TWI542696B - HLA - C genotyping and its related primers - Google Patents

Description

HLA-C genotyping method and related primers

The present invention relates to the field of molecular biology, and in particular, to a method for HLA-C genotyping, and specific primers for use in the method.

Human leucocyte antigen (HLA) is an important component of the human immune system and is the most complex genetic polymorphism system known to the human body. According to its structure, tissue distribution and function, HLA molecules can be divided into three types of molecules: HLA-I, HLA-II and HLA-III. Among them, HLA-I and HLA-II molecular functions are mainly immune. Rejection-related, HLA-III molecular function is mainly related to the synthesis of immune-related partial complement system and inflammation-related factors; clinical transplantation studies found that the long-term survival rate of grafts and HLA-I and HLA of both donors and recipients The degree of matching of the -II molecules is positively correlated, and the higher the degree of matching between the donor and recipient HLA molecules, the higher the long-term survival rate of the graft.

The HLA-C locus belongs to the classical HLA-I gene, located between the HLA-A and B loci, encoding the HLA-C molecule, and its gene length is about 3 Kb, consisting of 7 introns and 8 explicit Sub-composition. In recent years, with the continuous development of HLA genotyping technology, HLA-C has been found to be highly polymorphic like HLA-A and B molecules, and confirmed the importance of HLA-C molecules in clinical transplantation.

The current international standard HLA typing techniques include PCR-SSP (PCR-Sequence Specific Primer), PCR-SSO (PCR-Sequence Specific Oligonucleotide, polymerase chain reaction-sequence specific oligo Glycosylate) and PCR-SBT (PCR-Sequencing Based Typing, polymerase chain reaction - sequencing based typing).

The principle of HLA-SSP is to design a set of allele-specific primers, and obtain HLA-type specific amplification products by PCR technology, and determine the HLA type by electrophoresis analysis. The principle of HLA-SSO is to design an HLA-type specific oligonucleotide sequence as a probe, label the PCR product, and hybridize with the PCR product (gene DNA to be detected) and the probe. The HLA type is judged by detecting the fluorescent signal. The detection signals of HLA-SSP and HLA-SSO are analog signals, and the resolution generally can only reach the medium and low levels and can not detect new alleles.

HLA-SBT based on the Sanger legal sequence is a high-resolution typing method for determining the HLA genotype by directly performing Sanger sequencing (capillary microelectrophoresis) on the DNA product after PCR amplification, thereby determining the HLA genotype. Highly analytical and capable of detecting new alleles. However, the HLA-SBT based on Sanger's legal sequence has complicated shortcomings, low throughput and high experimental cost, making it difficult to apply to large-scale HLA high-resolution typing projects.

HLA-SBT based on the second-generation sequencing technology represented by Illumina Solexa and Roche 454 (hereinafter referred to as the new sequencing technology) is also a method for determining the HLA genotype by directly measuring the nucleic acid sequence of the PCR-amplified DNA product. The high-resolution typing method, in addition to the original intuitive, high-resolution and detection of new alleles, has the characteristics of single-molecule sequencing, simple experimental procedure, high throughput and low cost. However, compared to the first-generation sequencing technique (sequencing technology based on the Sanger principle principle), the DNA length that can be used for the sequencing of the new sequencing technology gene bank cannot be too long (the current maximum length of Illumina Solexa) It is 700 bp), and the new sequencing technology is generally short. The current Illumia GA bidirectional read length can reach 300 bp.

In view of the characteristics of the new sequencing technology, the length of the PCR product should not exceed 700 bp, and the PCR primer of HLA-SBT based on the Sanger legal method is no longer applicable. Therefore, it is necessary to design a set of primers with good conservation and specificity and the length of the PCR product to meet the requirements of the new sequencing technology.

Summary of invention

In order to use the second generation sequencing technology for HLA-C genotyping, the present invention provides two sets of three pairs of PCR primers for amplifying the exon 2, 3 and 4 of the HLA-C gene, which are respectively The sequence identification numbers shown in Table 1 are: 1 and 2, 3 and 4, and 5 and 6; the sequence identification numbers shown in Table 2 are: 7 and 8, 9 and 10, and 11 and 12. These two sets of 3 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 locus. The length of PCR products are less than 700 bp, which meets the requirements of normal Illumina Solexa. Order requirements. In addition, the primer of the present invention is also applicable to the Sanger legal order. Further, the primer of the present invention can also be used together with other HLA gene amplification primers.

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 sequences of the amplification primer pairs are shown in Table 1 and Table 2.

