WO2012000445A1 - 新的的pcr测序方法及其在hla基因分型中的应用 - Google Patents
新的的pcr测序方法及其在hla基因分型中的应用 Download PDFInfo
- Publication number
- WO2012000445A1 WO2012000445A1 PCT/CN2011/076688 CN2011076688W WO2012000445A1 WO 2012000445 A1 WO2012000445 A1 WO 2012000445A1 CN 2011076688 W CN2011076688 W CN 2011076688W WO 2012000445 A1 WO2012000445 A1 WO 2012000445A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- hla
- pcr
- primer
- seq
- sequencing
- Prior art date
Links
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
-
- 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
- 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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
-
- 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
-
- 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
- C12Q2600/16—Primer sets for multiplex assays
-
- 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
- C12Q2600/172—Haplotypes
Definitions
- the invention relates to the field of nucleic acid sequencing technology, in particular to the field of PCR sequencing technology.
- the invention also relates to DNA molecular tagging techniques and DNA incomplete disruption strategies.
- the method of the present invention is particularly useful for second generation sequencing techniques, particularly Pair-end sequencing in second generation sequencing techniques, and can also be used for HLA genotyping.
- the present invention provides methods for HLA genotyping, particularly for HLA-A, HLA-B, HLA-C and HLA-DQB1 genotyping, and PCR amplification for use in the methods Add primer pairs. Background technique
- the PCR sequencing method is a technique for obtaining a DNA fragment of a target gene by PCR, and then performing DNA sequence detection on the obtained DNA fragment of the target gene to obtain DNA sequence information of the target gene, which has been widely used for a long time. Gene mutation detection and genotyping.
- the DNA sequencing technology is mainly divided into the first generation DNA sequencing technology represented by Sanger sequencing and the second generation DNA sequencing technology represented by Illumina GA, Roche 454, ABI Solid and the like.
- Sanger DNA sequencing technology has experimental exercises Simple, accurate, accurate, and short experimental period, it has a wide range of applications in the field of clinical gene mutation detection and genotyping, which require high timeliness. However, its small flux and high cost limit its application in the field of large-scale genotyping.
- the second generation DNA sequencing technology has the characteristics of large sequencing throughput, low cost, high degree of automation and single molecule sequencing.
- Illumina GA single molecule sequencing technology an experimental process can be used. Produces 50G (about 50 billion) bases of data, generating an average of 5 billion bases of data per day. The average cost per base is less than 1/1000 of the cost of Sanger's sequencing, and the results can be processed directly by computer. , is a technology that is very suitable for large-scale sequencing projects. However, the serial sequencing length of the second-generation sequencing technology is generally short.
- the current maximum measurement length of Illumina GA is 200 bp; although the maximum length of Roche 454 GS-FLX can reach 500 bp, the sequencing cost is higher and the flux is smaller. .
- the PCR method can be directly used to test the entire PCR product, and the entire DNA sequence information of the PCR product to be detected cannot be obtained.
- the short sequencing read length limits the application of second-generation sequencing technology in PCR sequencing methods. In addition to the progressive improvement of sequencing technology to obtain longer actual sequencing reads, development can overcome the existing sequencing of second-generation DNA sequencers. New technologies that are lacking in PCR sequencing applications have become a top priority.
- HLA Human leukocyte antigen
- the international standard HLA high-resolution genotyping technology is a PCR sequencing method based on Sanger sequencing technology.
- the method is to amplify the corresponding HLA gene region by PCR, and sequence the amplified product.
- the sequencing result is divided into professional typing software. Type, and finally get the HLA genotype information of the sample. It is intuitive, highly resolved and capable of detecting new alleles.
- the high cost and small flux of Sanger sequencing limits its use in institutions such as the Hematopoietic Stem Cell Volunteer Registry (Bone Marrow Bank), which requires large-scale HLA typing.
- Illumina GA has ultra-high sequencing throughput, but its long measurement 200bp; Although the length of Roche 454 GS-FLX can reach 500bp, its sequencing cost is higher and the flux is smaller. Although the length of the first-generation sequencer can reach more than lOObp, its flux and cost cannot be compared. Compared to the second generation of sequencers.
- the second generation sequencing technology adopted by the present invention includes Pair-end sequencing technology in the second generation sequencing technology, and PCR sequencing technology with a DNA reference sequence for the PCR template.
- the present invention provides a method for PCR sequencing, which reduces the limitation caused by the short read length of the self-sequencing and expands the application of the second generation DNA sequencing technology in the field of PCR sequencing applications.
- the primer primer with the primer primer sequence added at the end can be used to interrupt the amplified PCR product, and the product is terminated after the disruption. Repair and connect deoxyadenosine (A) at its 3' end, then connect different PCR-free linkers.
- a sequence of tags is attached to the front end of the amplification primer to achieve simultaneous sequencing of multiple samples. Specifically, it can be combined with PCR-index/barcode technology.
- a primer primer was added to the primer primer at the 5' end of the PCR primer to introduce a unique primer tag for each sample during the PCR process. In this way, in the detection process using the second generation DNA sequencing technology, in addition to the PCR step must be processed one by one, other experimental links can be mixed together and processed simultaneously, and finally the detection result of each sample can pass its unique primer label. The sequence is retrieved.
- adapter or “library adapter”
- tag technology refers to the addition of different library linkers to multiple sequencing libraries (different library linkers have different composition sequences, and different sequences are called link tags ( Adapter index ) ) , a tag-sequencing library is constructed, so that a plurality of different tag sequencing libraries can be mixed and sequenced, and finally the sequencing results of each tag sequencing library can be distinguished from each other by a library tag technology.
- PCR-Free library linker refers to a designed set of bases whose primary function is to assist in the immobilization of DNA molecules on a sequencing chip and to provide a binding site for universal sequencing primers.
- PCR-Free library linkers can be passed The DNA ligase directly ligates it to both ends of the DNA fragment in the sequencing library, and the introduction process of the linker is called PCR-Free library linker because there is no PCR involved.
- the PCR-FREE library linker is used in the embodiment of the present invention. ILLUMIA.
- the PCR-FREE library construction method combining the library linker technology means that the library linker is directly ligated to both ends of the DNA fragment in the sequencing library, and the introduction process of the library linker is called PCR-Free library construction because there is no PCR involved. .
- the access method can be ligated using DNA ligase. There is no PCR participation in the whole library construction process, which avoids the inaccuracy of the final result caused by the introduction of errors in PCR during the construction of the PCR product pooling library with high sequence similarity.
- the DNA amplification method, the method of extracting DNA from a sample, the DNA purification method, and the method of DNA sequence alignment involved in the present invention may be any available in the art. Method. The method can be selected by a person skilled in the art according to the specific circumstances. The method for DNA sequencing can be carried out by a person skilled in the art according to a conventional method or according to the instruction manual of the sequencing instrument.
- the design of the primer label varies depending on the experimental platform used.
- the characteristics of the Illumina GA sequencing platform itself mainly considers the following points in the design of primer tags: 1: avoid more than 3 (including 3) single-base repeat sequences in the primer tag sequence, 2: the same identity of all primer tags The total content of base A and base C in the site is between 30% and 70% of the total base content, 3: the GC content of the primer tag sequence itself is between 40 and 60%, 4: between the primer tags Sequence differences greater than 4 bases, 5: Sequences with high similarity to Illumina GA sequencing primers are avoided in the primer tag sequence, 6: Reduced primers are added to PCR primers, causing severe hairpins on PCR primers (hairpin) ), the emergence of the dimer (dimer) situation.
- the present invention introduces a primer tag at each end of the PCR product by PCR reaction (the primer tag sequence may be the same or different), so that the primer tag at either end of the PCR product can specifically label the sample information of the PCR product.
- the resulting PCR product was incompletely disrupted.
- the so-called incomplete interruption means that the final product contains a complete unbroken PCR product and a partially interrupted PCR product.
- the breaking method includes, but is not limited to, a chemical breaking method (for example, enzymatic cleavage) and a physical breaking method, and the physical breaking method includes an ultrasonic breaking method or a mechanical breaking method.
- Interrupted DNA is electrophoresed on 2% agarose, and the gel is purified to recover all DNA bands from the maximum read length of the sequencer to the longest DNA length range that the sequencer can use (the longest DNA available for the Illumina GA sequencer) It is 700 bp, which is the length of the original DNA, and does not include the length of the library linker). Purification and recovery methods include, but are not limited to, electrophoretic tapping recovery, magnetic bead recovery, and the like.
- the recovered DNA fragment is then constructed according to the second-generation sequencer sequencing library construction process, and then sequenced, preferably The sequencing library was constructed using the PCR-FREE sequencing library construction process, and the sequencing method was preferably sequenced by the Pair-End method.
- IUumina GA sequencing (IUumina's Genome Analyzer sequencer, IUumina GA for short) is a DNA sequence analysis using the principle of sequencing while synthesizing, and can detect haplotypes.
- the final output data is a series of sequences, which can be directly It is used for direct comparison with the reference sequence in the HLA database.
- the sequencing throughput of IUumina GA is large, and a current experimental procedure can produce 50G (50 billion) base-based data, generating an average of 5 billion bases per day. High data throughput allows for high sequencing depth for each sequence, ensuring the reliability of sequencing results, as the number of sequencing sequences is determined.
- the present invention applies IUumina GA sequencing to the field of HLA typing for the first time, combining DNA molecular tagging technology, DNA incomplete interruption and PCR-FREE database construction. Technology, achieving low cost, high throughput, high accuracy, high resolution typing of HLA.
- a PCR-based sequencing technique based on DNA molecular tagging technology, incomplete DNA inactivation, and PCR-FREE is used to group the samples to be analyzed, and the primers labeled by the bidirectional primer tags are used for each group of samples.
- Fragment amplification the maximum length of the PCR product depends on the maximum DNA length that the sequencer can bind to, At present, the maximum DNA length for Illumina GA is 700 bp, which is the length of the original DNA, and the length of the library linker is not included.
- the obtained PCR products are mixed in equal amounts, and the PCR-Free tag sequencing library is constructed by incompletely breaking the DNA.
- the different tag sequencing libraries obtained from each sample set were equimolar mixed, and all DNA fragments with a fragment length greater than the maximum sequencing length of the sequencer were selectively recovered, and subsequently sequenced using an Illumina GA sequencer.
- the sequence index of the adapter index, primer label and PCR primers By screening the sequence index of the adapter index, primer label and PCR primers, the DNA sequence information of each sample can be obtained, and the obtained DNA sequence is assembled and compared with the corresponding database in the IMGT HLA professional database, and finally The HLA genotype of the sample is available.
- DNA of different sets of samples is ligated with different library adaptors during the construction of the PCR-Free tag library, so that in the subsequent typing step,
- the obtained sequencing results correspond one-to-one with the samples based on the primer label and the linker label used for each sample.
- the alignment sequence of each sample is mapped to the known DNA reference sequence (reference Sequence) of the PCR product by the alignment program, and the complete PCR product sequence is spliced from the sequence of the broken DNA by sequence overlap and linkage.
- the invention provides a high-resolution typing method of HLA gene based on illumina GA sequencing technology, thereby realizing haplotype sequencing, software typing automation, increasing flux of HLA genotyping and reducing cost.
- the PCR primers originally used for the HLA-SBT method are no longer suitable for the HLA high-resolution typing method based on the new sequencing technology.
- the present invention designs a novel PCR primer which separately amplifies the specificity and conservation of the exons 2, 3 and 4 of the HLA- ⁇ , ⁇ gene, and the PCR product is not more than 700 bp in length, and is particularly suitable for Illumina GA ( The maximum DNA length currently available for Illumina GA is 700bp).
- the set of PCR primers provided by the present invention can be used for large-scale, high-throughput and low-cost HLA genotyping of subjects, particularly humans.
- the technical solution adopted by the present invention is to download all the latest HLA-A/B gene sequences from the IMGT/HLA Internet site (http://www.ebi.ac.uk/imgt/hla/) and save them to the local disk.
- For the HLA-A data set simultaneously download all the latest non-HLA-A HLA-I gene sequences as comparative data sets.
- the two data sets were compared, and the conserved and specific sequences of each gene locus were searched for both ends and inside of exons 2, 3, and 4, and the designed PCR primer sequences were compared with the human genome sequence for homology.
