EP4153778A1 - Procede de genotypage adapte au traitement simultane d'un grand nombre de patients - Google Patents
Procede de genotypage adapte au traitement simultane d'un grand nombre de patientsInfo
- Publication number
- EP4153778A1 EP4153778A1 EP21732473.0A EP21732473A EP4153778A1 EP 4153778 A1 EP4153778 A1 EP 4153778A1 EP 21732473 A EP21732473 A EP 21732473A EP 4153778 A1 EP4153778 A1 EP 4153778A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- sequence
- sequencing
- primers
- sequences
- index
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000003205 genotyping method Methods 0.000 title claims abstract description 23
- 238000000034 method Methods 0.000 claims abstract description 34
- 238000012165 high-throughput sequencing Methods 0.000 claims abstract description 7
- 238000012163 sequencing technique Methods 0.000 claims description 60
- 238000003752 polymerase chain reaction Methods 0.000 claims description 32
- 230000000087 stabilizing effect Effects 0.000 claims description 32
- 238000007481 next generation sequencing Methods 0.000 claims description 28
- 230000003321 amplification Effects 0.000 claims description 23
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 23
- 239000002773 nucleotide Substances 0.000 claims description 19
- 125000003729 nucleotide group Chemical group 0.000 claims description 19
- 230000000692 anti-sense effect Effects 0.000 claims description 14
- 239000003381 stabilizer Substances 0.000 claims description 11
- 239000000203 mixture Substances 0.000 claims description 9
- 239000011541 reaction mixture Substances 0.000 claims description 7
- 230000000295 complement effect Effects 0.000 claims description 5
- 206010068051 Chimerism Diseases 0.000 claims description 2
- 230000004544 DNA amplification Effects 0.000 claims 1
- 238000012544 monitoring process Methods 0.000 claims 1
- 238000012408 PCR amplification Methods 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 8
- 238000005516 engineering process Methods 0.000 description 7
- 108091093088 Amplicon Proteins 0.000 description 5
- 230000035772 mutation Effects 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 238000009396 hybridization Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- PCDQPRRSZKQHHS-XVFCMESISA-N CTP Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 PCDQPRRSZKQHHS-XVFCMESISA-N 0.000 description 2
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Chemical class Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 2
- 239000012472 biological sample Substances 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000001747 exhibiting effect Effects 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000001226 triphosphate Substances 0.000 description 2
- 235000011178 triphosphate Nutrition 0.000 description 2
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- KBDWGFZSICOZSJ-UHFFFAOYSA-N 5-methyl-2,3-dihydro-1H-pyrimidin-4-one Chemical compound N1CNC=C(C1=O)C KBDWGFZSICOZSJ-UHFFFAOYSA-N 0.000 description 1
- PGSPUKDWUHBDKJ-UHFFFAOYSA-N 6,7-dihydro-3h-purin-2-amine Chemical compound C1NC(N)=NC2=C1NC=N2 PGSPUKDWUHBDKJ-UHFFFAOYSA-N 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 208000024556 Mendelian disease Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 229960001456 adenosine triphosphate Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000008366 buffered solution Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 230000000771 oncological effect Effects 0.000 description 1
- 238000003793 prenatal diagnosis Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
Classifications
-
- 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
- C12Q1/686—Polymerase chain reaction [PCR]
Definitions
- the invention relates to a genotyping method for simultaneously treating a large number of patients. More particularly, it relates to a genotyping method comprising a first step of amplification by indexed PCR then a second step of high throughput sequencing, this method is simple, rapid and less expensive than the methods of the state of the art.
- High throughput sequencing (also called NGS for Next Generation Sequencing) allows a very large number of short sequences to be operated in parallel.
- the principle of this technique is as follows: the sequences to be analyzed are first of all prepared by adding adapter sequences to the ends. They are then fixed on a solid support via these adapter sequences and each fragment is enriched to form clonal “clusters” which will be sequenced subsequently. At each cycle, the addition of a nucleotide results in a fluorescent signal associated with each of the four nucleotides of DNA. These signals are then analyzed by a data processing software which restores the result of the reading of the sequences. This method is fast and precise.