Since the exons 2, 3 and 4 of HLA-C can be amplified by a PCR reaction, the method of the 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 to be 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 exons 2, 3 and/or 4 in a sample, comprising the steps of:

(1) providing a sample and extracting the DNA of the sample;

(2) The pair of primers of the present invention is preferably selected from the sequence identification number: 1 and the sequence identification number: 2. the sequence identification number: 3 and the sequence identification number: 4, the sequence identification number: 5, and the sequence identification number: 6 , or sequence identification number: 7 and sequence identification number: 8, sequence identification number: 9 and sequence identification number: 10, sequence identification number: 11 and sequence identification number: 12 primer pair for amplifying the DNA to obtain a PCR product Preferably, the PCR product is purified;

(3) The PCR product is sequenced, preferably by a second generation sequencing method such as Illumina Solexa or Roche 454.

The invention also provides a HLA-C genotyping method, comprising:

(1) Using the primer pair of the present invention, preferably selected from the sequence identification number: 1 and the sequence identification number: 2, the sequence identification number: 3, and the sequence identification number: 4, the sequence identification number: 5, and the sequence identification number: 6 , or sequence identification number: 7 and sequence identification number: 8, sequence identification number: 9 and sequence identification number: 10, sequence identification number: 11 and sequence identification number: 12 primer pair for PCR amplification, amplification of the sample to be tested HLA-C gene exons 2, 3 and/or 4;

(2) Sequencing the amplified exons and comparing the sequencing results with the standard sequences in the database to determine genotyping results, which are determined by Sanger sequencing, or The second generation sequencing method is, for example, Illumina Solexa or Roche 454 by a second generation sequencing method.

In the above method of the present invention, PCR amplification is further carried out using other HLA gene amplification primers.

In another aspect, the invention provides a kit for performing HLA-C genotyping, the kit comprising a PCR amplification primer pair of the invention, preferably selected from the sequence identification number: 1 and sequence identification No.: 2. Sequence identification number: 3 and sequence identification number: 4. Sequence identification number: 5 and sequence identification number: 6, or sequence identification number: 7 and sequence identification number: 8. Sequence identification number: 9 and sequence identification number : 10, sequence identification number: 11 and sequence identification number: 12 pairs of primers. In the kit of the present invention, other HLA gene amplification primers may further be included. In one embodiment, the kit further comprises other reagents, such as reagents for DNA amplification, DNA purification, and/or DNA sequencing.

The amplification primer pair and 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 simplify the process while saving time and cost.

Detailed description of the preferred embodiment

The following examples are presented to enable those of ordinary skill in the art to understand the present invention, which is intended to be illustrative only, and is not intended to limit the scope of the disclosure.

The present invention employs the following method to design three pairs of PCR primers that amplify exons 2, 3 and 4 of HLA-C. Download all the latest HLA-C gene sequences from the IMGT/HLA Internet site and save them to your local disk as an HLA-C database. Also download all the latest non-HLA-C HLA-I gene sequences as a comparison database. . The two databases were compared, and the conserved and specific sequences of each locus were searched at 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. Since the HLA-C gene has high sequence similarity to other genes belonging to the HLA class I molecule, the PCR primer is designed to ensure the specificity of the 3' end of the primer, and to ensure the specificity of the primer to amplify the HLA-C gene. At the same time, the length of the PCR product is less than 700 bp, and the bonding temperature of the positive and negative primers is basically the same. Multiple pairs of candidate HLA-C primers that meet the design requirements were used to amplify a small number of template DNAs with common serotypes of HLA-C, and the most conservative and specific ones were selected for amplification of HLA-C 2 2 sets of 3 pairs of PCR primers for exons 3 and 4.

The DNA amplification method, the method of extracting DNA from a sample, the DNA purification method, and the DNA sequence alignment method involved in the present invention may be any available methods in the art. These methods can be selected by a person skilled in the art in the art, depending on the circumstances. For methods of DNA sequencing, those skilled in the art can perform the methods according to conventional methods or according to the instructions for use of the sequencing instrument.

For example, in the sequencing process using the second-generation sequencing technique, a label primer with a primer index sequence added at the 5' end can be used to interrupt the amplified PCR product and interrupt the product. End repair was performed and deoxyadenosine (A) was ligated at its 3' end, followed by a different PCR-free adaptor.

A sequence of tags is attached to the front end of the amplification primer to achieve simultaneous sequencing of multiple samples. Specifically, a primer primer can be synthesized by adding a primer index sequence at the 5' end of the PCR primer in combination with the PCR-index/barcode technique, and a unique primer tag is introduced for each sample in 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. The tag sequence is retrieved. The design of the primer label varies according to the experimental platform applied. Considering the characteristics of the Illumina GA sequencing platform itself, the present invention mainly considers the following points when designing the primer label: 1: Avoid more than 3 in the sequence of the primer label (including 3) single base repeats, 2: the total content of base A and base C in the same address of all primer tags is between 30% and 70% of the total base content, 3: the primer tag sequence itself The GC content is between 40-60%, the sequence difference between 4: primer tags is greater than 4 bases, 5: the sequences with high similarity to the Illumina GA sequencing primer are avoided in the primer tag sequence, and the primer label is reduced. After the sequence was added to the PCR primer, the hairpin and dimer conditions caused by the PCR primer were observed.