- the HLA-A/B gene has high sequence similarity to other genes belonging to the HLA class I molecule, try to ensure primer 3 and terminal specificity when designing PCR primers, and ensure that the primers amplify the HLA-A/B gene. Specificity.
- the length of the PCR product is less than 700 bp, and the annealing temperature of the positive and negative primers is substantially the same.
- Two sets of 6 pairs of PCR primers were selected as primers, and 95 sets of label primers were designed to amplify 95 and 950 DNA templates with common serotypes of HLA-A/B (HLA type of the template) Do not include all common serotypes of HLA-A/B). All PCR products were mixed in equal amounts and sequenced with Illumina GA Pair-End 100. The assembled sequencing results were compared to the original typing results to verify the conservation and specificity of the PCR primers.
- HLA- ⁇ , ⁇ -site primers designed by the present invention that is, two sets of 6 pairs of HLA-A/B PCR primers for amplifying exons 2, 3, and 4, respectively, are shown in Tables 1 and 2.
- degenerate primers refer to a mixture of different sequences representing the possibility of encoding all the different bases of a single amino acid.
- R A/G
- Y C/T
- M A/C
- K G/T
- S C/G
- W A/T
- H A/C/T
- B C/G /T
- V A/C/G
- D A/G /T
- N A/C/G/T.
- the present invention employs a method for designating PCR primers for amplifying HLA-A/B gene exons 2, 3 and 4 as described above, and designing 2 sets of 3 pairs of PCR for amplifying exons 2, 3 and 4 of HLA-C. Primer.
- the present invention provides two sets of three pairs of PCR primers for amplifying exons 2, 3 and 4 of the HLA-C gene, which are SEQ ID NOs: 25 and 26, 27 and 28 shown in Table 3, respectively. 29 and 30, and SEQ ID NOS: 31 and 32, 33 and 34, and 35 and 36 shown in Table 4.
- the 6 pairs of PCR primers have good conservation and specificity, and can cover the full length sequences of exons 2, 3 and 4 of HLA-C sites, and the PCR products are less than 700 bp in length, which satisfies the requirements of normal Illumina Solexa sequencing.
- the primers of the present invention are also suitable for Sanger sequencing.
- the present invention provides PCR primers for amplifying exon 2 and/or exon 3 of HLA-DQB1, which are tables. SEQ ID NO: 37-40 shown in 5.
- the PCR primers have good conservation and specificity, and can cover the full length sequences of exons 2 and 3 of HLA-DQB1, and the length of the PCR products are less than 700 bp, which satisfies the requirements of normal Illumina Solexa sequencing.
- the primers of the invention are also suitable for sequencing by the Sanger method.
- the amplification primer pair and the genotyping method provided by the present invention can be amplified
- Genotyping was performed based on exon 2 and/or exon 3 of HLA-DQB1.
- the typing method utilizes Illumina Solexa sequencing technology, which has the characteristics of obtaining high-resolution HLA typing results with high throughput and low cost. detailed description
- the invention provides a method of determining a nucleotide sequence of a nucleic acid of interest in a sample, comprising:
- n samples n being an integer greater than or equal to 1, the sample preferably being from a mammal, more preferably a human, in particular a human blood sample; alternatively, dividing the n samples to be analyzed into m groups , m is an integer and n > m > l;
- Amplification For each sample, one or more pairs of label primers are used, and in the presence of a template from the sample, PCR amplification is performed under conditions suitable for amplifying the nucleic acid of interest, wherein each pair of label primers
- the forward label primer and the reverse label primer (both may be degenerate primers) including the primer label, wherein the forward label primer and the reverse label primer may contain the same or different primer labels; the label primer pair used for different samples The primer labels in each other are different from each other;
- Sequencing The recovered DNA mixture is sequenced using a second-generation sequencing technique, preferably a Pair-End technique (eg, Illumina GA, Illumina Hiseq 2000), to obtain a sequence of the broken DNA;
- a second-generation sequencing technique preferably a Pair-End technique (eg, Illumina GA, Illumina Hiseq 2000), to obtain a sequence of the broken DNA;
- the sequencing results obtained are based on the unique primer labels of each sample, and the sequencing sequences (for example, Blast, BWA program) are used to locate each sequencing sequence on the corresponding DNA reference sequence of the PCR product, by overlapping the sequences. And the linkage relationship, the complete target nucleic acid is spliced from the sequence of the interrupted DNA.
- the sequencing sequences for example, Blast, BWA program
- each pair of primer tags and PCR primer pairs are combined into a pair of tag primers, and the 5th ends of the forward and reverse PCR primers have (or are optionally joined by a linker sequence) a forward primer tag and a reverse primer tag, respectively.
- the PCR primer is a PCR primer for amplifying an HLA gene, particularly a PCR primer for amplifying an HLA-A/B gene, preferably HLA-A/B.
- PCR primers for exons 2, 3, 4 and HLA-DRB1 exon 2 preferably PCR primers for amplifying exons 2, 3 and 4 of HLA-A/B are shown in Table 1 or Table As shown in Figure 2, the preferred PCR primers for amplifying the HLA-DRB1 exon 2 are shown in Table 7.
- the PCR primer is a PCR primer for amplifying an HLA gene, particularly a PCR primer for amplifying an HLA-C gene, preferably for amplifying HLA-C.
- PCR primers for exons 2, 3 and/or 4, preferably the PCR primers are shown in Table 3 or Table 4.
- the PCR primer is a PCR primer for amplifying an HLA gene, in particular, a PCR primer for amplifying the HLA-DQB1 gene, preferably for amplifying HLA- PCR primers for DQB1 gene 2 and/or exon 3, preferably the PCR primers are shown in Table 5.
- the primer tag is designed for a PCR primer, preferably for a PCR primer for a specific gene for amplifying HLA, more preferably for amplifying HLA-A/B 2 3, exon 4 and PCR primers for exon 2 of HLA-DRB1, in particular, PCR primers as shown in Table 1 or Table 2 or Table 7, which specifically includes 95 pairs shown in Table 6.
- At least 10 pairs of primer labels or at least 20 pairs, or at least 30 pairs, or at least 40 pairs, or at least 50 pairs, at least 60 pairs, or at least 70 pairs, or at least 80 pairs, or at least 90 pairs, or 95 pairs (or the set of primer labels is 10 - 95 pairs of 95 pairs of primer labels shown in Table 1 (eg, 10 - 95 pairs, 20 - 95 pairs, 30 - 95 pairs, 40 - 95 pairs, 50 - 95 pairs, 60-95 pairs, 70-95 pairs, 80-95 pairs, 90-95 pairs, or 95 pairs)), and
- the set of primer labels preferably comprises at least 95 pairs of primer labels as shown in Table 6.
- the DNA disruption includes a chemical breaking method and a physical breaking method, wherein the chemical method includes an enzyme cutting method, and the physical breaking method includes an ultrasonic breaking method or mechanical punching Break method.
- all DNA bands from the maximum read length of the sequencer to the longest DNA length range applicable to the sequencer are recovered and recovered, wherein the purification is recovered.
- Methods include, but are not limited to, electrophoretic tapping recovery, or magnetic bead recovery.
- the method of determining the nucleotide sequence of the nucleic acid of interest in the sample may further comprise the steps 1) to 4) of claim 1, and the following steps:
- Sequencing The recovered DNA mixture is sequenced using a second-generation sequencing technique, preferably a Pair-End technique (eg, Illumina GA, Illumina Hiseq 2000), to obtain a sequence of the interrupted DNA;
- a second-generation sequencing technique preferably a Pair-End technique (eg, Illumina GA, Illumina Hiseq 2000)
- An aspect of the invention also provides the use of the method described above for HLA typing, characterized by comprising: sequencing a sample (especially a blood sample) from a patient using the method, and sequencing the result with an HLA database (eg IMGT HLA professional database) HLA exons, preferably HLA-A/B exons 2, 3, 4, HLA-C exons 2, 3 and / or 4, HLA-DQB1 gene 2 Sequence data alignment with exon 3 and/or exon 2 of HLA-DRB1, and 100% matching of the sequence alignment results is the HLA genotype of the corresponding sample.
- an HLA database eg IMGT HLA professional database
- HLA exons preferably HLA-A/B exons 2, 3, 4, HLA-C exons 2, 3 and / or 4, HLA-DQB1 gene 2
- Sequence data alignment with exon 3 and/or exon 2 of HLA-DRB1 is the HLA genotype of the corresponding sample.
- Another aspect of the invention provides a set of primer tags comprising at least 10 pairs, or at least 20 pairs, or at least 30 pairs, or at least 40 pairs, or at least 50 pairs, at least 60 of the 95 pairs of primer labels shown in Table 6. Yes, or at least 70 pairs, or at least 80 pairs, or at least 90 pairs, or 95 pairs (or the set of primer labels is 10 - 95 pairs of 95 pairs of primer labels shown in Table 1 (eg, 10 - 95 pairs, 20-95 pairs, 30-95 pairs, 40-95 pairs, 50-95 pairs, 60-95 pairs, 70-95 pairs, 80-95 pairs, 90-95 pairs, or 95 pairs), and
- the set of primer labels preferably comprises at least PI-1 to PI-10, or PI-11 to PI-20, or PI-21 to PI-30, or PI-31 in 95 pairs of primer labels shown in Table 6.
- PI-40 or PI-41 to PI-50, or PI-51 to PI-60, or PI-61 to PI-70, or PI-71 to PI-80, or PI-81 to PI-90, Or PI-91 to PI-95, or a combination of any two or more of them.
- the invention further provides the use of the set of primer tags for PCR sequencing methods, wherein, in particular, each pair of primer tags is combined with a PCR primer pair for amplifying a sequence of interest to be tested into a pair of tag primers, positive and negative
- the 5th ends of the PCR primers have (or are optionally joined by a linker sequence) a forward primer tag and a reverse primer tag, respectively.
- the PCR primer is a special for amplifying HLA A genetically modified PCR primer, preferably a PCR primer for amplifying exons 2, 3, and 4 of HLA-A/B and an exon 2 of HLA-DRB1, preferably for amplifying HLA-A/
- the PCR primers for the exons 2, 3 and 4 of B are shown in Table 1 or Table 2.
- the preferred PCR primers for amplifying the exon 2 of HLA-DRB1 are shown in Table 7; or preferably PCR primers for amplifying exons 2, 3 and/or 4 of HLA-C, preferably the PCR primers are shown in Table 3 or Table 4; or preferably for amplifying HLA-DQB1 gene 2 and / Or PCR primers for exon 3, preferably the PCR primers are shown in Table 5.
- a set of label primers comprising a set of primer labels and a PCR primer pair for amplifying a sequence of interest to be tested are provided, wherein each pair of primer labels and a pair of PCR primers are combined into a pair
- the label primer, the 5th end of the positive and negative PCR primers each have (or optionally be joined by a ligation sequence) a primer tag.
- the PCR primer is a PCR primer for amplifying a specific gene of HLA, preferably exon 2, 3, 4 for amplifying HLA-A/B and HLA- PCR primers for DRB1 exon 2, preferably PCR primers for amplifying exon 2, 3, and 4 of HLA-A/B are shown in Table 1 or Table 2, preferably for expansion PCR primers for increasing the exon 2 of HLA-DRB1 are shown in Table 7; or preferably PCR primers for amplifying exons 2, 3 and/or 4 of HLA-C, preferably the PCR primers are as follows 3 or Table 4; or preferably PCR primers for amplifying HLA-DQB1 gene 2 and/or exon 3, preferably the PCR primers are shown in Table 5.
- Another aspect of the invention also provides the use of the above-described tag primers for a PCR sequencing method.
