that the amount of PCR product produced is approximately equivalent as long as 30 ng of template DNA is added to the gelled simple reaction (FIG. 1 1) and to the gelled multiplex reaction. (FIG. 12). Slight differences appear due to errors in the pipetting, during the assembly of the PCR, or when the gel is loaded.
  • the signal strength of the PCR products is such that it is difficult to distinguish between the band of the PCR product of 278bp and the band of the PCR product of 246bp however a uniformity is maintained between the products obtained in the simple reaction and in the multiplex in all wells. Since the amount of PCR products remains very similar, any difference in genotyping results is due to simple versus multiple genotyping methods. The quality of the PCR products remains similar in all cases and is not variable, which is evident to one skilled in the art.
  • results obtained in FIG. 13 show high quality data in genotyping reactions as expected.
  • the blue circles appear to confirm the high quality obtained.
  • a reaction failure would have been indicated by a red circle and a poor quality result.
  • the inconsistency between the reference sequence and the results obtained or poor signal / noise ratios would have appeared yellow. It is clear to one skilled in the art that all reactions in this case pass the quality test regardless of the number of gelled sequencing oligos added.
  • the results illustrated in FIG. 14 describe the genotypes obtained by multiple sequencing in accordance with the genotypes obtained by simple sequencing.
  • the genotype of SNP 1 is shown in wells C1, C2, C3, C7, C8 and C9 with position 1, and is GG / in all cases.
  • the genotype of SNP 2 is shown in wells C4, C5 and C6 with position 1 and wells C7, C8 and C9 with position 2 and is T / C in all cases.
  • Mutations in the EmbB gene can result in strains of Mycobacterium ethambutol-resistant. There are two regions in the EmbB gene that appear to be critical targets for ethambutol activity. These two regions of the EmbB gene are separated by approximately 300 DNA bases. These two regions can be amplified by a simple PCR reaction generating a product of more than 300bp. Both regions are small, covering a maximum of 3 bases, and are likely to be sequenced by microsequencing techniques. Microsequencing normally yields information on 40 base sequences with good quality, so that both regions related to ethambutol resistance within the EmbB gene should be sequenced with two sequencing oligos individually.
  • the conventional method for sequencing the two regions of the EmbB gene mentioned would involve carrying out the same PCR reaction twice for each sample, and sequencing the two products with different sequencing oligos independently. With the conventional method it is clear that two PCR reactions and two sequencing reactions are needed. In order to reduce the cost per sample, both in economic terms and in terms relative to the volume of sample needed, it would be preferable to make a single PCR reaction and a single sequencing reaction to read both sequences at the same time. This method would reduce the costs of the test and reduce the workload necessary to complete said test, as well as allow a greater number of samples to be sequenced at the same time.
  • the fundamental difference between the simple conventional protocol and the multiple sequencing protocol is that in the latter case only a single PCR reaction to cover both regions and a single sequencing reaction would be carried out to solve both sequences simultaneously.
  • a total coverage of the two regions related to ethambutol resistance is obtained by using two sequencing oligonucleotides in a single sequencing reaction.
  • the resulting sequence is an overlap of the sequences read by each of the sequencing oligonucleotides. This overlapping sequence can be predicted by knowing all possible combinations of mutations. sought and the order in which dexosinucleotides are dispensed during the sequencing reaction.
  • DNA from a wild-type Mycobacterium fortuitum reference strain was amplified using gelled reagents. After two rounds of PCR amplification, the sample is sequenced individually with the gelled TB2a and TB2b sequencing oligos and is also sequenced in a single reaction with a mixture of the two gelled TB2a and TB2b oligos. The same information is generated both with the multiple sequencing reactions that contain the gelled sequencing oligos and with the two simple sequencing reactions each containing one of the gelled sequencing oligos.
  • DNA isolated from a wild reference strain of Mycobacterium fortuitum is prepared at a concentration of 10 ng / ⁇ . This DNA is previously characterized and the sequence of the EmbB gene in the wild strain is confirmed. The colonies from which this DNA is obtained grew under standard Mycobacterium culture conditions and their growth was inhibited by the presence of ethambutol. Growth inhibition in the presence of ethambutol as well as DNA sequencing undoubtedly confirms that this DNA does not contain mutations in any of the two regions of the EmbB gene studied in this experiment. The DNA is stored at 4 5 C at high concentration and diluted to a concentration of 10ng / ⁇ just before use.
  • a pre-amplification reaction was made to enrich the PCR templates used to amplify the regions related to ethambutol resistance. Although this is not essential since in this experiment the concentration of the isolated DNA is known, it is considered that it should be a standard step in the protocol when working with unknown samples, in which the quality and quantity of DNA in the cells is normally unknown. same. This step in this experiment is done to demonstrate that the pre-amplification phase does not interfere with the sequencing reaction either in its simple mode or in its multiple mode.