The term "PCR-Free gene bank adapter" refers to a designed set of bases whose primary function is to assist in the immobilization of DNA molecules on a sequencing wafer and to provide a binding site for a universal sequencing primer, PCR-Free The gene bank adapter can be directly ligated to the DNA fragment in the sequencing gene pool by DNA ligase. The introduction process of the adapter is called PCR-Free gene library adapter because there is no PCR involved. "adapter" or "library adapter" tagging technique refers to the addition of different gene bank adapters to multiple sequencing gene banks [different gene pool adapters have different composition sequences The different parts of the sequence are called adapter indices, and the tag sequencing gene pool is constructed, so that multiple different tag sequencing gene pools can be mixed and sequenced, and finally the sequencing of each tag sequencing gene bank is completed. The result is a gene library tagging technique that can be distinguished from each other. For example, a PCR-FREE gene bank adapter is used in the embodiment of the invention from ILLUMIA.

In the following examples, 95 pairs of known HLA common genotypes were subjected to HLA-C locus PCR amplification using the selected 3 pairs of PCR primers, and the amplified products were subjected to Sanger method and second generation sequencing. The method is sequenced. The sequencing results were used for HLA-C typing and the conservatism and specificity of the PCR primers were verified by comparison with the original typing results.

Example 1 HLA-C genotyping using the second generation sequencing technique (Illumina GA) Sample DNA extraction

DNA was extracted from blood samples from 95 known HLA genotypes using a KingFisher automatic extractor. The main steps are as follows: Take out 6 deep-hole plates and 1 shallow-hole plate of Kingfisher automatic extractor, add a certain amount of matching reagents and mark according to the instructions, and place all the well-added well plates as required. Position, select the program "Bioeasy_200ul Blood DNA_KF.msz" and press "start" to execute the program for nucleic acid extraction. At the end of the program, 100 ul of the eluted product in the plate Elution was collected as the extracted DNA as a template for the next PCR.

2. PCR amplification

Different PCR tag primers were made by synthesizing PCR primers with different primer tags at the 5' end. These different PCR tag primers can be used for different samples. These PCR primers are for exons 2, 3 and 4 of HLA-C. PCR primers are shown in Table 1. A primer tag is then introduced at both ends of the PCR product by a PCR reaction to specifically label PCR products from different samples.

95 sets of PCR primers were used to amplify 95 DNA samples, each set of PCR primers by PCR primers for exon 2, 3 and 4 of HLA-C (Table 1) and a pair of bidirectional primer tags. (Table 3) Composition in which a forward primer label of a pair of primer tags is attached to the 5' end of each forward PCR primer, and a reverse primer tag of a pair of primer tags is attached to the 5' end of the reverse PCR primer. The primer tag was added directly to the 5' end of the PCR primer when the primer was synthesized.

The 95 DNAs obtained in the sample extraction step were sequentially numbered 1-95, and the PCR reaction was carried out in a 96-well plate. The total number was 3 plates, numbered HLA-P-C2, HLA-P-C3, and HLA-P-C4. (C2/3/4 indicates the amplified locus), a negative control without a template was placed in the plate, and the primer used in the negative control was the same as the primer PI-96. At the same time of the experiment, record the sample number information corresponding to each pair of primer labels.

The DNA extracted by the KingFisher automatic extractor in step 1 was used as a template, and the single-tube HLA-C exon primers with 5' ends were PCR-amplified. The PCR procedure was as follows:

C2: 96 ° C for 2 minutes

95°C 30 seconds → 62°C 30 seconds → 72°C 20 seconds (35 cycles) 15°C standing

C3: 96 ° C for 2 minutes

95 ℃ 30 seconds → 56 ℃ 30 seconds → 72 ℃ 20 seconds (35 cycles) 15 deg.] C was allowed to stand

C4: 96 ° C 2 minutes

95 ℃ 30 seconds → 60 ℃ 30 seconds → 72 ℃ 20 seconds (35 cycles) 15 deg.] C was allowed to stand

The PCR reaction system of HLA-C is as follows:

Wherein PI _nf -CF _2/3/4 represents the F-introduction of HLA-C with the nth forward primer tag sequence (Table 3) at the 5' end of the primer, and PI _nf -CR _2/3/4 represents the primer 5' The R-introduction of HLA-C (here n≦96) with the nth reverse primer tag sequence (Table 3) at the end, and so on. And each sample corresponds to a specific set of PCR primers.