- an HLA typing method comprising:
- n samples n being an integer greater than or equal to 1, the sample preferably being from a mammal, more preferably a human, in particular a human blood sample;
- each pair of label primers comprises primers
- the label's forward label primer and reverse label primer both may be degenerate primers, wherein the forward label primer and the reverse label primer may contain the same or different primer labels; the primers in the label primer pair used for different samples Labels are different from each other;
- Adapter to distinguish between different PCR-Free sequencing libraries, recovering all DNA bands between the maximum read length of the sequencer used and the longest DNA length range used by the sequencer used, specifically 450-750 bp length range DNA fragment;
- the recovered DNA mixture is sequenced using a second-generation sequencing technique, preferably a Pair-End technique (eg, Illumina GA, Illumina Hiseq 2000), to obtain the sequence of the interrupted DNA;
- a second-generation sequencing technique preferably a Pair-End technique (eg, Illumina GA, Illumina Hiseq 2000), to obtain the sequence of the interrupted DNA;
- HLA exons in the sequencing results and HLA database (such as IMGT HLA professional database), preferably exons 2, 3, 4 of HLA-A/B, HLA-C 2, 3 and / Or the exon 4, HLA-DQB1 gene 2 and / or exon 3 and / or HLA-DRB1 exon 2 sequence data alignment, the sequence alignment results 100% match is the corresponding sample HLA genotype do not.
- HLA database such as IMGT HLA professional database
- each pair of primer tags and PCR primer pairs are combined into a pair of tag primers, and the 5th ends of the forward and reverse PCR primers respectively have (or are optionally connected by a link sequence) positive Primer label and reverse primer label.
- the PCR primer is a PCR primer for amplifying a specific gene of HLA, preferably exon 2, 3, 4 for amplifying HLA-A/B and HLA- PCR primers for DRB1 exon 2, preferably PCR primers for amplifying exon 2, 3, and 4 of HLA-A/B are shown in Table 1 or Table 2, preferably for expansion PCR primers for increasing the exon 2 of HLA-DRB1 are shown in Table 7; or preferably PCR primers for amplifying exons 2, 3 and/or 4 of HLA-C, preferably the PCR primers are as follows 3 or Table 4; or preferably PCR primers for amplifying HLA-DQB1 gene 2 and/or exon 3, preferably the PCR primers are shown in Table 5.
- the primer tag is a set of primer tags as described above.
- the DNA disruption comprises a chemical breaking method and a physical breaking method, wherein the chemical method comprises an enzyme cutting method, and the physical breaking method comprises Ultrasonic breaking method or mechanical breaking method.
- the purification and recovery method includes, but is not limited to, electrophoresis tapping recovery, and may also be magnetic bead recovery.
- the binding library linker technology the PCR product library constructed by breaking the PCR product library refers to m PCRs obtained by using m library linkages to 2) A product library plus a linker, wherein each PCR product library uses a different library linker to construct m linker tag sequencing libraries; m linker tag sequencing libraries are equimolarly mixed to construct a hybrid linker tag sequencing library.
- the method in which the library linker is ligated means that the DN A ligase is directly ligated without a PCR program.
- the invention provides PCR primers for HLA genotyping, characterized in that the PCR primers are exons 2, 3, 4 for HLA-A/B and HLA-DRB1 2
- the PCR primer of the exon preferably the PCR primer for amplifying the exon 2, 3, and 4 of HLA-A/B is shown in Table 1 or Table 2, and the above is preferably used for amplifying HLA.
- PCR primers for exon 2 of DRB1 are shown in Table 7; or PCR primers for amplifying exons 2, 3 and/or 4 of HLA-C, preferably the PCR primers are shown in Table 3 or Table Or a PCR primer for amplifying the HLA-DQB1 gene 2 and/or exon 3, and the PCR primers are preferably shown in Table 5.
- the invention further provides a method for sequencing of the above PCR primers, comprising:
- the PCR primer is used to amplify DNA derived from a blood sample to obtain a PCR product, and the PCR product is purified;
- Sequencing the PCR product which can be Sanger sequencing, or can be a second-generation sequencing method (eg HiSeq 2000, Illumina GA) And Roche454).
- Another aspect of the present invention provides the use of the above PCR primer for HLA genotyping, characterized in that the PCR primers described above are used for assembly and comparative analysis according to the results obtained by the above sequencing method, and the sequencing results are in a database. The standard sequences were compared to obtain HLA genotyping results.
- kits for performing HLA genotyping which comprises the above PCR primers, is further provided.
- the invention provides a set of PCR primers for HLA-A, B genotyping, characterized in that the PCR primers are as shown in Table 1 or Table 2.
- Another aspect of the present invention provides a method for sequencing of a PCR primer for HLA-A, B genotyping of the present invention, comprising:
- the PCR primer is used to amplify DNA derived from a blood sample to obtain a PCR product, and the PCR product is purified;
- the PCR product is sequenced, either by Sanger sequencing or by second generation sequencing (eg HiSeq 2000, Illumina GA and Roche454)
- Another aspect of the present invention provides the use of the PCR primer for HLA-A, B genotyping of the present invention for HLA genotyping, characterized in that the PCR primers described above are used for assembly according to the sequencing results obtained by the above method. The analysis was compared and the sequencing results were compared with the standard sequences in the database to obtain HLA genotyping results.
- the present invention provides a kit for performing HLA genotyping, which comprises the PCR for HLA- ⁇ , ⁇ genotyping of the present invention. Primer. PCR primers for HLA-C genotyping
- the present invention also provides a novel method for amplifying exon 2, 3 and 4 of HLA-C gene, characterized in that PCR amplification is carried out using the amplification primer pair of the present invention, and the sequence of the amplification primer pair is shown. In Table 3 or Table 4.
- the method of the present invention is particularly advantageously useful for HLA-C genotyping. Compared with the existing HLA-C genotyping method, since the method using the method of the present invention and the product of the amplification primer are controlled within 700 bp, HLA based on Illumina Solexa sequencing technology can be utilized for further typing. -SBT.
- the present invention also provides a method of sequencing HLA-C gene 2, 3 and/or exon 4 in a sample, comprising the steps of:
- a PCR primer pair of the present invention for HLA-C genotyping preferably selected from the group consisting of SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO : 29 and SEQ ID NO: 30, or primer pairs of SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36 are used Amplifying the DNA to obtain a PCR product, preferably purifying the PCR product;
- sequencing the PCR product preferably by second generation sequencing, such as Illumina Solexa or Roche 454.
- the invention also provides a HLA-C genotyping method, the method comprising: 1) using a PCR primer pair of the invention for HLA-C genotyping, preferably selected from the group consisting of SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, or SEQ
- the primer pairs of ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36 were subjected to PCR amplification, and the HLA of the sample to be tested was amplified.
- the present invention provides a kit for performing HLA-C genotyping, which comprises the PCR primer pair of the present invention for HLA-C genotyping, preferably selected from the group consisting of SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, or SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 33 and primer pairs of SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36.
- the kit further comprises other reagents, such as reagents for DNA amplification, DNA purification, and/or DNA sequencing.
- the amplification primer pair and the genotyping method provided by the present invention can be used for genotyping based on the amplification of exons 2, 3 and 4 of HLA-C. Therefore, compared to the prior art, this type of classification utilizes Illumina Solexa sequencing technology to increase throughput and streamline processes while saving time and cost.
- the present invention also provides a novel method for amplifying exon 2 and/or exon 3 of HLA-DQB1, characterized in that PCR amplification is performed using the amplification primer pair of the present invention, and the sequence of the amplification primer pair is shown. In Table 5.
- the method of the present invention is particularly advantageously applicable to the HLA-DQB1 group. Due to classification. Compared with the existing HLA-DQB1 genotyping method, since the method using the method of the present invention and the product of the amplification primer are controlled between 300 and 400 bp, the Illumina Solexa-based sequencing technology can be utilized for further typing. HLA-SBT.
- the present invention also provides a method of sequencing exons 2 and/or exon 3 of HLA-DQB1 in a sample, comprising the steps of:
- the PCR primer pair of the present invention for HLA-DQB1 genotyping preferably the PCR primer pair in Table 5 is used to amplify the DNA to obtain a PCR product, preferably a PCR product;
- the PCR product is sequenced, and the sequencing method can be a second generation sequencing method such as Illumina Solexa or Roche454.
- the invention provides an improved HLA-DQB1 genotyping method, the method comprising:
- PCR primer pair for HLA-DQB1 genotyping of the present invention preferably the PCR amplification primer pair in Table 5 to amplify exon 2 and / or exon 3 of HLA-DQB1 to be tested,
- the sequencing method may be Sanger sequencing, or may be a second generation sequencing method such as Illumina Solexa or Roche 454.
- the present invention provides a kit for performing HLA-DQB1 genotyping, which comprises the PCR primer pair of the present invention for HLA-DQB1 genotyping, preferably in Table 5. PCR amplification of primer pairs.
- the kit further comprises other reagents, such as reagents for DNA amplification, DNA purification, and/or DNA sequencing.
- Figure 1 Sequence primers for primer labeling, DNA disruption, and DNA sequencing.
- the positive and negative primer tag sequences Index-NF/R (1) were introduced into the PCR product of the No. N sample.
- the products of the PCR product interrupted by physical methods include: a product with a primer tag sequence at one end, both ends a product without a primer tag sequence, a product that is completely unbroken, and a tapping purification to recover all DNA bands located between the maximum read length of the sequencer and the longest DNA length range applicable to the sequencer for sequencing (2), Using the Index-NF/R to retrieve the sequencing result of the PCR product belonging to the No. N sample in the sequencing result, and using the known reference sequence information of the PCR product to locate the relative reference position of each sequencing sequence, and according to the overlap between the sequencing sequences And the linkage relationship assembled into a complete PCR product (3, 4).
- Figure 2 The electrophoresis results of the corresponding exon PCR products of sample No. 1 in HLA-A/B/DRB1 in Example 2, from the electropherogram, the PCR product is a series of single bands with a fragment size of 300bp-500b, Lane M is a molecular weight marker (DL 2000, Takara), and lanes 1-7 are HLA-A/B/DRB1 exons of sample No. 1 (A2, A3, A4, B2, B3, B4, DRB1-2) PCR amplification product, negative control (N) without amplification bands. The results for the other samples are similar.
- Figure 3 For Example 4, the DNA electrophoresis after HLA-Mix was interrupted (before and after cutting), and the tapping area was 450-750 bp.
- Lane M is a molecular weight marker (NEB-50bp DNA Ladder)
- lane 1 is the electrophoresis of HLA-Mix before cutting
- lane 2 is the gel of HLA-Mix after tapping.
- Figure 4 is a screenshot of the consensus sequence construction procedure for sample No. 1 in Example 6, illustrating an example of splicing out the complete sequence of the PCR product based on the overlapping relationship between the primer tag and the DNA fragment.
- the query for HLA typing naming can be found at http://www.ebi.ac.uk/imgt/hla/align.htmlo from the left result output column We see the results of all coding sequences for A*02:03:01 A*ll:01:01, where the sequence of exon 2 is identical to the original known result of template No. 1.
- Figure 5 Schematic representation of the PCR product after labeling the primer and adaptor indices.
- primer tags were simultaneously introduced at both ends of the PCR product of each sample by PCR; a plurality of PCR products with different primer tags were mixed together to construct a sequencing library.
- each sequencing library can be labeled by adding a library linker with a different linker tag.
- multiple sequencing libraries with different linker tags can be mixed together and sequenced with Illumina GA (primer tags between sequencing libraries labeled with different linkers can be identical).
- the DNA sequence information of each sample can be obtained by screening the linker and primer tag sequence information in the sequencing result.
- Figure 6 is a result of electrophoresis of the HLA-C 2, 3, and 4 exon PCR products of a part of the sample in Example 8. From the electropherogram, the PCR product fragment size is a single strip between 400 and 500 b, respectively. Band, wherein lane M is a DNA standard molecular weight reference (DL 2000, Takara).
- Fig. 7 shows the case where the DNA gel electrophoresis gel was cut after HLA-Mix was interrupted in Example 8, and the tapping area was 450-750 b.
- Lane M is the molecular weight standard (NEB-50bp DNA Ladder)
- lane 1 shows the HLA-Mix gel map before cutting
- lane 2 shows the HLA-Mix gel map after tapping.
- Figure 8 A screenshot showing the construct of the exon 2 consensus sequence of the HLA-C site of sample No. 2 in Example 8.
- the sequencing sequence of the C site of the sample is aligned to the reference sequence by BWA software, and the consensus sequence of exons 2, 3, and 4 of the sample is constructed respectively, and then according to the linkage relationship between the SNPs.
- the haplotype sequence for each exon of the C site and finally, the type of the sample is determined by the intersection of each exon haplotype sequence.
- sample C gene sequence 695 - The 764 region contains two heterozygous SNPs.
- the SNP linkage relationship can be determined by readl and read2: AC, GA ("" in the figure indicates the same base as the reference sequence).