- LNA TM Locked Nucleic Acid
- This NGS technology is widely used in research and diagnostics. It makes it possible to sequence entire genomes. It thus constitutes a very powerful tool and represents an aid in the diagnosis of genetic and oncological diseases for example.
- a library of sequences is prepared upstream of the sequencing in order to enrich the library with sequences of interest.
- This sequence amplification is carried out by polymerase chain reaction (PCR).
- PCR polymerase chain reaction
- NGS sequencing allows a large number of sequences to be read simultaneously, it is beneficial to mix samples from different patients.
- these can be labeled during the amplification step by adding label or “index” sequences.
- the indexed PCR method is known to those skilled in the art and described for example in the article by Stahlberg A. et al. (Nucleic Acid Research, 2016, vol.44, No. 11)
- Document EP 2599877 describes a sequencing method comprising a first PCR step during which the sequences are marked using an index then a second second generation sequencing step. During sequencing, a strategy of incomplete DNA shearing is implemented to increase the amplitude of the reading of the sequences.
- NGS sequencing although very powerful in terms of sequencing capacity has drawbacks.
- the main obstacle is the time it takes to perform genotyping from sample preparation to obtaining the result, which takes several days.
- the present invention relates to a genotyping method suitable for the simultaneous processing of a large number of DNA samples from patients comprising: a. a step of amplifying the DNA from each patient by an indexed polymerase chain reaction (PCR), in which the specific primers comprise in 3 'a stabilizing sequence comprising between 8 and 12 nucleotides among which 45% to 65 % are G or C; b. a high-throughput NGS-type sequencing step in which the sequencing primers do not contain an LNA TM base but a sequence complementary to said stabilizing sequence.
- PCR polymerase chain reaction
- the inventors introduced sequences of around ten nucleotides at the ends of the primers. This addition makes it possible to dispense with modified bases while maintaining high reading reliability during sequencing.
- the present invention also relates to a genotyping kit for the successive implementation of an indexed PCR and high throughput sequencing of the NGS type, as well as a reaction mixture for NGS sequencing without the use of LNA TM bases.
- the genotyping method according to the invention has several advantages: it is simple, rapid and inexpensive and makes it possible to process, simultaneously, a large series of samples from different patients.
- This method is simple, it does not require any other heavy equipment other than a PCR machine and equipment intended for NGS sequencing. It is offered as a ready-to-use kit. This format limits the manipulations by the experimenter and therefore the risks of error. It also saves time by reducing the number of actions to be performed.
- This method is fast because the amplification and indexing are carried out in one and the same step. For example, the result can be obtained in less than 48 hours if you process up to 96 samples at the same time and have a single PCR machine. It is understood that the processing time depends on the technical environment (number of machines available) and on the number of samples to be processed, but that the method according to the invention will nevertheless be faster than the methods existing until then.
- This method is inexpensive and makes it possible to propose a genotyping solution in a single locus or a small number of loci (generally between 1 and 5), simple to implement and very reliable, accessible to the greatest number of people. It is intended to be deployed in all laboratories where genotyping is carried out, for example in research centers, hospitals, blood banks and blood transfusion centers where it is part of a process of controlling public health and productivity costs.
- This method is particularly applicable to the genotyping of diseases in which a single gene is involved. This may involve diagnosing hereditary diseases as part of a family diagnosis, prenuptial or prenatal diagnosis, a post-transplant chimerism follow-up study or diagnosing a cohort of cancer patients on a few key mutations. . DETAILED DESCRIPTION OF THE INVENTION
- a first object of the invention relates to a genotyping method suitable for the simultaneous treatment of a large number of patients comprising: a. a step of amplifying the DNA by an indexed polymerase chain reaction (PCR), in which the specific primers comprise at 3 ′ a stabilizing sequence comprising from 8 to 12 nucleotides among which 45 to 65% are G or VS ; b. a high-throughput NGS-type sequencing step in which the sequencing primers do not contain an LNA TM base but a sequence complementary to said stabilizing sequence.
- PCR polymerase chain reaction
- the term “stabilizer sequence” means a sequence of 8 to 12 nucleotides, namely 8, 9, 10, 11 or 12 nucleotides.