The PCR reaction was run on a Bio-Rad PTC-200 PCR machine. After the PCR was completed, 2 ul of the PCR product was detected by 1.5% agarose gel electrophoresis. Figure 1 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). The gel map has a series of single bands with a fragment size of 400 bp to 500 bp, indicating that this Some samples of HLA-C exons (C2, C3, C4) were successfully amplified by PCR. The results for the other samples are similar.

3. PCR product mixing and purification

From the remaining PCR products of the 96-well plate HLA-P-C2 (except the negative control), 20 ul each was mixed in a 3 ml EP tube, labeled HLA-C2-Mix, and the same for the other two 96-well plates. The operations were labeled as 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 and mix at 1.5 ml. In the EP tube, labeled as HLA-Mix, 500 ul of DNA mixture from HLA-Mix was purified by Qiagen DNA Purification kit (QIAGEN) column (see the instructions for specific purification steps), and the purified 200 ul DNA was purified. Nanodrop 8000 (Thermo Fisher Scientific) determined the HLA-Mix DNA concentration to be 50 ng/ul.

4. Construction of Illumina GA PCR-Free sequencing gene library 4.1 Interruption of PCR products

A total of 5 ug of DNA from the purified HLA-Mix was disrupted on Covaris S2 (Covaris) using Covaris microTube with AFA fiber and Snap-Cap. The breaking conditions are as follows:

4.2 Purification of PCR products after interruption

All interrupted products of HLA-Mix were recovered by QIAquick PCR Purification Kit and dissolved in 37.5 ul of EB (QIAGEN Elution Buffer).

4.3 End repair response

The DNA end-repairing reaction was carried out on the purified product, and the system was as follows (reagents were purchased from Enzymatics):

The reaction conditions were: a temperature bath at 20 ° C for 30 minutes in a Thermomixer (Eppendorf).

The reaction product was recovered by QIAquick PCR Purification Kit and dissolved in 32 μl of EB (QIAGEN Elution Buffer).

4.4 3' end plus A reaction

In the previous step, the 3' end of the DNA was recovered and the A reaction was carried out. The system was as follows (reagents were purchased from Enzymatics):

The reaction conditions were: a temperature bath at 37 ° C for 30 minutes in a Thermomixer.

The reaction product was recovered and purified by MiniElute PCR Purification Kit (QIAGEN) and dissolved in 38 μl of EB solution (QIAGEN Elution Buffer).

4.5 Connecting the Illumina GA PCR-Free Gene Bank Adapter (adaptor)

The product after addition of A was linked to the Illumina GA PCR-Free gene bank adapter, and the system was as follows (reagents were purchased from Illumina):

The reaction conditions were: overnight at a temperature of 16 ° C in a Thermomixer.

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 fluorescent quantitative PCR (QPCR) as follows:

4.6 tapping recycling

30 μL of HLA-Mix was recovered with 2% low melting point agarose gel. The electrophoresis conditions were 100 V for 100 minutes. The DNA standard molecular weight reference is a 50 bp DNA Ladder from NEB. The gel was recovered to recover DNA fragments ranging in length from 400 to 750 bp (Fig. 2). The recovered product was recovered and purified by QIAquick PCR Purification Kit (QIAGEN). The purified volume was 32 ul, and the DNA concentration was 17.16 nM by fluorescent quantitative PCR (QPCR).

5. Illumina GA sequencing

According to the QPCR test results, 10 pmol of DNA was sequenced using the Illumina GA PE-100 program. The specific procedure is described in the Illumina GA operating instructions (Illumina GA II x).

6. Analysis of results

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 sequence in the sequencing results, the data of the sequencing results of the PCR products of the HLA-C exons corresponding to each primer tag are established. Library; locate the sequenced results of each exon by BWA (Burrows-Wheeler Aligner) on the reference sequence of the corresponding exon (reference sequence source: http://www.ebi.ac.uk/imgt/hla/ And constructing a consensus sequence for each database; combining base sequencing quality values and the degree of difference between the sequence and the consensus sequence, screening and sequencing error correction of the sequence; and correcting DNA sequences can be assembled into corresponding sequences of each of the HLA-C exons by sequence overlap and pair (Pair-End linkage) relationships. The screenshot of Figure 3 exemplifies the process of constructing the exon 2 consensus sequence of the HLA-C locus of sample No. 1.

The DNA sequence of the sequenced HLA-C intron is aligned with the sequence library of the corresponding exons of HLA-C in the IMGT HLA professional database. The 100% match of the sequence alignment results is the HLA of the corresponding sample. C genotype. For all 95 samples, the obtained classification results were completely consistent with the original known classification results. The specific results of the samples No. 1-32 were compared with the original classification results of the samples as follows: (As shown in Table 4, all the test results are the same as the original The test results are the same).