- the sequences correspond to the shaded portions of the C*010201 and C*07020101 type sequences, respectively.
- the determination of the linkage relationship in other regions is similar.
- Figure 9 shows the electrophoresis pattern of PCR products of 26 samples of HLA-C sites 2, 3 and 4 in Example 9. 26 samples of HLA-C site 2, 3 and 4 exon PCR product electropherogram, as shown in the figure, all PCR products are less than 500 bp, and the electrophoresis bands are single, no obvious non-specific bands, the same The amplification efficiency of the primers for each sample was uniform.
- Figure 10 shows the results of the analysis of the sequencing peak of the amplification PCR product No. 1 template by uType software in Example 9, and the result in the left result output column shows C*08:01:01 C*15:05:01, Template No. 1 was originally known to be the same type.
- Figure 11 Electrophoresis results of 94 samples of HLA-DQB1 2+3 exon PCR products in Example 10, from the electropherogram, the PCR product fragment size is a single band between 250 bp and 500b, wherein the lanes M is the DNA standard molecular weight reference (DL 2000, Takara), and the lanes PI-1 to PI-94 are 94 samples of HLA-DQB1 2+3 exon PCR amplification products, and the negative control (N) is not amplified. Bands.
- the lanes M is the DNA standard molecular weight reference (DL 2000, Takara)
- N negative control
- Fig. 12 is a view showing the case where the DNA gel electrophoresis gel was cut after the HLA-Q-Mix was interrupted in Example 10, and the tapping area was a 350-550 bp region.
- Lane C is the DNA standard molecular weight reference (NEB-50bp DNA Ladder)
- lane 1 shows the HLA-Q-Mix gel map before cutting
- lane 2 shows the HLA-Q-Mix gel map after tapping.
- Figure 13 shows a partial screenshot of the sample consensus sequence builder in the tenth embodiment, exemplifying the main flow of data analysis.
- the sequencing sequence of the DQB1 locus of the sample is aligned to the reference sequence by BWA software to construct a consensus sequence of the exon 2, 3 of the sample DQB1, and then according to the SNP The linkage relationship between the DQB1 2,3 exon haplotypes was determined.
- readl can determine the linkage relationship of SNP1-SNP5 to TGTCC, and the binding relationship of another set of SNP1-SNP5 can be determined by read2 CCAGT, the binding relationship of SNP3-SNP6 can be determined by read3 as AGTG, and the linkage relationship of another group of SNP3-SNP6 can be determined by read4 as TCCA.
- the linkage relationship between SNP and read4 can be determined by the linkage relationship of SNP, and read2 is linked with read3.
- the complete SNP combinations in this region are: TGTCCA and CCAGTG, the sequences of which correspond to the shaded parts of the DQB1*0303 and DQB1*0602 types. The determination of the linkage relationship of other regions is similar.
- Figure 14 exemplarily shows an electrophoresis pattern of two pairs of PCR primers for separately amplifying HLA-DQB1 sites 2 and 3 exons and simultaneously amplifying exons 2 and 3 exons, respectively, in Example 11, which shows from 7
- the negative control (N) has no amplified bands, and the lane M is the DNA standard molecular reference (DL 2000, Takara).
- Fig. 15 exemplarily shows the results of analysis of the sequencing peaks of the PCR products of the HLA-DQB1 amplification No. 7 template 2 and exon 3 in the Example 11 by the uType software, and the left result output column shows DQB1*03: 03 The result of DQB1*06:02 is the same as the original known result of template No. 7.
- PCR product 16 is a result of electrophoresis of the corresponding exon PCR product of sample No. 1 in HLA-A/B/C/DQB1 in Example 12. From the electropherogram, the PCR product is a series of single bands with a fragment size of 300 bp to 500 bp. Lanes M are molecular weight markers (DL 2000, Takara), and lanes 1-10 are HLA-A/B/C/DQB1 exons of sample No. 1 (A2, A3, A4, B2, B3, B4, C2, C3, C4, DQBl) PCR amplification products, negative control (N) no amplification bands. The results for the other samples are similar.
- Lane 17 is HLA-l-Mix, HLA-2-Mix, HLA-3-Mix, HLA-4-Mix, HLA-5-Mix, HLA-6-Mix, HLA-7-Mix, in Example 12, HLA-8-Mix, HLA-9-Mix and HLA-10-Mix were collected by equimolar mixing of agarose gel.
- Lanes M are molecular weight markers
- lane 1 is the result of electrophoresis of the mixture
- lane 2 is an electropherogram after the gel fraction recovery of the 450-750 bp length fragment.
- Fig. 18 is a view showing a screenshot of the construct of the exon 2 consensus sequence of the HLA-C site of the sample No. 1 in Example 12.
- the sequencing sequence of the C site of the sample is aligned to the reference sequence by the BWA software, and the consensus sequence of the exons 2, 2, and 4 of the sample C, respectively, is constructed, and then according to the linkage relationship between the SNPs.
- the haplotype sequence for each exon of the C site and finally, the type of the sample is determined by the intersection of each exon haplotype sequence. As shown in the figure, there are two heterozygous SNPs in the 695-764 region of sample C gene sequence No.
- Examples 1-6 of the present invention 95 samples of HLA-A/B 2, 3, 4 were taken by a combination of primer label + DNA incomplete disruption strategy + Illumia GA sequencer Pair-End 100 sequencing technique.
- the exon number and the genotyping of exon 2 of HLA-DRB1 proved that this strategy can fully utilize the high-throughput and low-cost characteristics of the second-generation sequencer.
- the classification of gene fragments that exceed the length of the sequencer itself can be achieved.
- the primers to be analyzed are introduced into the primers of the HLA-A/B 2, 3, 4 exons and the HLA-DRB1 exon 2 PCR product by PCR, so that the specific PCR products are labeled.
- Sample information The PCR amplification products of the three sites of HLA-A/B/DRB1 in each sample were mixed together to obtain a PCR product library; the PCR product library was not completely interrupted by ultrasound, and a PCR-Free sequencing library was constructed.
- the sequencing library was subjected to 2% low melting point agarose electrophoresis, and all the DNA bands between 450 bp and 750 bp in length were recovered by tapping purification (the library linker was added to both ends of the DNA fragment during the construction of the PCR-Free sequencing library.
- the length of the DNA fragment on the electropherogram is about 250 bp larger than the actual length. Therefore, the recovery of the 450 bp to 700 bp fragment is actually equivalent to the recovery of a DNA fragment of 200 bp to 500 bp in length.
- the recovered DNA was sequenced by Illumina GA PE-100.
- the sequence information of all the tested samples can be found by the primer tag sequence, and the sequence of the entire PCR product is assembled by the overlapping and linkage relationship between the reference sequence information of the known DNA fragment and the sequence of the DNA fragment, and then passed through HLA-A/
- the alignment of the standard database of the corresponding exons of B/DRB1 can assemble the entire sequence of the original PCR product to achieve genotyping of HLA-A/B/DRB1.
- DNA was extracted from 95 blood samples of known HLA-SBT typing (Chinese Hematopoietic Stem Cell Donor Database (hereinafter referred to as "Zhonghua Marrow Bank") using a KingFisher Automatic Extractor (Thermo Corporation, USA).
- the main steps are as follows: Take out the deep hole plate and one shallow hole plate of 6 Kingfisher automatic extractor. Add a certain amount of matching reagents according to the instructions and mark them. Place all the well plates with reagents as required. In the corresponding position, select the program "Bioeasy_200ul Blood DNA KF.msz", and press “star” to execute the program for nucleic acid extraction. At the end of the program, approximately 100 ⁇ L of the eluted product in the plate Elution was collected as the extracted DNA.
- Example 2 Example 2
- PCR tag primers can be made by synthesizing PCR primers with different primer tags at the 5' end, such that different PCR tag primers can be used for different samples, the PCR primers are for HLA-A/B 2, 3, 4 Exon exon and PCR primers for exon 2 of HLA-DRB1. Primer tags are then introduced at both ends of the PCR product by PCR to specifically label PCR products from different samples.
- each set of PCR tag primers consisting of a pair of bidirectional primer tags (Table 6) and exons 2, 3, 4 for amplifying HLA-A/B ( Table 1) and PCR primers (Table 7) of exon 2 of HLA-DRB1, wherein each forward PCR primer has a forward primer label attached to a pair of primer tags at the 5' end, and 5 of the reverse PCR primers. , a reverse primer label that links a pair of primer tags at the end.
- the primer tag is added directly to the 5, end of the PCR primer when the primer is synthesized.
- the 95 DNAs obtained in the sample extraction step of Example 1 were sequentially numbered 1-95, and the PCR reaction was carried out in a 96-well plate for 7 plates, numbered HL AP-A2, HLA-P-A3, HLA-, respectively.
- P-A4, HLA-P-B2, HLA-P-B3, HLA-P-B4 and HLA-P-DRB1-2 (A2/3/4, B2/B3/B4, DRB1-2 indicate the amplified position Point)
- a negative control without a template was set in the plate, and the primer used in the negative control was the same as the corresponding primer of template 1.
- Table 6 Primer Label Information
- D2-F1, D2-F2, D2-F3, D2-F4, D2-F5, D2-F6, D2-F7 are forward primers for amplifying HLA-DRB1 exon 2
- D2-R is for amplifying HLA- Reverse primer for exon 2 of DRB1.
- HLA-A/B PCR reaction system All reagents were purchased from Promega ⁇ (Beijing) Biotechnology Co., Ltd. (Promega)
- the PCR reaction system of HLA-DRB1 is as follows:
- PI nr A/B/D2-F 1/2/3/4/5/6/7 represents primer 5, and HLA-A/B/DRB1 with the nth forward primer tag sequence (Table 6) at the end F primer, PI nr -A/B/D2-R 2/3/4 denotes primer 5, and the R primer of HLA-A/B/DRB1 with the nth reverse primer tag sequence at the end (here n ⁇ 95 ), others and so on. And each sample corresponds to a specific set of PCR primers ( PI nr A/B/D2-F 1/2 PI nr --A/B/D2-R 2/3/4 )
- FIG. 1 shows the electrophoresis results of the corresponding exon PCR products of sample No. 1 HLA-A/B/DRB1.
- the DNA molecular marker is DL 2000 ( Takara), and a series of fragments with a size of 300bp-500b on the gel map indicates The HLA-A/B/DRB1 exons of sample No. 1 (A2 A3 A4 B2 B3 B4 DRB1-2 ) were successfully amplified by PCR, and the negative control (N) had no amplified bands.
- the results of other samples are similar
- PCR product mixing and purification From the remaining PCR product of 96-well plate HLA-P-A2 (except negative control) 20 ul was mixed in a 3 ml EP tube, labeled as HLA-A2-Mix, and the same operation was performed on the other 6 96-well plates, respectively.
- HLA-A3-Mix Marked as HLA-A3-Mix, HLA-A4-Mix, HLA-B2-Mix, HLA-B3-Mix, HLA-B4-Mix and HLA-D2-Mix, oscillating hooks, from HLA-A2-Mix, HLA 200 ⁇ l of each of -A3-Mix, HLA-A4-Mix, HLA-B2-Mix, HLA-B3-Mix, HLA-B4-Mix, and HLA-D2-Mix were mixed in a 3 ml EP tube, labeled HLA -Mix, 500ul DNA mixture from HLA-Mix was purified by column using Qiagen DNA Purification kit (QIAGEN) (see the instructions for specific purification procedure), and purified 200ul DNA was obtained by Nanodrop 8000 (Thermo Fisher Scientific) The HLA-Mix DNA concentration was determined to be 48 ng/ul.
- QIAGEN Qiagen DNA Purification kit
- DNA end-repairing reaction was performed on the purified HLA-Mix after the disruption, and the system was as follows (reagents were purchased from Enzymatics):
- T4 DNA Polymerase (T4 DNA Polymerase) 5 ⁇
- thermomixer Thermomixer, Eppendorf 20
- reaction product was recovered by QIAquick PCR Purification Kit and dissolved in 34 ⁇ M EB (QIAGEN Elution Buffer).
- the DNA was recovered in the previous step, and the end was added with A reaction.
- the system was as follows (reagents were purchased from Enzymatics):
- the reaction conditions were: Constant Temperature Mixer (Thermomixer, Eppendorf) 37 "C warm bath for 30 min.