- these sequences are rich in G and C, that is to say they contain between 45% and 65% of G and / or C bases.
- An amplicon therefore contains two stabilizing sequences which will play different roles during the sequencing: one hybridizing to a sequencing primer and the other hybridizing to a read primer.
- Examples of stabilizing sequences which can be used according to the invention are the sequences SEQ ID No. 1 and SEQ. ID No.2. These examples are non-limiting since those skilled in the art will know how to combine the bases in order to modify the sequence of the stabilizing sequence.
- the stabilizer sequences are sequences of 10 nucleotides. In another preferred embodiment, the stabilizer sequences contain 50% to 60% G and / or C bases. For example, a 10 nucleotide sequence contains 5 to 6 G and / or C bases.
- Step a amplification of the sequences of interest is based on an indexed PCR technology making it possible to simultaneously generate the amplicons and to label them using a specific label of the sample. It can be summarized as follows:
- Amplification reaction of the sequences by PCR with addition of specific labels of the sample (index) on each amplified sequence uses two types of primers simultaneously: o pairs of primers specific for the sequences to be amplified, these sense and antisense primers each comprising a universal sequence located at their 3 ′ end, characterized in that the universal sequences contain at the 5 ′ end (between the specific primer and the universal sequence) a stabilizing sequence of around ten nucleotides (generally between 8 and 12 nucleotides) consisting of 45% to 65% of G or C nucleotides; o pairs of sense and antisense primers comprising at least a first part capable of hybridizing with the universal sense and antisense sequences respectively and a second part comprising a sequence for an adapter necessary for sequencing, one of the sense or antisense primers comprising in in addition to an index positioned between these two parts.
- the specific primers further comprise a universal sequence located 3 'of said stabilizer sequence.
- universal sequence within the meaning of the invention, is meant a defined sequence which will be used in all the amplicons. These sequences play two roles: on the one hand, they allow the hybridization of the indexing primers with a view to adding the index during the amplification step, and on the other hand they allow the amplification of the indexing primers. sequences for reading during the sequencing step. The method actually implements two types of universal sequences, a sense sequence and an antisense sequence so as to be able to define the orientation of the amplified sequences. Examples of universal sequences which can be used according to the invention are the sequences SEQ. ID No.3 and SEQ ID No.4. These examples are non-limiting since those skilled in the art will know how to use other sequences as universal sequences.
- the amplification step also implements pairs of primers intended for indexing (called “indexing primers”) of the amplified sequences, said primers comprising: a. at least a first part capable of hybridizing with the universal sequences amplified by virtue of the specific primers and b. a second part comprising a sequence for an adapter necessary for sequencing, one of the sense or antisense primers further comprising an index positioned between these two parts.
- indexing primers pairs of primers intended for indexing
- index within the meaning of the invention, is meant a sequence used as label or bar code.
- indexes are polymorphic short sequences. A unique particular index is associated with each patient. The samples from different patients, in particular from 2 to 384 patients, being mixed during the sequencing, these indexes are used to know to which patient to associate the sequences after analysis. Examples of indexes which can be used according to the invention are the sequences SEQ ID No.5, SEQID No.6 and SEQ ID No.7. These examples are non-limiting because those skilled in the art will know how to modify these indexes to provide a multitude of unique sequences.
- adapter necessary for sequencing within the meaning of the invention, is meant a sequence allowing the fixing of the sequences on a support with a view to their sequencing in accordance with the NGS sequencing protocol used.
- binding sequences necessary for the sequencing which can be used according to the invention are the sequences SEQ. ID No.8 and SEQ ID No.9.
- the amplification of the DNA sequences by indexed PCR makes it possible to obtain PCR products each bearing a particular label specific to each patient. Thus, it is possible to pool and mix all the PCR products in order to perform a single sequencing reaction. The result of the sequencing then provides two pieces of information per sequence: the reading of the sequence of interest and the identification of the patient to whom this sequence corresponds.