  • the pre-amplification reaction mixture consists of 511 of ultrapure DNA polymerase enzyme, manufactured by Biotools Biotechnologial & Medical Laboratories SA, reaction buffer at a final concentration 1 x that is marketed together with the enzyme, between 200 ⁇ and 250 ⁇ concentration final of each of the four dexosiribonucleotides that form the dexosiribonucleic acid chain (dATP, dTTP, dGTP, dCTP), and between 50nM and 75nM final concentration of each of the amplification oligonucleotides designed for pre-amplification of the EmbB target gene .
  • dATP dexosiribonucleotides that form the dexosiribonucleic acid chain
  • dATP dexosiribonucleotides that form the dexosiribonucleic acid chain
  • dATP dexosiribonucleotides that form the dexosiribonucleic acid
  • amplification oligonucleotides included in the pre-amplification reaction are not biotinylated and cannot be used for sequencing. They are used only to improve the amount of template DNA available for the subsequent amplification reaction.
  • the pre-amplification reaction mixture including all the reagents described above is stabilized by the gelation process.
  • a gelation mixture composed of trehalose, melezitose, glycogen and lysine is added to the reaction mixture described above which contains all the reagents necessary to carry out the pre-amplification PCR reaction.
  • the gelation process is completed after drying under vacuum conditions and at a temperature below 40 5 C, resulting in a stabilized reaction mixture containing between 10% and 30% water.
  • a gelled PCR reaction is prepared to amplify the two regions of the EmbB gene related to ethambutol resistance.
  • This reaction includes all the reagents necessary to amplify a 409bp region of the EmbB gene that incorporates the two mutation points related to ethambutol resistance.
  • the reverse amplification oligonucleotide TB2 is biotinylated to allow separation of the strands of the PCR product obtained and thus have a single stranded template DNA for sequencing.
  • the PCR reaction is carried out 9 times so that the PCR product obtained can be sequenced three times with the TB2a gelled sequencing oligo, three times with the TB2b gelled sequencing oligo and three times with the mixture of both TB2a and Tb2b sequenced oligos gelled in a multiplex format.
  • the composition of the mastermix is composed of 5U of ultrapure DNA polymerase enzyme, manufactured by Biotools Biotechnologial & Medical Laboratories SA, reaction buffer at a final concentration 1 x that is marketed together with the enzyme, between 200 ⁇ and 250 ⁇ final concentration of each of the four dexosiribonucleotides that form the dexosiribonucleic acid chain (dATP, dTTP, dGTP, dCTP), and between 100nM and 200nM final concentration of each of the amplification oligonucleotides designed for amplification of the EmbB target gene.
  • the amplification reaction mixture including all the reagents described above is stabilized by the gelation process.
  • a gelation mixture composed of trehalose, melezitose, glycogen and lysine is added to the reaction mixture described above which contains all the reagents necessary to carry out the amplification PCR reaction.
  • the gelation process is completed after drying under vacuum conditions and at a temperature below 40 5 C, resulting in a stabilized reaction mixture containing between 10% and 30% water.
  • PCR product is mixed with 2 ⁇ of loading buffer and electrophoresis is performed on a 2% agarose gel in 0.5X TBE buffer, stained with 1 x SYBRsafe. Run the gel for 30 minutes at 100V in 0.5x TBE buffer, and load in a lateral well next to the PCR product 2 ⁇ of 100 bp DNA marker as standard for the band size.
  • the sequencing oligo TB2a covers the resistance mutations Met306Val, Met306Leu, and two mutations Met306lle (one is the substitution of G by T and the other is the substitution of G for A) and read SEQ ID NO: 17 (CATGGCCCG AGTCGCCG ACCACGCC) ⁇
  • the sequencing oligo TB2b covers the resistance mutations Arg406Cys, Arg406Ser, Arg406Ala, and Arg406Asp and reads SEQ ID NO: 18 (CCGGAGGGCATCATCGCGCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCAT.
  • the mixture of both oligo of sequencing TB2a and TB2b in the multiple genotyping sequence mode reads SEQ ID NO: 19
  • the remaining product of the PCR reaction is used to prepare single stranded DNA for pyrosequencing. 44 ⁇ of a 10: 1 mixture of binding buffer and streptavidin sepharose are added to the PCR products and mixed for 5 minutes. The mixture is then aspirated on the filters of the Q96 vacuum work station and immersed in 70% ethanol, denaturation buffer and wash solution all under vacuum conditions.