- PCR-Free library linker refers to a designed base that is designed to assist in the immobilization of DNA molecules on sequencing chips and to provide universal sequencing primers.
- the binding site of the PCR-Free library can be directly ligated to the DNA fragments in the sequencing library by DNA ligase.
- the introduction process of the library linker is called PCR-Free library linker because there is no PCR involved.
- PCR-free oligonucleotide linker mix (30mM) (PCR-free Adapter ⁇
- the reaction conditions were: Constant Temperature Mixer (Thermomixer, Eppendorf) 20 Warm bath for 15 min.
- the reaction product was purified by Ampure Beads (Beckman Coulter Genomics) and dissolved in 50 ul of deionized water.
- the DNA concentration was determined by real-time PCR (QPCR) as follows:
- the sequencing result of Illumina GA is a series of DNA sequences. By searching the sequence of the positive and negative primers and the primer sequences in the sequencing results, the sequencing results of the PCR products of the HLA-A/B/DRB1 exons corresponding to each primer label are established. database. The sequencing results of each exon are mapped to the reference sequence of the corresponding exon by BWA (Burrows-Wheeler Aligner) (reference sequence source: http://www.ebi.ac.uk/imgt/hla/) Build consistency across databases
- the (pair-End linkage) relationship can be assembled into the corresponding sequence of each exon of HLA-A/B/DRB1.
- the obtained DNA sequence was aligned with the sequence database of the corresponding exons of HLA-A/B/DRB1 in the IMGT HLA professional database, and the 100% match of the sequence alignment results was the HLA-A/B/DRB1 genotype of the corresponding sample. do not.
- a screenshot of the exon 2 consensus sequence builder for the HLA-A site of sample No. 1 illustrated in Figure 4 can be seen.
- DRB1*1201 in HLA-DRB1 type does not exclude the possibility of DRB1*1206/1210/1217
- DRB1*1454 does not exclude the possibility of DRB1*1401 because the above alleles have identical sequences in the exon of HLA-DRB12.
- Example 7 HLA-A, B and DRB1 genotyping using second-generation sequencing technology (Illumina GA)
- the 950 DNAs obtained in the sample extraction step were sequentially numbered 1-950, and were divided into 10 groups of 95 DNAs each labeled HLA-1, HLA-2, HLA-3, HLA-4, HLA-5, HLA-6, HLA-7, HLA-8, HLA-9, HLA-10.
- 95 sets of PCR primers with exon amplifying HLA-A/B 2, 3, and 4 exons (Table 1) and HLA-DRB1 exon 2 were used.
- PCR primers (Table 7) were used to amplify 95 DNA samples, respectively.
- the PCR reaction was carried out in a 96-well plate with a total of 70 plates, numbered HLA-XP-A2, HLA-XP-A3, HLA-XP-A4, HLA-XP-B2, HLA-XP-B3, HLA-XP- B4 and HLA-XP-DRB1-2 ("X” indicates sample group number information 1/2/3/4/5/6/7/8/9/10, "A2/3/4, B2/3/4 , DRB1-2" indicates the amplified site), one negative control was added to each plate without template, and the primer used for the negative control was PI-1 (Table 1) labeled primer. At the same time of the experiment, record the sample group number information and primer label information corresponding to each sample. The specific method is as described in Example 2.
- DNA concentration 500 ul of DNA mixture was purified by Qiagen DNA Purification kit (see the instructions for specific purification steps), and 200 ul of DNA was purified, and the DNA concentration was determined by Nanodrop 8000 (Thermo Fisher Scientific). The other groups were operated identically.
- the DNA concentration is:
- the reaction product was purified by Ampure Beads (Beckman Coulter Genomics) and dissolved in 50 ul of deionized water.
- the results of DNA molar concentration detected by fluorescent quantitative PCR (QPCR) were as follows:
- HLA-1-Mix, HLA-2-Mix, HLA-3-Mix, HLA-4-Mix, HLA-5-Mix, HLA-6-Mix, HLA-7-Mix, HLA-8-Mix, HLA -9-Mix It was mixed with HLA-10-Mix equimolar (final concentration 72.13 nM/ul), labeled as HLA-Mix-10, and 30 ⁇ HLA-Mix-10 was recovered with 2% low melting agarose strand.
- the electrophoresis conditions were 100V, 100min.
- the DNA marker is NEB's 50 bp DNA marker net cut strand to recover a DNA fragment of 450-750 bp in length.
- the strand recovered product was recovered and purified by QIAquick PCR Purification Kit (QIAGEN), and the volume after purification was 32 ul, which was subjected to real-time PCR (QPCR).
- the DNA concentration was detected to be 9.96 nM.
- Example 8 HLA-C genotyping using second generation sequencing technology (Illumina GA)
- PCR primers used are exon 2, 3 and 4 PCR primers for HLA-C, as shown in Table 3.
- each forward PCR primer has a forward primer label attached to a pair of primer tags at the 5th end, and a reverse primer label to a pair of primer tags at the 5th end of the reverse PCR primer. .
- the primer tag is added directly to the 5, end of the PCR primer when the primer is synthesized.
- the 95 DNAs obtained in the sample extraction step were numbered 1-95 in turn, and the PCR ⁇ should be performed in a 96-well plate, totaling 3 plates, numbered HLA-P-C2, HLA-P-C3, HLA-P- C4 (C2/3/4 indicates the amplified site), and a template is not added in the plate.
- the primer used for the negative control was identical to the primer PI-96. At the same time as the experiment, record the sample number information corresponding to each pair of primer labels.
- primer labels used are the same as the primer labels PI-1 to PI-95 listed in Table 6, and the negative control primer label PI-96 (Table 8) below.
- the DNA extracted by the KingFisher automatic extractor in step 1 was used as a template, and the HLA-C exon primers with 5, end-end primers were single-tube PCR amplified.
- the PCR procedure was as follows:
- the PCR system of HLA-C is as follows
- PI nf -CF 2/3/4 represents Primer 5
- PI nr -CR 2/3/4 represents Primer 5
- each sample corresponds to a specific set of PCR primers.
- PCR ⁇ should be run on Bio-Rad's PTC-200 PCR machine. After the PCR was completed, 2 ul of the PCR product was detected by 1.5% agarose gel electrophoresis.
- Figure 6 shows the results of electrophoresis of the corresponding exon PCR products of the first 20 samples of HLA-C.
- the DNA molecule is labeled as DL 2000 ( Takara), and a series of fragments with a size of 400 bp to 500 bp are shown on the gel image, indicating that Some samples of HLA-C exons (C2, C3, C4) were successfully amplified by PCR. The results for the other samples are similar.
- HLA-C2-Mix From the remaining PCR products of 96-well plate HLA-P-C2 (except the negative control), 20 ul each was mixed in a 3 ml EP tube, labeled as HLA-C2-Mix, and the same for the other two 96-well plates. The operations were labeled HLA-C3-Mix and HLA-C4-Mix, respectively, and shaken and mixed. Take 200 ul from HLA-C2-Mix, HLA-C3-Mix and HLA-C4-Mix in a 1.5 ml mixture.
- HLA-Mix 500 ul of DNA mixture from HLA-Mix was purified by Qiagen DNA Purification kit (QIAGEN) (see the instructions for specific purification steps), and the purified 200 ul DNA was obtained. Nanodrop 8000 (Thermo Fisher Scientific) determined the HLA-Mix DNA concentration to be 50 ng/ul.
- a total of 5 ug of DNA from the purified HLA-Mix was interrupted on Covaris S2 (Covaris) using Covaris microTube with AFA fiber and Snap-Cap.
- the breaking conditions are as follows:
- Cycles/Burst (Cycles/Burst) 200
- the purified product was subjected to DNA end-repairing, and the system was as follows (reagents were purchased from Enzymatics):
- the reaction conditions are: at 20. Under C, warm bath in Thermomixer (Eppendorf) for 30 minutes.
- reaction product was purified by QIAquick PCR Purification Kit and dissolved in 32 ⁇ M EB (QIAGEN Elution Buffer).
- the DNA was recovered in the previous step, and the end was added with A reaction.
- the system was as follows (reagents were purchased from Enzymatics):
- the reaction conditions are: at 37. Under C, warm bath in a Thermomixer for 30 minutes.
- the reaction product was recovered and purified by MiniElute PCR Purification Kit (QIAGEN) and dissolved in 38 ⁇ M of EB solution (QIAGEN Elution Buffer).
- reaction conditions are: at 16. Under C, warm in a Thermomixer overnight.
- the reaction product was purified by Ampure Beads (Beckman Coulter Genomics) and dissolved in 50 ul of deionized water.
- the DNA concentration was determined by quantitative PCR (QPCR) as follows:
- HLA-Mix 30 ⁇ HLA-Mix was recovered using 2% low melting point agarose gel. The electrophoresis conditions were 100V for 100 minutes.
- the DNA standard molecular weight reference is NEB's 50 bp DNA Ladder faced tapping recovery DNA fragment of 400-750 bp length (Fig. 7).
- the recovered product was recovered and purified by QIAquick PCR Purification Kit (QIAGEN). Ul, the DNA concentration was detected by real-time PCR (QPCR) to be 17.16 nM.
- the sequencing result of Illumina GA is a series of DNA sequences.
- a database for sequencing results of each exon PCR product corresponding to each HL AC sample is established.
- (Burrows-Wheeler Aligner) locates the sequencing results of each exon on the reference sequence of the corresponding exon (reference sequence source: http://www.ebi.ac.uk/imgt/hla/) and constructs each Consensus sequence of the database; combined with the quality of the base sequencing and the difference between the sequencing sequence and the consensus sequence, screening and sequencing error correction of the sequencing sequence; and the alignment of the corrected DNA sequence by sequence overlap and linkage ( The Pair-End linkage relationship can be assembled into the corresponding sequences of each of the HLA-C exons.
- the screenshot of Fig. 8 exemplifies the process of constructing the exon 2 consensus sequence of the HLA-C site of sample No. 2.
- the DNA sequence of the sequenced HLA-C intron was aligned with the sequence database of the corresponding exons of HLA-C in the IMGT HLA professional database, and the HLA-C gene of the corresponding sample was matched 100% of the sequence alignment results. Type. For all 95 samples, the obtained classification results are completely consistent with the original known classification results. The specific results of the samples No. 1-32 are compared with the original classification results of the sample as follows: (See Table 9, all test results and original test The result is the same).
- the PCR system for HLA-C is as follows:
- the PCR product was detected by agarose gel electrophoresis (Fig. 9) and prepared for purification.
- PCR product purification was performed using a millipore purification plate. The basic steps are: Mark the required wells on the 96-well PCR product purification plate with a marker, add 50 ⁇ l of ultrapure water to the wells to be used, attach the remaining pores to the sealing membrane, let stand for 15 minutes at room temperature or connect to the pumping On the filter system, take -10 Pa for 5 minutes. Each time the purification plate is removed from the suction system, the liquid remaining in the bottom drain of the purification plate is blotted on the absorbent paper.
- the PCR product to be purified was centrifuged at 4000 rpm for 1 minute; the lid or silica gel pad of the PCR product to be purified was opened, and 100 ul of ultrapure water was added to each PCR reaction system. Then, the purification plate to which the PCR product to be purified is added is connected to the suction filtration system, the vacuum degree is adjusted to a barometer to display -10 Pa, and the microporous regenerated fiber membrane at the bottom of the purification plate is suction-filtered to have no liquid, and the light is observed without completeness. The liquid surface reflects the luster.
- the sequencing plate was placed in the dark for 30 minutes and air dried to an ethanol free odor. Add 10 ⁇ g of HI-DI formamide to each well of a 96-well plate (384 ⁇ L per well), cap the membrane, shake for 5 seconds, and centrifuge to 1000 rpm.
- the purified sequencing reaction product was subjected to capillary electrophoresis on ABI 3730XL, and the sequencing peak image was analyzed by uType software (Invitrogen) (Fig. 10) to obtain HLA typing results. All test results are the same as the original test results, as shown in Table 10.
- Example 8 According to the method of Example 8, 94 samples of known HL A-SBT typing results were subjected to HLA-DQB1 genotyping, except as described below.