- the method according to the invention it is possible to carry out genotyping at a particular locus for 1 patient or preferably 2 to 384, and even more preferably for a multiple of 96 patients simultaneously, namely 96, 192, 288 or 384 patients. It is also possible to detect several mutations per patient (located on distant regions or on the same region) by using several primers simultaneously in a multiplexing amplification system, therefore without reducing the same number of patients treated simultaneously.
- the genotyping method according to the invention can be carried out either from DNA of human or animal origin.
- Step b. sequencing is based on a high throughput sequencing technology such as the NGS technology developed in particular by the company Illumina ® .
- the inventors have demonstrated that sequences of around ten nucleotides, called “stabilizing sequences” make it possible to maintain a sufficient degree of hybridization for the NGS sequencing to take place correctly even when the half-hybridization temperature (Tm) is high.
- Tm half-hybridization temperature
- they have shown that the stabilizing effect of LNA TM bases can be replaced by the addition of a stabilizing sequence and that this sequence is sufficient for the result of the sequencing to be as reliable as when LNA TM bases are used.
- the fact of dispensing with the use of LNA TM bases makes the method simpler and less expensive.
- the present invention proposes for the first time to combine an indexed PCR with an NGS sequencing in a method that is simple to implement, and the cost of which is controlled.
- a ready-to-use kit allows a simplified implementation of the proposed method.
- Step b. of the method according to the invention is carried out using the sequencing and index reading primers, none of which contains LNA TM bases but each of which contains a stabilizing sequence, namely: a pair of sequencing primers hybridizing to the universal sequences and comprising a stabilizer sequence positioned at the 5 'end; these primers make it possible to amplify the sequences of interest for the sequencing; an index read primer hybridizing to the universal sequence juxtaposing the index and comprising a stabilizer sequence; this primer allows the amplification of the index to identify the patient from which the analyzed sequence originates.
- the sequencing and index reading primers none of which contains LNA TM bases but each of which contains a stabilizing sequence, namely: a pair of sequencing primers hybridizing to the universal sequences and comprising a stabilizer sequence positioned at the 5 'end; these primers make it possible to amplify the sequences of interest for the sequencing; an index read primer hybridizing to the universal sequence juxtaposing the index and comprising a stabilizer sequence; this primer allows the
- stabilizing sequences of the sequencing primers and of the index reading primer are complementary to the stabilizing sequences present on the sequences amplified with a view to sequencing. In other words, these stabilizing sequences have the same sequences as those present in the specific primers.
- the index reading primer is complementary to the universal antisense sequence and the stabilizing sequence is positioned at its 3 ′ end.
- the reverse primer is used as a sense primer upstream of the index.
- sequences of sequencing primers which can be used according to the invention are the sequences SEQ. ID No.10 and SEQ ID No.11. These examples are non-limiting because a person skilled in the art can propose other sequencing primers as a function of the universal sequences flanking the sequences to be amplified.
- a second object of the invention relates to a genotyping kit for the successive implementation of an indexed PCR and high throughput sequencing of the NGS type without the use of an LNA TM base comprising: a first series of containers (of the NGS type. PCR tube) each comprising at least one pair of primers specific for a locus of interest to be amplified, these primers comprising in 3 'a stabilizing sequence comprising 8 to 12 nucleotides among which 45% to 65% are G or C, said stabilizing sequence being preceded by a universal sequence; a second series of containers (of PCR tube type) each comprising: hybridizing with said universal sequences amplified by virtue of the specific primers and at least a second part comprising a sequence for an adapter necessary for sequencing, one of the sense or antisense primers further comprising an index positioned between these two parts; and o an amplification mix a third series of containers (of the tube type) comprising a reaction mixture for NGS sequencing without LNA TM base comprising a pair of
- the tubes of the first and second series are used for indexed PCR, so their number is the same.
- the mixture of the third series is used for sequencing and is the same for all reactions; it can be supplied in a single tube or in different tubes, in particular as many tubes as for each of the first and second series.
- this kit further comprises at least one of the following elements: reaction mixtures for NGS sequencing without LNA TM base, preferably presented in tubes, a user manual.