Note: C*0303 in HLA-C type does not exclude the possibility of C*0320N, C*0401 does not exclude the possibility of C*0409N/0430, C*0702 does not exclude the possibility of C*0750, C*0801 does not Excluding the possibility of C*0822, C*1505 does not rule out the possibility of C*1529 because the above alleles have identical sequences in exons 2, 3 and 4 of HLA-C.

Example 2 HLA-C genotyping using the Sanger legal sequence Sample DNA extraction

Similar to the one described in Example 1, 26 of the 95 samples of the HLA genotype were extracted from the 95 samples taken by the KingFisher automatic extractor.

2. PCR amplification

The DNA extracted by the above KingFisher automatic extractor was used as a template, and three pairs of PCR primers were randomly amplified by C-F2, C-R2, C-F3, C-R3, C-F4 and C-R4, respectively. The primer PCR program is as follows:

C2: 96 ° C for 2 minutes

95 ℃ 30 seconds → 2 ℃ 30 seconds → 72 ℃ 20 seconds (35 cycles) 15 deg.] C was allowed to stand

C3: 96 ° C for 2 minutes

95°C 30 seconds → 56°C 30 seconds → 72°C 20 seconds (35 cycles) 15°C standing

C4: 96 ° C 2 minutes

95°C 30 seconds → 60°C 30 seconds → 72°C 20 seconds (35 cycles) 15°C standing

The PCR reaction system of HLA-C is as follows:

The PCR product was detected by agarose gel electrophoresis (Fig. 4) and prepared for purification.

3. PCR product purification

PCR product purification was performed using a millipore purification plate. The basic procedure is to mark the wells to be used on the 96-well PCR product purification plate with a marker, and add 50 ul of ultrapure water to the wells to be used. The remaining holes are adhered to the parafilm, allowed to stand at room temperature for 15 minutes or connected to On the suction filtration system, remove it at -10 Pa for 5 minutes. Each time the purification plate is removed from the suction filtration system, the liquid remaining in the liquid discharge port at the bottom of the purification plate is blotted on the absorbent paper.

The PCR product to be purified was centrifuged at 4000 rpm for 1 minute; a lid or a silicone 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 be added to the PCR product to be purified 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.

Add 50 ul of ultrapure water or TE to the microporous regenerated fiber membrane to the wells to be purified. At room temperature, use a micro-oscillator to shake the plate for 5 minutes, transfer all the liquid in the corresponding well to the new 96-well. The PCR plate corresponds to the well.

4. Perform sequencing reactions and purify the sequencing reaction products

Using the above purified PCR product as a template for the sequencing reaction sequence conditions

96 ° C 2 minutes

96°C 10 seconds → 55°C 5 seconds → 60°C 2 minutes (25 cycles)

15 ° C standing

The system for sequencing reactions is:

The sequencing reaction product was purified by the following procedure: The sequencing plate was removed and centrifuged at 3000 g for 1 minute. 96-well plates were added with 2 ul of 0.125 mol/L EDTA-Na2 solution and 33 μL of 85% ethanol per 5 μL reaction system, covered with a silicone pad, shaken well for 3 minutes, and centrifuged at 3000 g for 30 minutes at 4 °C. After the end of the centrifugation, the sequencing plate was taken out, the silicone pad was opened, the sequencing reaction plate was inverted on the absorbent paper, and the mixture was centrifuged until the centrifugal force reached 185 g, and immediately stopped. A 96-well plate was filled with 50 ul of 70% ethanol per well, covered with a silicone pad, shaken for 1.5 minutes, and centrifuged at 3000 g for 15 minutes at 4 °C. The sequencing plate was placed in the dark for 30 minutes and air dried until it had no ethanol smell. Add 10 μL per well to a 96-well plate (8 μL per well in a 384-well plate), seal the membrane with HI-DI, shake for 5 seconds, and centrifuge to 1000 rpm.

5. Sequencing results and analysis

The purified sequencing reaction product was subjected to capillary electrophoresis sequencing on ABI 3730XL, and the sequencing peak map was analyzed by uType software (Invitrogen) (Fig. 5) to obtain HLA typing results. All test results are the same as the original test results (Table 5).