- each set of PCR primers consisting of PCR primers for exon 2 or 3 of HLA-DQB1 (Table 5) and a pair of bidirectional primer tags (see below)
- the composition wherein each of the forward PCR primers has a forward primer label attached to a pair of primer tags at the 5th end, and a reverse primer label of a pair of primer tags attached to the 5th end of the reverse PCR primer.
- Primer tags were added directly to the 5' end of the PCR primers during primer synthesis, and the primers were synthesized by Shanghai Invitrogen.
- the 94 DNAs obtained in the sample extraction step were numbered 1-94 in turn, and the PCR ⁇ should be performed in a 96-well plate.
- the DQB1 2,3 exons of each sample were amplified in the same reaction well.
- Two negative controls without template were set in the plate, and the primers used for the negative control corresponded to the label numbers PI-95 and PI-96.
- primer labels used were the same as the primer labels PI-1 to PI-94 listed in Table 6, and the following negative control primer labels PI-95 and PI-96 (Table 11) Table 11, information on the negative control primer labels used
- the PCR system of HLA-DQBl is as follows
- PInf-Q-F2/3 represents primer 5
- PInf-QR 2/3 represents the primer 5
- the end has the nth The R primer of HLA-DQB1 of the reverse primer tag sequence (where n ⁇ 96), and so on.
- each sample corresponds to a specific set of PCR primers.
- PCR ⁇ should be run on Bio-Rad's PTC-200 PCR machine. After the PCR was completed, 2 ul of the PCR product was detected by 1.5% agarose gel electrophoresis.
- Figure 11 shows the results of electrophoresis of 94 samples of HLA-DQB1 2+3 exon PCR products.
- the DNA standard molecular weight reference (M) is DL 2000 ( Takara). 3. PCR product mixing and purification
- the breaking conditions are as follows:
- reaction product after the end-repair reaction was recovered by QIAquick PCR Purification Kit and dissolved in 34 ⁇ M of EB (QIAGEN Elution Buffer).
- reaction product after the end of the A reaction was recovered and purified by a MiniElute PCR Purification Kit (QIAGEN), and dissolved in 13 ⁇ M of EB solution (QIAGEN Elution Buffer).
- the reaction product after ligation of the library linker was purified by Ampure Beads (Beckman Coulter Genomics) and dissolved in 50 ul of deionized water.
- the DNA concentration was determined by fluorescent quantitative PCR (QPCR) as follows:
- the 350-550 bp DNA fragment was recovered by tapping (Fig. 12). After purification of the gel recovery product, the DNA concentration was 18.83 nM by real-time PCR (QPCR).
- the sequencing result of Illumina GA is a series of DNA sequences. Each primer pair is created by looking up the positive and negative primer tag sequences and primer sequences in the sequencing results.
- the screenshot of Figure 13 exemplifies the process of constructing the exon 2 consensus sequence of the HLA-DQB1 site of sample No. 7.
- the DNA sequence of the sequenced HLA-DQB1 2,3 exon is aligned with the sequence database of the corresponding exon of HLA-DQB1 in the IMGT HLA professional database.
- the 100% match of the sequence alignment results is the HLA of the corresponding sample. DQB1 genotype.
- Table 12 Classification results for samples 1-32.
- the PCR system of HLA-Q is as follows:
- the PCR product was detected by agarose gel electrophoresis and prepared for purification.
- the purified sequencing reaction product was subjected to capillary electrophoresis on ABI 3730XL, and the sequencing peak image was analyzed by uType software (Invitrogen) (Fig. 15) to obtain HLA typing results. All test results are the same as the original test results (Table 13).
- DQB1*05:03 DQB1*04:01 DQB1*05:03 DQB1*04:01 is the embodiment 12.
- HLA-A/B/C 2, 3, 4 exons and HLA-DQB1 for 950 samples Genotyping of exon 2, exon 3
- HLA-A/B/C 2 is used for 950 samples based on primer labeling, DNA incomplete disruption, library tagging, and PCR-FREE library Illumia GA Pair-End sequencing. Genotyping of exon 3, exon 4 and HLA-DQB1 exon 2 (the length of PCR product is between 300bp and 500bp), which proves that this strategy can achieve gene fragmentation beyond the length of the sequencer itself. Genotyping, and demonstrating that the invention enables low cost, high throughput, high accuracy and high resolution HLA genotyping.
- the samples to be analyzed are divided into 10 groups on average, and each group of samples is subjected to PCR reaction at both ends of the PCR products of HLA-A/B/C 2, 3, 4 exons and HLA-DQB1 2, exon 3.
- a primer tag is introduced to specifically label the sample information of the PCR product.
- the PCR amplification products of the four sites of HLA-A/B/C/DQB1 in each sample were mixed together to obtain a PCR product library; the obtained PCR product library was constructed after different incomplete interruption by ultrasound.
- PCR-Free tag sequencing library (where the PCR product library of each set of samples uses a different linker to construct 10 tag sequencing libraries); Mix 10 tag sequencing libraries in equimolar to construct a hybrid tag sequencing library, mix Tag sequencing library with 2% low melting point Agarose electrophoresis, tapping purification recovers all DNA bands between 450-750p length.
- the recovered DNA was sequenced by Illumina GA PE-100.
- the sequence information of all the tested samples can be found by the library tag and the primer tag sequence, and the sequence of the entire PCR product is assembled by the overlapping and linkage relationship between the reference sequence information of the known DNA fragment and the sequence of the DNA fragment, and then passed through HLA.
- -A/B/C/DQB1 The alignment of the standard database of the corresponding exons can assemble the entire sequence of the original PCR product to achieve genotyping of HLA-A/B/C/DQB1.
- the 950 DNAs obtained in the sample extraction step were sequentially numbered 1-950, and were divided into 10 groups of 95 DNAs each labeled HLA-1, HLA-2, HLA-3, HLA-4, HLA-5, HLA-6, HLA-7, HLA-8, HLA-9, HLA-10.
- 95 sets of bidirectional primer tags (Table 6) were used to amplify HLA-A/B2, exons 3, 4 (Table 2), and exons HLA-C 2, 3, 4 ( Table 4) and PCR primers for HLA-DQB1 exon 2, exon 3 (Table 5) were used to amplify 95 DNA samples, respectively.
- the PCR reaction was carried out in a 96-well plate with a total of 100 plates, numbered HLA-XP-A2, HLA-XP-A3, HLA-XP-A4, HLA-XP-B2, HLA-XP-B3, HLA-XP- B4, HLA-XP-C2, HLA-XP-C3, HLA-XP-C4 and HLA-XP-DQB1 ("X" indicates sample group number information 1/2/3/4/5/6/7/8/ 9/10, "A2/3/4, B2/3/4, C2/3/4, DQBl" indicates the amplified site), each plate is provided with a negative control without template, Primers used for sex control were primers labeled with PI-1 (Table 6). At the same time of the experiment, the sample group number information and the primer label information corresponding to each sample are recorded. Such as PI-1 and
- the information about the PI-2 primer label is as follows, and so on.
- the PCR program and PCR reaction system of HLA-A/B/C are the same as in Example 2, and the PCR primers used to amplify the corresponding exons of HLA-A/B are shown in Table 2, which are used to amplify HLA.
- the PCR primers for the corresponding exons of -C are shown in Table 4.
- the multiplex (2,3 exon amplification together) PCR reaction system of HLA-DQB1 was the same as in Example 10, and the PCR primers used to amplify the corresponding exons of HLA-DQB1 are shown in Table 5.
- PI nr A/B/CF 2/3/4 and PI nf -Q-F2/F3 represent primer 5, and HLA-A/B/C/ with the nth forward primer tag sequence (Table 6) at the end.
- the F primer of DQB1, PI nr -A/B/CR 2/3/4 and PI nr -Q-R2/R3 represent primer 5, HLA-A/B/C with the nth reverse primer tag sequence at the end /RQB1 R primer (here n ⁇ 95), others and so on.
- Each sample corresponds to a specific set of PCR primers (PInfA/B/CF, PI nr -A/B/CR, PI nf -Q-F2/F3 , PI nr -Q-R2/R3 ).
- the PCR reaction was run on a Bio-Rad PTC-200 PCR machine. After the PCR was completed, 3 ul of the PCR product was detected by 2% agarose gel electrophoresis.
- Figure 16 shows the results of electrophoresis of the corresponding exon PCR product of sample No. 1 HLA-A/B/C/DQB1.
- the DNA molecule is labeled as DL 2000 ( Takara).
- the gel image has a series of single bands with a fragment size of 300bp-500bp. , indicating that the exon (A2, A3, A4, B2, B3, B4, C2, C3, C4, DQB1) of HLA-A/B/C/DQB1 of sample No. 1 was successfully amplified by PCR, negative control (N) No amplified bands. The results for the other samples are similar.
- the reaction product was purified by Ampure Beads (Beckman Coulter Genomics) and dissolved in 50 ul of deionized water.
- the molar concentration of DNA detected by real-time PCR (QPCR) was as follows:
- HLA-1-Mix, HLA-2-Mix, HLA-3-Mix, HLA-4-Mix, HLA-5-Mix, HLA-6-Mix, HLA-7-Mix, HLA-8-Mix, HLA -9-Mix and HLA-10-Mix were equimolar mixed (final concentration 70.86 nM/ul), labeled HLA-Mix-10, and 30 ⁇ HLA-Mix-10 was recovered with 2% low melting agarose gel.
- the electrophoresis conditions were 100V, 100min.
- the DNA marker is a 50 bp DNA marker from NEB. The gel was recovered to recover a DNA fragment of 450-750 bp in length (Fig. 17).
- Glue recovery product by QIAquick PCR Purification Kit QIAGEN The company was recovered and purified, and the volume after purification was 32 ul.
- the DNA concentration was 10.25 nM by real-time PCR (QPCR).
- ⁇ 0:90* should be ZO:W)* should be zo : W*3 zo ⁇ L0*3 ⁇ TO ⁇ ⁇ ⁇ ⁇ * ⁇ ⁇
- Tiercy J M Molecular basis of HLA polymorphism: implications in clinical transplantation. [J]. Transpl Immunol, 2002, 9: 173-180.