  • the resulting single strand of DNA is dispensed in a multi-well plate Q96 containing 20 ⁇ of hybridization buffer and 3 ⁇ of sequencing oligonucleotides at 10 ⁇ concentration (once they have been re-hydrated by adding 100 ⁇ of Milli-Q water to its gelled form) in the case of simple reactions, or 3 ⁇ of the mixture of sequencing oligos at a concentration of 10 ⁇ (once they have been re-hydrated by adding 100 ⁇ of Milli-Q water to its gelled form) in the case of multiple reactions.
  • the multiwell plate Q96 already containing single stranded DNA, oligonucleotides and annealing buffer is then sequencing incubated at 80 5 C for 2 minutes and then cooled to room temperature. While the samples are cooling, the Q96ID pyrosequencer is programmed including the names of the samples, the choice of the cartridge as well as the dispensing order, which in this case are 10 TGCA dispensations. Deoxynucleotides, enzyme mixing and substrate mixing are then added to the cartridge in the volumes indicated by the program and thus sequencing begins.
  • FIG. 15 shows the results of the agarose gel. 10 ⁇ of the PCR product was run on a 2% agarose gel. Each replication of the PCR reaction must contain approximately equivalent amounts of the PCR product. The theoretical size of the PCR product is 409bp
  • FIG. 16 shows the overall quality of pyrosequencing reactions. The blue circles represent the high quality of the sequence obtained.
  • Table 1 shows the sequence data obtained by using the oligo of sequencing of the EmbB gene in its simple mode.
  • the reference sequence for this region of the EmbB gene of Mycobacter ⁇ um fortuitum is shown in black, with sites related to the mutation responsible for ethambutol resistance underlined.
  • the text below shows the high quality of the sequence data obtained in the three sequencing replicas carried out with the oligos TB2a and TB2b separately.
  • Table 2 shows the sequence data obtained, in triplicate, using the EmbB sequencing oligos in their multiple mode.
  • the upper lines of the text show the expected multiple sequence, the sequence obtained with the sequencing oligo TB2a is shown in red and the sequence obtained with the sequencing oligo TB2b is shown in blue.
  • the underlined bases show the positions where they are located sites related to mutation related to ethambutol resistance.
  • Below the expected sequence the sequence data obtained for the three replicates of the multiple sequencing reactions are shown.
  • the color code to indicate the sequence quality is, blue for high quality sequences, orange if the sequence quality is acceptable and red if the sequence is of poor quality.
  • FIG. 15 shows that the amount of PCR product produced when 10 ⁇ of PCR product is added to the reactions is equivalent in all PCR reactions. Slight differences appear due to errors in the pipetting, during the assembly of the PCR, or when the gel is loaded. The expected size of the band, 409 bp is achieved in all reactions. In no case the difference between the PCR reactions is apparent. The quality of the PCR products remains similar in all cases and is not variable, which is evident to one skilled in the art.
  • results illustrated in FIG. 16 show high quality data in the genotyping reactions of sequences as expected.
  • the blue circles appear to confirm the high quality obtained.
  • a reaction failure would have been indicated by a red circle and a poor quality result.
  • the inconsistency between the reference sequence and the results obtained or poor signal / noise ratios would have appeared yellow. It is evident to an expert in the field that all reactions in this case pass the quality test regardless of the number of sequencing oligos added.
  • Table 2 show that the multiple sequencing mode is possible through the use of a single sequencing reaction and that the information that can usually only be obtained with two microsequencing reactions is achieved with the genotyping of multiple mode sequences. .
  • the sequence obtained through the multiple mode can be foreseen in advance and generate consistent results. Then, it is evident that the same genotypic sequences are obtained as a result of both sequencing in its single and multiple mode.

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Abstract

Méthode pour la détermination du génotype de séquences multiples. L'invention concerne une méthode pour la détermination du génotype de séquences multiples d'intérêt, notamment de polymorphismes d'un nucléotide simple (SNP). Ladite méthode utilise une réaction unique d'amplification visant à amplifier les séquences cibles qui comprennent des séquences multiples d'intérêt, en particulier des sites de SNP, et une réaction unique du génotype pour le séquençage de séquences multiples d'intérêt, en particulier de sites de SNP de forme simultanée. Ladite méthode diminue le nombre de réactions nécessaires pour séquencer de petites régions d'intérêt en extensions plus grandes d'ADN de faible intérêt. Ladite méthode diminue également le coût des essais qui autrement nécessitent une grande quantité de réactions de séquençage, réduit la quantité d'ADN matrice de départ nécessaire au séquençage de multiples régions d'intérêt, et débouche sur un produit qui est techniquement moins compliqué.
PCT/ES2011/070565 2011-08-01 2011-08-01 Méthode pour la détermination du génotype de séquences multiples WO2013017702A1 (fr)

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