- amplification mix is meant a mixture comprising Taq polymerase, a buffered solution containing MgCh, dNTPs (mixture of the four deoxyribonucleotides: dATP (deoxy-adenine tri-phosphate), dCTP (deoxy-cytosine tri phosphate) ), dGTP (deoxy-guanine tri-phosphate), dTTP (deoxy-thymine tri-phosphate), a solution of DMSO and a buffer which reduces the adhesion of molecules to the plastic.
- the reaction mixture for NGS sequencing without the use of LNA TM base comprises primers which are configured as follows: a forward sequencing primer hybridizing to the universal sequence located 5 'of the sequence to be analyzed and comprising a stabilizing sequence at its 5'end; an antisense sequencing primer hybridizing to the universal sequence located 3 'of the sequence to be analyzed and comprising a stabilizing sequence at its 5'end; an index read primer hybridizing to the universal sequence juxtaposing the index and comprising a stabilizer sequence positioned at one of its ends.
- the index reading primer is a forward primer hybridizing to the universal sequence located at 3 ′ of the sequence to be analyzed and comprising a stabilizing sequence positioned at its 3 ′ end.
- the two sequencing primers capable of hybridizing to the universal sequences located on either side of the sequence to be analyzed allow the amplification of the latter while the sense primer hybridizes downstream of the sequence. to be analyzed allows the amplification of the index to identify the patient from which the analyzed sequence originates.
- the reaction mixture for NGS sequencing without the use of LNA TM base comprises: a sense sequencing primer of sequence SEQ ID No.10; an antisense sequencing primer of sequence SEQ. ID No.11; and a SEQ ID No. 12 sequence index read primer.
- the method according to the invention are particularly suitable for the genotyping of a large number of patients, in particular from 2 to 384 patients, in a single reaction.
- the amplicons should not be too long (preferably ⁇ 500bp) and the multiplexing should be moderate (between 1 and 5, preferably 2 to 3 targets per PCR reaction). It allows laboratories to answer recurring genotyping questions by simultaneously treating a large number of patients.
- FIGURES Figure 1 Schematic representation of the primers used and sequences amplified according to a particular embodiment of the present invention.
- A Specific Primers
- B Universal and Stabilizer Sequences
- C P5-P7 and Index Adapters
- D Sense Sequencing Primer
- E Antisense Sequencing Primer
- E Index Read Primer .
- Figure 2 Representation of an amplified sequence as a description of a particular embodiment.
- sequences of interest are identified for genotyping.
- Primer pairs specific for the allelic or mutant sequences to be amplified are designed and validated. These primers will allow the amplification of the sequences of interest.
- the biological samples to be analyzed are prepared for PCR, the DNA is extracted and purified. This preparation step is well known to those skilled in the art and results in samples exhibiting standardized DNA concentrations.
- the actual amplification reaction is carried out by indexed PCR, according to a standard protocol.
- the specific primers as well as the primers intended for indexing and the amplification mix are mixed in tubes suitable for the PCR equipment used.
- the amplification reaction takes place under standard conditions.
- sequences are recovered, purified and quantified in order to be sequenced.
- the sequencing is carried out in an NGS sequencer from the company Illumina.
- Sequencing is carried out using the pair of sequencing primers to amplify the sequences to be analyzed and the index reading primer to identify the patient to which the sequence relates.