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Management, Administration, Business Operations System, And Electronic Commerce (AREA)
Description
Claims
Priority Applications (10)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1020137002332A KR101709826B1 (ko) | 2010-06-30 | 2011-06-30 | 신규 pcr 서열화 방법 및 hla 제노타이핑에서의 상기 방법의 사용 |
US13/807,660 US9957564B2 (en) | 2010-06-30 | 2011-06-30 | Application of a PCR sequencing method, based on DNA barcoding technique and DNA incomplete shearing strategy, in HLA genotyping |
DK11800190.8T DK2599877T3 (da) | 2010-06-30 | 2011-06-30 | Hidtil ukendt pcr sekvenseringsfremgangsmåde og anvendelse deraf i hla-genotypebestemmelse |
JP2013516983A JP5968879B2 (ja) | 2010-06-30 | 2011-06-30 | Dna分子タグ技術及びdna不完全断片化技術に基づいたpcrシークエンシング法及びそれを用いたhla遺伝子タイピング法 |
SG2012096616A SG186876A1 (en) | 2010-06-30 | 2011-06-30 | New pcr sequencing method and use thereof in hla genotyping |
EP11800190.8A EP2599877B1 (en) | 2010-06-30 | 2011-06-30 | New pcr sequencing method and use thereof in hla genotyping |
CA2803940A CA2803940C (en) | 2010-06-30 | 2011-06-30 | Application of a pcr sequencing method, based on dna barcoding technique and dna incomplete shearing strategy, in hla genotyping |
AU2011274090A AU2011274090B2 (en) | 2010-06-30 | 2011-06-30 | New PCR sequencing method and use thereof in HLA genotyping |
BR112012032586-8A BR112012032586B1 (pt) | 2010-06-30 | 2011-06-30 | Métodos para determinar a sequência nucleotídica de um ácido nucléico de interesse e para determinar o genótipo hla em uma amostra |
RU2013103795/10A RU2587606C2 (ru) | 2010-06-30 | 2011-06-30 | Новый способ пцр-секвенирования и его применение в генотипировании hla |
Applications Claiming Priority (10)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201010213721.2 | 2010-06-30 | ||
CN201010213719.5A CN101921841B (zh) | 2010-06-30 | 2010-06-30 | 基于Illumina GA测序技术的HLA基因高分辨率分型方法 |
CN201010213719.5 | 2010-06-30 | ||
CN201010213717.6A CN101921840B (zh) | 2010-06-30 | 2010-06-30 | 一种基于dna分子标签技术和dna不完全打断策略的pcr测序方法 |
CN201010213717.6 | 2010-06-30 | ||
CN 201010213721 CN101921842B (zh) | 2010-06-30 | 2010-06-30 | Hla-a,b基因分型用pcr引物及其使用方法 |
PCT/CN2010/002149 WO2012083505A1 (zh) | 2010-12-24 | 2010-12-24 | Hla-c基因分型的方法及其相关引物 |
CNPCT/CN2010/002149 | 2010-12-24 | ||
CNPCT/CN2010/002150 | 2010-12-24 | ||
PCT/CN2010/002150 WO2012083506A1 (zh) | 2010-12-24 | 2010-12-24 | Hla-dqb1基因分型的方法及其相关引物 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2012000445A1 true WO2012000445A1 (zh) | 2012-01-05 |
Family
ID=45401405
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2011/076688 WO2012000445A1 (zh) | 2010-06-30 | 2011-06-30 | 新的的pcr测序方法及其在hla基因分型中的应用 |
Country Status (12)
Country | Link |
---|---|
US (1) | US9957564B2 (zh) |
EP (1) | EP2599877B1 (zh) |
JP (1) | JP5968879B2 (zh) |
KR (1) | KR101709826B1 (zh) |
AU (1) | AU2011274090B2 (zh) |
BR (1) | BR112012032586B1 (zh) |
CA (1) | CA2803940C (zh) |
DK (1) | DK2599877T3 (zh) |
MY (1) | MY173793A (zh) |
RU (1) | RU2587606C2 (zh) |
SG (1) | SG186876A1 (zh) |
WO (1) | WO2012000445A1 (zh) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2986741A4 (en) * | 2013-04-17 | 2016-12-14 | Agency Science Tech & Res | METHOD FOR GENERATING ADVANCED SEQUENCE READINGS |
EP3006571A4 (en) * | 2013-05-09 | 2017-02-22 | Genodive Pharma Inc. | Hla gene multiplex dna typing method and kit |
CN108699600A (zh) * | 2016-02-23 | 2018-10-23 | 诺维信公司 | 改进的新一代测序 |
US10233490B2 (en) | 2014-11-21 | 2019-03-19 | Metabiotech Corporation | Methods for assembling and reading nucleic acid sequences from mixed populations |
Families Citing this family (26)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2016528913A (ja) * | 2013-08-26 | 2016-09-23 | ザ・トランスレーショナル・ジェノミクス・リサーチ・インスティチュート | 病原体試料におけるマイナー変異体突然変異検出のための単一分子重複読み取り分析 |
GB201409282D0 (en) | 2014-05-23 | 2014-07-09 | Univ Sydney Tech | Sequencing process |
EP3842544A1 (en) * | 2014-09-05 | 2021-06-30 | QIAGEN GmbH | Preparation of adapter-ligated amplicons |
ES2726149T3 (es) * | 2014-09-12 | 2019-10-02 | Mgi Tech Co Ltd | Oligonucleótido aislado y su uso en la secuenciación de ácidos nucleicos |
CN105400864B (zh) * | 2014-09-12 | 2020-04-14 | 深圳华大基因股份有限公司 | 用于基于血液样品构建测序文库的方法及其在确定胎儿遗传异常中的用途 |
CA2971589C (en) | 2014-12-18 | 2021-09-28 | Edico Genome Corporation | Chemically-sensitive field effect transistor |
US9857328B2 (en) | 2014-12-18 | 2018-01-02 | Agilome, Inc. | Chemically-sensitive field effect transistors, systems and methods for manufacturing and using the same |
US9859394B2 (en) | 2014-12-18 | 2018-01-02 | Agilome, Inc. | Graphene FET devices, systems, and methods of using the same for sequencing nucleic acids |
US10020300B2 (en) | 2014-12-18 | 2018-07-10 | Agilome, Inc. | Graphene FET devices, systems, and methods of using the same for sequencing nucleic acids |
US9618474B2 (en) | 2014-12-18 | 2017-04-11 | Edico Genome, Inc. | Graphene FET devices, systems, and methods of using the same for sequencing nucleic acids |
US10006910B2 (en) | 2014-12-18 | 2018-06-26 | Agilome, Inc. | Chemically-sensitive field effect transistors, systems, and methods for manufacturing and using the same |
CN107002153B (zh) * | 2015-02-04 | 2020-10-27 | 深圳华大生命科学研究院 | 一种构建长片段测序文库的方法 |
KR101651817B1 (ko) * | 2015-10-28 | 2016-08-29 | 대한민국 | Ngs 라이브러리 제작용 프라이머 세트 및 이를 이용한 ngs 라이브러리 제작방법 및 키트 |
WO2017201081A1 (en) | 2016-05-16 | 2017-11-23 | Agilome, Inc. | Graphene fet devices, systems, and methods of using the same for sequencing nucleic acids |
IL266197B2 (en) | 2016-10-24 | 2024-03-01 | Geneinfosec Inc | Hiding information contained within nucleic acids |
MX2019005684A (es) * | 2016-11-15 | 2019-10-30 | Quest Diagnostics Invest Llc | Métodos para detectar mutaciones de fibrosis quística mediante el uso de extracción con puntas mitra. |
WO2018093724A1 (en) | 2016-11-15 | 2018-05-24 | Quest Diagnostics Investments Llc | Methods for detecting dna mutations using mitra tip extraction |
EP3626835A1 (en) | 2018-09-18 | 2020-03-25 | Sistemas Genómicos, S.L. | Method for genotypically identifying both alleles of at least one locus of a subject's hla gene |
RU2703394C1 (ru) * | 2018-10-24 | 2019-10-16 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Кубанский государственный аграрный университет имени И.Т. Трубилина" | Способ выявления и генотипирования РНК вируса репродуктивно-респираторного синдрома свиней |
RU2703401C1 (ru) * | 2018-10-24 | 2019-10-16 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Кубанский государственный аграрный университет имени И.Т. Трубилина" | Тест-система для выявления и генотипирования РНК вируса репродуктивно-респираторного синдрома свиней |
FR3100820B1 (fr) | 2019-09-12 | 2024-06-28 | Bionobis | Procédé de génotypage adapté au traitement d’un grand nombre d’échantillons, notamment en cas de polymorphisme élevé |
FR3106596B1 (fr) | 2020-01-27 | 2022-12-02 | Bionobis | Procede de genotypage hla simple et rapide |
CN111235266B (zh) * | 2020-03-10 | 2023-12-01 | 广州医科大学附属第二医院 | Hla亚型检测试剂盒及其应用 |
FR3110178B1 (fr) | 2020-05-18 | 2022-05-27 | Bionobis | Procédé de génotypage adapté au traitement simultané d’un grand nombre de patients |
CN112553313A (zh) * | 2020-12-30 | 2021-03-26 | 武汉康圣达医学检验所有限公司 | 用于Sanger法的测序反应产物的变性方法 |
KR102507110B1 (ko) * | 2022-02-15 | 2023-03-07 | 주식회사 네오젠티씨 | 주조직 적합성 복합체의 타입들을 분석하기 위한 방법 및 장치 |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7300755B1 (en) * | 2003-05-12 | 2007-11-27 | Fred Hutchinson Cancer Research Center | Methods for haplotyping genomic DNA |
CN101313078A (zh) * | 2005-09-29 | 2008-11-26 | 科因股份有限公司 | 突变群体的高通量筛选 |
CN101921841A (zh) * | 2010-06-30 | 2010-12-22 | 深圳华大基因科技有限公司 | 基于Illumina GA测序技术的HLA基因高分辨率分型方法 |
CN101921840A (zh) * | 2010-06-30 | 2010-12-22 | 深圳华大基因科技有限公司 | 一种基于dna分子标签技术和dna不完全打断策略的pcr测序方法 |
CN101921842A (zh) * | 2010-06-30 | 2010-12-22 | 深圳华大基因科技有限公司 | Hla-a,b基因分型用pcr引物及其使用方法 |
Family Cites Families (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1993009245A1 (en) | 1991-10-31 | 1993-05-13 | University Of Pittsburgh | Reverse dot blot hybridization using tandem head-to-tail monomers containing probes synthesized by staggered complementary primers |
US5580730A (en) * | 1994-08-19 | 1996-12-03 | Olympus America, Inc. | Enzyme digestion method for the detection of amplified DNA |
US6287764B1 (en) * | 1997-02-11 | 2001-09-11 | William H. Hildebrand | Class I sequence based typing of HLA-A, -B, and -C alleles by direct DNA sequencing |
EP0953650A1 (en) * | 1998-04-20 | 1999-11-03 | Innogenetics N.V. | Method for typing of HLA alleles |
EP1594950A4 (en) * | 2003-01-29 | 2007-07-25 | 454 Corp | PRODUCT FOR THE PREPARATION OF SIMPLE STRANDED DNA BANKS |
US20070111213A1 (en) * | 2003-10-28 | 2007-05-17 | Lu Wang | Primers, methods and kits for amplifying or detecting human leukocyte antigen alleles |
AU2006244042B2 (en) * | 2005-05-10 | 2010-07-29 | State Of Oregon Acting By & Through The State Board Of Higher Education On Behalf Of The University Of Oregon | Methods of mapping polymorphisms and polymorphism microarrays |
EP2477029A1 (en) * | 2005-06-02 | 2012-07-18 | Fluidigm Corporation | Analysis using microfluidic partitioning devices |
CN101310024B (zh) | 2005-11-14 | 2012-10-03 | 科因股份有限公司 | 高通量筛选转座子标记群体和大量平行的插入位点的序列鉴定方法 |
CN101374963B (zh) * | 2005-12-22 | 2014-06-04 | 凯津公司 | 用于基于aflp的高通量多态性检测的方法 |
AU2007257340A1 (en) * | 2006-06-09 | 2007-12-13 | Celera Corporation | Identification of a nucleic acid molecule |
US20100086914A1 (en) * | 2008-10-03 | 2010-04-08 | Roche Molecular Systems, Inc. | High resolution, high throughput hla genotyping by clonal sequencing |
CA2701411A1 (en) * | 2007-10-16 | 2009-04-23 | F. Hoffmann-La Roche Ag | High resolution, high throughput hla genotyping by clonal sequencing |
RU2393231C1 (ru) * | 2008-12-29 | 2010-06-27 | Федеральное государственное учреждение здравоохранения "Российский научно-исследовательский противочумный институт "Микроб" Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека ("РосНИПЧИ "Микроб") | Способ определения генетического родства штаммов холерных вибрионов методом секвенирования генов, фланкирующих кластер генов биосинтеза о-антигена |
-
2011
- 2011-06-30 SG SG2012096616A patent/SG186876A1/en unknown
- 2011-06-30 BR BR112012032586-8A patent/BR112012032586B1/pt active IP Right Grant
- 2011-06-30 DK DK11800190.8T patent/DK2599877T3/da active
- 2011-06-30 CA CA2803940A patent/CA2803940C/en active Active