Example 3 Genotyping of HLA-A/B/C Exons 2, 3, and 4 and HLA-DQB1 Exon 2 and 3 in 950 samples

In the embodiment of the present invention, a sequencing method based on primer labeling, incomplete DNA inactivation, gene library labeling, and PCR-FREE library Illumia GA Pair-End is used, and HLA-A/B/ of 950 samples is simultaneously used. Genotyping of exons C 2, 3, and 4 and exons 2 and 3 of HLA-DQB1 (the length of the PCR product is between 300 bp and 500 bp), which proves that the strategy can be measured beyond the sequencer itself. More than one gene fragment was genotyped, and it was proved that the invention can achieve low-cost, high-throughput, high-accuracy and high-resolution HLA genotyping.

Principle: The samples to be analyzed were divided into 10 groups on average, and each group of samples was subjected to PCR reaction at both ends of the PCR products of HLA-A/B/C exons 2, 3 and 4 and HLA-DQB1 2 and 3 exons. A primer tag is introduced to specifically label the sample information of the PCR product. The PCR amplification products of the four loci of HLA-A/B/C/DQB1 in each group were mixed together to obtain a PCR product gene pool; after the obtained PCR product gene library was incompletely interrupted by ultrasonic waves, Construct different PCR-Free tag sequencing gene pools (the PCR product gene pool of each set of samples uses a different adaptor to construct 10 tag sequencing gene banks); 10 tag sequencing gene banks, etc. The molars were mixed together to construct a mixed-tag sequencing gene pool, and the mixed-tag sequencing gene library was subjected to 2% low-melting agarose electrophoresis, and the gel was purified to recover all the DNA bands located between 450-750 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 gene 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 DNA fragment sequence, and then The alignment of the standard library of the corresponding exons of HLA-A/B/C/DQB1 can assemble the entire sequence of the original PCR product and realize the genotyping of HLA-A/B/C/DQB1.

Sample extraction

DNA was extracted from 950 blood samples of known HLA-SBT typing results [Chinese Hematopoietic Stem Cell Donor Database (hereinafter referred to as "China Bone Marrow Bank") using KingFisher Automatic Extractor (Thermo Corporation, USA). The main steps are the same as in the first embodiment.

2. PCR amplification

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. For each set of samples, 95 sets of bidirectional primer tags (Table 3) were used to amplify HLA-A/B exons 2, 3, and 4 (Table 6), exons HLA-C 2, 3, and 4 (Table 2) and PCR primers for exons of HLA-DQB1, 2 (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 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, DQB1" indicates the amplified locus), each plate is provided with a negative control without a template, and the negative control is used as a negative control. Primers labeled with PI-1 (Table 3). At the same time of the experiment, the sample group number information and the primer label information corresponding to each sample are recorded. For example, the PI-1 and PI-2 primer tags are as follows, and so on.

The PCR program and PCR reaction system of HLA-A/B/C are as follows. The PCR primers used to amplify the corresponding exons of HLA-A/B are shown in Table 6, and are used to amplify HLA-C. The PCR primers for exons are shown in Table 2.

96 ° C 2 minutes

95°C 30 seconds → 60°C 30 seconds → 72°C 20 seconds (32 cycles)

15 ° C standing

HLA-A/B/C PCR Reaction System All reagents were purchased from Promega (Beijing) Biotechnology Co., Ltd. (Promega).

The PCR procedure for HLA-DQB1 is as follows, and the PCR primers used to amplify the corresponding exons of HLA-DQB1 are shown in Table 5.

96 ° C 2 minutes

95°C 30 seconds → 55°C 30 seconds → 72°C 20 seconds (32 cycles)

15 ° C standing

The PCR reaction system of HLA-DQB1 is as follows:

Wherein, PI _nf -A/B/CF _2/3/4 and PI _nf -Q-F2/F3 represent HLA-A/B/C with the nth forward primer tag sequence (Table 3) at the 5' end of the primer. F-introduction of /DQB1, PI _nr -A/B/CR _2/3/4 and PI _nr -Q-R2/R3 represent HLA-A/B/ with the nth reverse primer tag sequence at the 5' end of the primer The R primer of C/DQB1 (here n≦95), and so on. And each sample corresponds to a specific set of PCR primers (PI _nf -A/B/CF _2/3/4 , PI _nr -A/B/CR _2/3/4 , 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 6 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 map has a series of fragments ranging from 300 bp to 500 bp. The band indicates that the exons (A2, A3, A4, B2, B3, B4, C2, C3, C4, DQB1) of HLA-A/B/C/DQB1 of sample No. 1 were successfully amplified by PCR, and the negative control (negative control) N) No amplified bands. The results for the other samples are similar.