- Table 1 Summary of the sequences described
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR2004917A FR3110178B1 (fr) | 2020-05-18 | 2020-05-18 | Procédé de génotypage adapté au traitement simultané d’un grand nombre de patients |
PCT/FR2021/050862 WO2021234269A1 (fr) | 2020-05-18 | 2021-05-18 | Procede de genotypage adapte au traitement simultane d'un grand nombre de patients |
Publications (1)
Publication Number | Publication Date |
---|---|
EP4153778A1 true EP4153778A1 (fr) | 2023-03-29 |
Family
ID=73013484
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP21732473.0A Pending EP4153778A1 (fr) | 2020-05-18 | 2021-05-18 | Procede de genotypage adapte au traitement simultane d'un grand nombre de patients |
Country Status (3)
Country | Link |
---|---|
EP (1) | EP4153778A1 (fr) |
FR (1) | FR3110178B1 (fr) |
WO (1) | WO2021234269A1 (fr) |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2599877B1 (fr) | 2010-06-30 | 2017-09-27 | BGI Genomics Co., Limited | Nouvelle méthode de séquençage par pcr et son utilisation dans le génotypage hla |
KR101672240B1 (ko) * | 2015-12-30 | 2016-11-07 | 대한민국 | 인간 객체 염색체 상의 str 분석방법 및 이를 이용한 분석 키트 |
-
2020
- 2020-05-18 FR FR2004917A patent/FR3110178B1/fr active Active
-
2021
- 2021-05-18 EP EP21732473.0A patent/EP4153778A1/fr active Pending
- 2021-05-18 WO PCT/FR2021/050862 patent/WO2021234269A1/fr unknown
Also Published As
Publication number | Publication date |
---|---|
WO2021234269A1 (fr) | 2021-11-25 |
FR3110178A1 (fr) | 2021-11-19 |
FR3110178B1 (fr) | 2022-05-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP2087134B1 (fr) | Methode de diagnostic et de suivi d'une vaginose bacterienne par quantification moleculaire | |
CN110719957B (zh) | 用于核酸靶向富集的方法和试剂盒 | |
JP7389551B2 (ja) | Metエキソン14欠失の検出と、関連する治療法 | |
Bi et al. | Rapid prenatal diagnosis using uncultured amniocytes and oligonucleotide array CGH | |
CN110392739B (zh) | 检测dna突变的测序方法 | |
JP2010081944A (ja) | 短いタンデム反復遺伝子座の多重増幅 | |
CN106912197A (zh) | 用于多重pcr的方法和组合物 | |
FR2639651A1 (fr) | Procede de caracterisation d'un echantillon analytique d'adn genomique par extension de sequences de nucleotides, et kits de diagnostic pour la mise en oeuvre de ce procede | |
US20240132962A1 (en) | Genetic test kit for detecting thalassemia | |
Bunce | PCR-sequence-specific primer typing of HLA class I and class II alleles | |
EP1179091B2 (fr) | Procede d'analyse de la predisposition genetique d'un patient a au moins une maladie et amplification adaptee a un tel procede | |
EP4097254A1 (fr) | Procede de genotypage hla simple et rapide | |
US20220145380A1 (en) | Cost-effective detection of low frequency genetic variation | |
EP4153778A1 (fr) | Procede de genotypage adapte au traitement simultane d'un grand nombre de patients | |
EP4028548A1 (fr) | Procédé de génotypage adapté au traitement d'un grand nombre d'échantillons, notamment en cas de polymorphisme élevé | |
EP3728644A1 (fr) | Méthode de diagnostic d'une vaginose bactérienne par détection de methanobrevibacter smithii | |
CA2228694A1 (fr) | Test co-dominant de diagnostic genetique | |
Caylor | Nonrandom X chromosome inactivation detection | |
WO2015001056A1 (fr) | Kit et methode de detection in vitro du rhesus d | |
Wijaya et al. | Nested PCR amplification secures DNA template quality and quantity in real-time mCOP-PCR screening for SMA | |
EP1137804B1 (fr) | Methode de detection in vitro d'une substance cable dans un echantillon comprenant le marquage de ladite substance par un gene rapporteur et par les sequences necessaires a l'expression dudit gene rapporteur in vitro | |
RU2806581C1 (ru) | Способ диагностики инфекции Хеликобактер пилори и ее устойчивости к кларитромицину и левофлоксацину | |
WO2023052735A1 (fr) | Méthode de détection de mutations rares sur biopsie liquide | |
Yau | Repeat Expansions in Movement Disorders: Disease Modification and New Horizon | |
KR20240041142A (ko) | 타액, 혈액 및 정액 동시 검출을 위한 다중분석 역전사 중합효소 연쇄반응용 프라이머 세트 및 이를 포함하는 검출 키트 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: UNKNOWN |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE |
|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE |
|
17P | Request for examination filed |
Effective date: 20221118 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR |
|
DAV | Request for validation of the european patent (deleted) | ||
DAX | Request for extension of the european patent (deleted) | ||
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: EXAMINATION IS IN PROGRESS |
|
17Q | First examination report despatched |
Effective date: 20231129 |