- 2011-06-30 JP JP2013516983A patent/JP5968879B2/ja active Active
- 2011-06-30 AU AU2011274090A patent/AU2011274090B2/en active Active
- 2011-06-30 KR KR1020137002332A patent/KR101709826B1/ko active IP Right Grant
- 2011-06-30 RU RU2013103795/10A patent/RU2587606C2/ru active IP Right Revival
- 2011-06-30 EP EP11800190.8A patent/EP2599877B1/en active Active
- 2011-06-30 US US13/807,660 patent/US9957564B2/en active Active
- 2011-06-30 WO PCT/CN2011/076688 patent/WO2012000445A1/zh active Application Filing
- 2011-06-30 MY MYPI2012005591A patent/MY173793A/en unknown
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7300755B1 (en) * | 2003-05-12 | 2007-11-27 | Fred Hutchinson Cancer Research Center | Methods for haplotyping genomic DNA |
CN101313078A (zh) * | 2005-09-29 | 2008-11-26 | 科因股份有限公司 | 突变群体的高通量筛选 |
CN101921841A (zh) * | 2010-06-30 | 2010-12-22 | 深圳华大基因科技有限公司 | 基于Illumina GA测序技术的HLA基因高分辨率分型方法 |
CN101921840A (zh) * | 2010-06-30 | 2010-12-22 | 深圳华大基因科技有限公司 | 一种基于dna分子标签技术和dna不完全打断策略的pcr测序方法 |
CN101921842A (zh) * | 2010-06-30 | 2010-12-22 | 深圳华大基因科技有限公司 | Hla-a,b基因分型用pcr引物及其使用方法 |
Non-Patent Citations (24)
Title |
---|
A. DORMOY; N. FROELICH; LEISENBACH ET AL.: "Mono-allelic amplification of exons 2-4 using allele group-specific primers for sequence-based typing (SBT) of the HLA-A, -B and -C genes: Preparation and validation of ready-to-use pre-SBT mini-kits", J]. TISSUE ANTIGENS, vol. 62, 2003, pages 201 - 216 |
AL- HUSSEIN K A; RAMA N R; BUTT A I ET AL.: "HLA class II sequence based typing in normal Saudi individuals", TISSUE ANTIGENS, vol. 60, 2002, pages 259 - 261 |
C.ANTOINE; S.MIILLER; A.CANT ET AL.: "Long-term survival and transplantation of haemopoietic stem cells for immunodeficiencies: report of the European experience", THE LANCET, vol. 9357, 2003, pages 553 - 560, XP004778553, DOI: doi:10.1016/S0140-6736(03)12513-5 |
C.E.M. VOORTER; M.C. KIK1; E.M. VAN DEN BERG-LOONEN ET AL.: "High-resolution HLA typing for the DQB1 gene by sequence-based typing", TISSUE ANTIGENS, vol. 51, 2008, pages 80 - 87 |
CAMPBELL, K.J. ET AL.: "Characterization of 47 MHC class I sequences in Filipino cynomolgus macaques", IMMUNOGENETICS, vol. 61, 2009, pages 177 - 187, XP019705365 |
CHRISTIAN HOFFMANNL; NANA MINKAHL; JEREMY LEIPZIG: "DNA barcoding and pyrosequencing to identify rare HIV drug resistance mutations", NUCLEIC ACIDS RESEARCH, 2007, pages 1 - 8 |
D. C. SAYER; D. M. GOODRIDGE: "Pilot study: assessment of interlaboratory variability of sequencing-based typing DNA sequence data quality", TISSUE ANTIGENS, vol. 69, 2007, pages 66 - 68 |
ELAINE R. MARDIS: "The impact of next-generation sequencing technology on genetics", TRENDS IN GENETICS, vol. 24, 2008, pages 133 - 141, XP022498431 |
G. BENTLEY; R. HIGUCHI; B. HOGLUND ET AL.: "High-resolution, high-throughput HLA genotyping by next-generation sequencing", TISSUE ANTIGENS, vol. 74, 2009, pages 393 - 403, XP055087325, DOI: doi:10.1111/j.1399-0039.2009.01345.x |
GOULDER, P.J.R.; WATKINS, D.I.: "Impact of MHC class I diversity on immune control of immunodeficiency virus replication", NAT. REV. IMMUNOL, vol. 8, 2008, pages 619 - 630 |
H. A. ERLICH; G. OPELZ; J. HANSEN ET AL.: "HLA DNA Typing and Transplantation", IMMUNITY, vol. 14, 2001, pages 347 - 356 |
HOFFMANN C; MINKAH N; LEIPZIG J; WANG G; ARENS MQ; TEBAS P; BUSHMAN FD: "DNA bar coding and pyrosequencing to identify rare HIV drug resistance mutations", NUCLEIC ACIDS RES., vol. 35, no. 13, 2007, pages E91, XP055059023, DOI: doi:10.1093/nar/gkm435 |
HORTON V; STRATTON I; BOTTAZZO G. F. ET AL.: "Genetic heterogeneity of autoimmune diabetes: age of presentation in adults is influenced by HLA DRB1 and DQB1 genotypes", DIABETOLOGIA, vol. 42, 1999, pages 608 - 616 |
IWANKA KOZAREWA; ZEMIN NING; MICHAEL A QUAIL: "Amplification-free Illumina sequencing-library preparation facilitates improved mapping and assembly of (G+C)-biased genomes", NATURE METHODS, vol. 6, 2009, pages 291 - 295, XP055087323, DOI: doi:10.1038/nmeth.1311 |
JEESUK: "Analysis of children with type 1 diabetes in Korea: high prevalence of specific anti-islet autoantibodies, immunogenetic similarities to Western populations with ''unique'' haplotypes, and lack of discrimination by aspartic acid at position 57 of DQB.", CLINICAL IMMUNOLOGY., vol. 113, September 2004 (2004-09-01), pages 318 - 325, XP002716063 * |
LILLO R; BALAS A; VICARIO JL: "Two new HLA class allele, DPB 1*02014, by sequence-based typing", TISSUE ANTIGENS, vol. 59, 2002, pages 47 - 48 |
MARSH, S.G.E.; PARHAM, P.; BARBER, L.D.: "The HLA Facts Book", vol. 3, 2000, ACADEMIC PRESS, pages: 91 |
O'LEARY, C.E. ET AL.: "Identification of novel MHC class I sequences in pig-tailed macaques by amplicon pyrosequencing and full-length cDNA cloning and sequencing", IMMUNOGENETICS, vol. 61, 2009, pages 689 - 701, XP019757439, DOI: doi:10.1007/s00251-009-0397-4 |
ROBINSON J; MALIK A; PARHAM P; BODMER JG; MARSH SGE: "IMGT/HLA database-a sequence database for the human major histocompatibility complex", TISSUE ANTIGENS, vol. 55, 2000, pages 80 - 7 |
SAYER D; WHIDBORNE R; BRESTOVAC B.: "HLA-DRB1 DNA sequencing based typing: an approach suitable for high throughput typing including unrelated bone marrow registry donors", TISSUE ANTIGENS, vol. 57, no. 1, 2001, pages 46 - 54 |
See also references of EP2599877A4 |
SHANNON J.ODELBERG; ROBERT B.WEISS; AKIRA HATA: "Template-switching during DNA synthesis by Thermus aquaticus DNA polymerase I", NUCLEIC ACIDS RESEARCH, vol. 23, 1995, pages 2049 - 2057 |
TIERCY J M.: "Molecular basis ofHLA polymorphism: implications in clinical transplantation", J]. TRANSPL IMMUNOL, vol. 9, 2002, pages 173 - 180, XP027388192 |
WU, D. L. ET AL.: "Comparative analysis of serologic typing and HLA-II typing by micro-PCR-SSP", DI YI JUN YI DA XUE XUE BAO, vol. 22, 2002, pages 247 - 249 |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2986741A4 (en) * | 2013-04-17 | 2016-12-14 | Agency Science Tech & Res | METHOD FOR GENERATING ADVANCED SEQUENCE READINGS |
EP3597772A1 (en) * | 2013-04-17 | 2020-01-22 | Agency For Science, Technology And Research | Method for generating extended sequence reads |
US11859171B2 (en) | 2013-04-17 | 2024-01-02 | Agency For Science, Technology And Research | Method for generating extended sequence reads |
EP3006571A4 (en) * | 2013-05-09 | 2017-02-22 | Genodive Pharma Inc. | Hla gene multiplex dna typing method and kit |
US10711306B2 (en) | 2013-05-09 | 2020-07-14 | Genodive Pharma Inc. | Method and kit for multiplex DNA typing of HLA gene |
US10233490B2 (en) | 2014-11-21 | 2019-03-19 | Metabiotech Corporation | Methods for assembling and reading nucleic acid sequences from mixed populations |
CN108699600A (zh) * | 2016-02-23 | 2018-10-23 | 诺维信公司 | 改进的新一代测序 |
Also Published As
Publication number | Publication date |
---|---|
CA2803940A1 (en) | 2012-01-05 |
SG186876A1 (en) | 2013-02-28 |
EP2599877A4 (en) | 2014-01-01 |
AU2011274090A1 (en) | 2013-02-07 |
RU2013103795A (ru) | 2014-08-20 |
US9957564B2 (en) | 2018-05-01 |
JP2013529472A (ja) | 2013-07-22 |
KR101709826B1 (ko) | 2017-02-24 |
EP2599877A1 (en) | 2013-06-05 |
KR20130038353A (ko) | 2013-04-17 |
EP2599877B1 (en) | 2017-09-27 |
DK2599877T3 (da) | 2017-11-20 |
JP5968879B2 (ja) | 2016-08-10 |
US20130237432A1 (en) | 2013-09-12 |
BR112012032586A2 (pt) | 2019-07-30 |
AU2011274090B2 (en) | 2015-04-09 |
MY173793A (en) | 2020-02-24 |
CA2803940C (en) | 2019-07-02 |
RU2587606C2 (ru) | 2016-06-20 |
BR112012032586B1 (pt) | 2021-08-17 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2012000445A1 (zh) | 新的的pcr测序方法及其在hla基因分型中的应用 | |
WO2012000152A1 (zh) | 一种基于dna分子标签技术和dna不完全打断策略的pcr测序方法 | |
WO2012000150A1 (zh) | Hla-a,b基因分型用pcr引物及其使用方法 | |
WO2012000153A1 (zh) | 基于illuminaga测序技术的hla基因高分辨率分型方法 | |
CN110218781B (zh) | 21个微单倍型位点的复合扩增体系、下一代测序分型试剂盒及分型方法 | |
CA2841060A1 (en) | Method and kit for dna typing of hla gene | |
WO2012037883A1 (zh) | 核酸标签及其应用 | |
WO2011035550A1 (zh) | Hla基因扩增和基因分型方法及其相关引物 | |
WO2012068955A1 (zh) | Mhc区域核酸文库及其构建方法和用途 | |
WO2020232635A1 (zh) | 基于甲基化dna目标区域构建测序文库及系统和应用 | |
CN116179671A (zh) | 一种用于hla基因分型的扩增引物组、试剂盒及方法 | |
CN103270170B (zh) | Hla-dqb1基因分型的方法及其相关引物 | |
Bettinotti et al. | Comprehensive method for the typing of HLA-A, B, and C alleles by direct sequencing of PCR products obtained from genomic DNA | |
WO2017135396A1 (ja) | Pcrを用いないキャプチャー法によるhla遺伝子タイピング用プローブセット及びそれを用いたタイピング方法 | |
CN116323979A (zh) | 用于hla分型的方法、组合物和试剂盒 | |
TWI542696B (zh) | HLA - C genotyping and its related primers | |
KR102475292B1 (ko) | Hla 유전자 증폭용 조성물 및 이의 용도 | |
Wu et al. | Strategies for unambiguous detection of allelic heterozygosity via direct DNA sequencing of PCR products: application to the HLA DRB1 locus | |
JP5143450B2 (ja) | Hla−bローカスにおける新規アリル | |
Kunkel et al. | Molecular methods for human leukocyte antigen typing: current practices and future directions | |
WO2023248997A1 (ja) | ヒトsting1遺伝子における一塩基多型のハプロタイプ解析法 | |
CN114540486A (zh) | 一种hla-dpa1基因全长扩增引物组、分型试剂盒 | |
CN113957141A (zh) | 检测与高血压相关基因scnn1b突变的寡核苷酸和其应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 11800190 Country of ref document: EP Kind code of ref document: A1 |
|
REEP | Request for entry into the european phase |
Ref document number: 2011800190 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2011800190 Country of ref document: EP |
|
ENP | Entry into the national phase |
Ref document number: 2803940 Country of ref document: CA |
|
ENP | Entry into the national phase |
Ref document number: 2013516983 Country of ref document: JP Kind code of ref document: A |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
ENP | Entry into the national phase |
Ref document number: 20137002332 Country of ref document: KR Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 2013103795 Country of ref document: RU Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 2011274090 Country of ref document: AU Date of ref document: 20110630 Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 13807660 Country of ref document: US |
|
REG | Reference to national code |
Ref country code: BR Ref legal event code: B01A Ref document number: 112012032586 Country of ref document: BR |
|
REG | Reference to national code |
Ref country code: BR Ref legal event code: B01E Ref document number: 112012032586 Country of ref document: BR |
|
ENP | Entry into the national phase |
Ref document number: 112012032586 Country of ref document: BR Kind code of ref document: A2 Effective date: 20121219 |