3. PCR product mixing and purification

For the "X" group ("X" is 1/2/3/4/5/6/7/8/9/10) sample, from the remaining PCR product of 96-well plate HLA-XP-A2 (negative control) Except for each) 20 ul of each is mixed in a 3 ml EP tube, labeled HLA-X-A2-Mix, and the same operation is performed on the other 9 96-well plates of the "X" group of samples, labeled HLA- X-A3-Mix, HLA-X-A4-Mix, HLA-X-B2-Mix, HLA-X-B3-Mix, HLA-X-B4-Mix, HLA-X-C2-Mix, HLA-X- C3-Mix, HLA-X-C4-Mix and HLA-X-DQB1-Mix, shake and mix, from HLA-X-A2-Mix, HLA-X-A3-Mix, HLA-X-A4-Mix, HLA -X-B2-Mix, HLA-X-B3-Mix, HLA-X-B4-Mix, HLA-X-C2-Mix, HLA-X-C3-Mix, HLA-X-C4-Mix and HLA-X 200 ul of each of the -DQB1-Mix was mixed in a 3 ml EP tube labeled HLA-X-Mix, and 500 ul of the DNA mixture was purified from the Qiagen DNA Purification kit column (see the instructions for specific purification steps). The purified 200 ul DNA was determined by Nanodrop 8000 (Thermo Fisher Scientific), and the other 9 samples were operated in the same manner. The DNA concentrations determined by the 10 samples were:

4. Illumina GA sequencing gene library construction

As in the method described in Example 1, a total of 5 ug of DNA was taken from the purified HLA-X-Mix for DNA disruption, disruption and purification, end-repair reaction, 3' end plus A reaction, and ligation. Illumina GA PCR-Free gene bank adapter.

The correspondence between the sample group and the gene bank adapter is as follows:

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 were detected by fluorescent quantitative PCR (QPCR) as follows:

5. Tapping recycling

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 Mixing of -9-Mix and HLA-10-Mix, etc. (final concentration 70.86 nM/ul), labeled as HLA-Mix-10, and 30 μL of HLA-Mix-10 was recovered with 2% low melting point agarose gel. The electrophoresis conditions were 100 V for 100 minutes. The DNA marker is a 50 bp DNA marker from NEB. The gel was recovered to recover DNA fragments ranging in length from 450 to 750 bp (Fig. 7). The recovered product was recovered and purified by QIAquick PCR Purification Kit (QIAGEN). After purification, the volume was 32 ul, and the DNA concentration was 10.25 nM by fluorescent quantitative PCR (QPCR).

6. Illumina GA sequencing and results analysis

According to the method of Example 1, sequencing and result analysis were performed.

A database of sequencing results of each exon PCR product corresponding to the sample HLA-A/B/C/DQB1 was established for each primer tag, and the obtained DNA sequence was used in accordance with HLA-A/B/C/DQB1 in the IMGT HLA professional database. The sequence library of exons is aligned, and the 100% match of the sequence alignment results is the HLA-A/B/C/DQB1 genotype of the corresponding sample. A screenshot of the exon 2 consensus sequence constructor of the HLA-C locus of sample No. 1 illustrated in Figure 8 can be seen. For all 950 samples, the results obtained were completely consistent with the original known classification results. The specific results of samples 1-32 are as follows:

Note: When encountering identical results for exon 2, 3, and 4 in the HLA-A/B/C locus, the common type is taken.

Using the technical route of the present invention, the samples of 950 known HLA-SBT typing results were genotyped at the HLA-A/B/C/DQB1 locus, and it was found that the genotype obtained by the technical route of the present invention was used. The results are exactly the same as the original results.

It will be apparent to those skilled in the art that various modifications and changes can be made in the present invention without departing from the scope and spirit of the invention. Other embodiments of the present invention will be apparent to those skilled in the art from this disclosure. The description and the examples are to be considered as illustrative only, and the true scope and spirit of the invention are described in the appended claims.

references

[1] Marsh, S.G.E., Parham, P. & Barber, L.D. The HLA Facts Book 3-91 (Academic Press, London, 2000).

[2] Campbell, KJ et al . Characterization of 47 MHC class I sequences in Filipino cynomolgus macaques. Immunogenetics 61, 177-187 (2009).

[3] Goulder, P.J.R. & Watkins, D.I. Impact of MHC class I diversity on immune control of immunodeficiency virus replication. Nat. Rev. Immunol. 8,619-630 (2008).

[4] 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 61,689-701 (2009).

[5] Robinson J, Malik A, Parham P, Bodmer JG, Marsh SGE. IMGT/HLA database-a sequence database for the human major histocompatibility complex. Tissue Antigens 55, 80-7 (2000).

[6] Elaine R. Mardis. The impact of next-generation sequencing technology on genetics. Trends in Genetics. 2008, 24: 133-141.

[7] 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. 2007;35(13):e91.

[8]. http://www.ebi.ac.uk/imgt/hla/stats.html

[9]. Tiercy J M. Molecular basis of HLA polymorphism: implications in clinical transplantation. Transpl Immunol, 2002, 9: 173-180.

[10] Wegner KM: Massive parallel MHC genotyping: titanium that shines. Molecular Ecology 2009, 18: 1818-1820.

[11] Ellegren H: Sequencing goes 454 and takes large-scale genomics into the wild. Molecular Ecology 2008, 17: 1629-1635.

[12] Shendure J, Ji H: Next-generation DNA sequencing. Nature Biotechnology 2008, 26: 1135-1145.

[13] Nagler A, Brautbar C, Slavin S, et al. Bone marrow transplantation using unrelated and family related donors: the mpact of HLA-C disparity. Bone Marrow Transplant, 1996, 18(5): 891-897.

[14] Ho VT, Kim HT, Liney D, et al. HLA-C mismatch is associated with inferior survival after unrelated donor non-myeloablative hematopoietic stem cell transplantation. Bone Marrow Transplant, 2006, 37(9): 845-850.

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<120> HLA-C genotyping method and related primers

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Claims (18)

A primer combination comprising a primer pair having a sequence such as sequence identification number: 1 and sequence identification number: 2, a primer pair having a sequence such as sequence identification number: 3 and sequence identification number: 4, and having sequence identification No.: 5 and sequence identification number: the pair of primers of the sequence shown in sequence 6, or the pair of primers having the sequence of sequence identification number: 7 and sequence identification number: 8, with sequence identification number: 9 and sequence identification number: 10 The pair of primers of the indicated sequence and the pair of primers having the sequence of sequence identification number: 11 and sequence identification number: 12. A method for amplifying exon 2, 3 and/or 4 of HLA-C gene, characterized in that it is selected from the group consisting of sequence identification number: 1 and sequence identification number: 2; sequence identification number: 3 and sequence identification number: 4; Sequence identification number: 5 and sequence identification number: 6, or sequence identification number: 7 and sequence identification number: 8, sequence identification number: 9 and sequence identification number: 10, sequence identification number: 11 and sequence identification number: 12 primer For PCR amplification, the HLA-C gene 2, 3 and/or 4 exons are amplified. A method for sequencing HLA-C gene exons 2, 3 and/or 4 in a sample, comprising the steps of: (1) providing a sample and extracting DNA of the sample; (2) being selected from the sequence Identification number: 1 and sequence identification number: 2; sequence identification number: 3 and sequence identification number: 4; sequence identification number: 5 and sequence identification number: 6, or sequence identification number: 7 and sequence identification number: 8, sequence identification Number: 9 and sequence identification number: 10, sequence identification number: 11 and sequence identification number: 12 primer The pair is used to amplify the DNA to obtain a PCR product; and (3) the PCR product is sequenced. The method of claim 3, wherein the PCR product obtained in the step (2) is further subjected to a purification treatment. The method of claim 3, wherein the sample is a blood sample. The method of claim 5, wherein the blood sample is from a mammal or a human. An HLA-C genotyping method comprising: (1) using a sequence identification number: 1 and sequence identification number: 2; sequence identification number: 3 and sequence identification number: 4; sequence identification number: 5 and sequence Identification number: 6, or sequence identification number: 7 and sequence identification number: 8, sequence identification number: 9 and sequence identification number: 10, sequence identification number: 11 and sequence identification number: 12 primer pair for PCR amplification, expansion Increasing the HLA-C gene exons 2, 3 and/or 4; and (2) sequencing the amplified exons and comparing the sequencing results to the standard sequences in the database To determine genotyping results. For example, the method of claim 3, wherein the ordering is by Sanger sequencing or by the second generation sequencing method. The method of claim 7, wherein the ordering is by Sanger sequencing or by a second generation sequencing method. The method of claim 8, wherein the second generation sequencing method is Illumina Solexa or Roche 454. For example, the method of claim 9 of the patent scope, wherein the second generation sequencing method is Illumina Solexa or Roche454. The method of any one of claims 2 to 11, which further comprises performing PCR amplification using other HLA gene amplification primers. A kit for performing HLA-C genotyping, the kit comprising a sequence identification number: 1 and a sequence identification number: 2. a sequence identification number: 3 and a sequence identification number: 4. a sequence identification number: 5 and a sequence Identification number: 6; or sequence identification number: 7 and sequence identification number: 8, sequence identification number: 9 and sequence identification number: 10, sequence identification number: 11 and sequence identification number: 12 PCR amplification primer pair. A kit according to claim 13 which further comprises an agent for DNA amplification, DNA purification and/or DNA sequencing. For example, the kit of claim 14 includes other HLA gene amplification primers. A use of a primer combination as claimed in claim 1 for HLA-C genotyping. A method of the method of claim 7 for HLA-C genotyping. A kit as claimed in claim 13 for use in HLA-C genotyping.