WO2018195765A1 - Method for use in simultaneously sequencing and genotyping 14 functional killer immunoglobulin-like receptor (kir) genes - Google Patents

Abstract

Provided is a method for use in simultaneously amplifying and simultaneously sequencing and genotyping the complete 14 functional killer immunoglobulin-like receptor (KIR) genes, which is suitable for high through-put processes.

Description

Method for simultaneous sequencing and typing of 14 functional killer cell immunoglobulin-like receptor KIRs genes Technical field

The invention relates to the field of biomedical, in particular DNA sequencing, typing. The invention provides a method for simultaneous amplification and simultaneous sequencing of all 14 functional KIRs genes suitable for high-pass quantification.

Background technique

Killer immunoglobulin-like receptors (KIRs) belong to the immunoglobulin superfamily and are expressed on the surface of natural killer cells (NK cells) and some T cells. According to the number of extracellular domains, it can be divided into KIR2D and KIR3D subfamilies. According to the length of cytoplasmic region and the presence or absence of immunoreceptor tyrosine inhibitory motif (ITIM), KIR is functionally divided into inhibitory type (L type). ) and activation type (S type). KIR molecules bind to HLA class antigens on the surface of target cells, conduct activation or inhibition signals, regulate the activity of NK cells, and play an important role in transplantation immunity, tumor immunity and anti-infection.

The KIR gene family is located on human chromosome 19 and is codominantly expressed. In addition to the two pseudogenes 2DP1 and 3DP1, it also includes 14 functional KIR genes (KIR2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1) ^[1] . The structure of KIR gene is complex: in addition to KIR3DL3 deletion of exon 6 in the functional KIR3D gene, the remaining functional KIR3D genes (3DL1, 3DL2, 3DS1) contain 9 exons, 8 introns and 5'- The promoter region and the 3'-UTR region. Each exon encodes a different peptide domain, wherein the first exon and the second exon encode a leader peptide, and the third exon, the fourth exon, and the fifth exon encode an extracellular region, respectively. The D0, D1, D2 domains, the 6th exon encodes the stem domain, the 7th exon encodes the transmembrane region, the 8th exon and the 9th exon encode the cytoplasmic region; in the KIR2D gene, The third exon of eight genes, such as KIR2DS1~3 and 2DS1~5, is a pseudoexon, which cannot encode the corresponding D0 extracellular domain; KIR2DL4 and 2DL5 all lack the fourth exon, and no D1 cell Outer domain.

In the international IPD-KIR database ^[2] (http://www.ebi.ac.uk/cgi-bin/ipd/kir/), the total length of the included KIR genome sequence is 9901 to 17009 bp. The coding sequence (CDS) of each functional KIR gene is only 915 to 1368 bp in length (see Table 3), while the non-coding region sequence is 8773 to 15641 bp (see Table 4). The lengths of the 5th intron and the 6th intron of the wings of the 6th exon accounted for 44.0% to 61.2% of the corresponding full length sequence of the KIR genome (see Table 4).

The first exon (34 or 40 bp in length) and the second exon (36 bp in length) of the functional KIR gene are short in length and limited in polymorphism of SNPs: 2DL2, 2DL4 and 2DS4 genes first and second There are no SNPs polymorphism in exons, and the remaining ones have 1 to 3 SNPs. The first intron between the first exon and the second exon has a length of 199 to 2280 bp. Due to the short length of the first and second exons and the limited polymorphism of SNPs, not every KIR gene needs to be detected during routine sequencing. For the necessary detection, PCR amplification should be performed separately, and the PCR amplification products cover the complete The 1 exon, the 1st intron and the 2nd exon do not cause the amplified product fragment to be too long.

For the third, fourth and fifth exons, each exon has a relatively long length (282-300 bp), and the SNPs are polymorphic, and should cope with exons and exons encoding the extracellular domain. The introns were subjected to separate PCR amplification. In view of the fact that the third exon of 8 genes of KIR2DL1~3 and KIR2DS1~5 is a pseudo exon, no amplification is required, and the amplified product covers the complete 4th exon, 4th intron and 5th exon. The 4th and 5th exons are sequenced bidirectionally; KIR2DL4 and 2DL5 have no exon 4, and the amplified product covers the complete exon 3, exon 5 and introns in between. Sequence, two-way sequencing of the third and fifth exons; the remaining four functional KIR genes (KIR3DS1, 3DL1~3), the amplification products covering the complete exon 3, exon 4, 5th The exons and the intron sequences in between are bidirectionally sequenced for the third, fourth and fifth exons.

The 6th exon (KIR3DL3 lacks the exon), the length is only 51 bp, based on the IPD-KIR Database (Release 2.6.0), the 4th exon of the 4 KIR genes (2DS4, 3DL1, 3DL2 and 3DS1) There are no SNPs, and the remaining functional KIR genes have 1 or 2 SNPs. Due to the limited distribution of the SNPs in the 6th exon, and located in the first The 5th and 6th intron sequences of the 6 exons are as long as 4937~9841bp (see Table 4). Therefore, the polymorphism of the 6th exon must be detected, and the exon 6 must be amplified by PCR. In order to avoid amplification of the entire sequence of the 5th intron and/or the 6th intron, the amplified product fragment is too long, the amplification time is too long, and the amplification efficiency is affected.

The lengths of the 7th, 8th, and 9th exons are 102 to 105 bp, 51 to 53 bp, and 8 to 270 bp, respectively, and the lengths of the 7th and 8th introns are 460 to 462 bp and 98 to 118 bp, respectively. Therefore, amplification of the entire exon 7, the 7th intron, the 8th exon, the 8th intron, and the 9th exon sequence does not cause the amplified product fragment to be too long.

The structure of the full-length sequence of the genome based on all of the above functional KIR genes, the distribution of SNPs polymorphisms, and the length of the exon two-wing introns are essential for the development of a scientific and efficient PCR amplification strategy.

The polymorphism of the KIR gene is reflected in the KIR allele polymorphism and haploid composition. As of April 2017, the total number of functional KIR alleles published by the International IPD-KIR Database (Release 2.6.0) has reached 698, of which 7 KIR alleles are not expressed; among the 14 functional KIR genes, The most polymorphic KIR3DL2 has 112 alleles (Table 5).

Identification of the KIR allele is functional. Different KIR gene expression levels are different, McErlean et al ^[3] found that the mRNA expression levels of different KIR genes from high to low are: KIR3DL2>KIR2DS2>KIR3DS1>KIR2DS5>KIR2DL5>KIR2DS3>KIR2DL1>KIR3DL1>KIR2DS1>KIR2DL2>KIR2DL4>KIR2DS4>KIR2DL3. There are also differences in the expression levels, affinities, and mediated activation/inhibition abilities of different alleles in the same KIR gene. Yawata et al ^[4] reported that the expression level of KIR3DL1 allele on NK cell membrane was from high to low: KIR3DL1*01502>*020>*001>*007>*005, and the mediated inhibitory effect was: KIR3DL1*001>*005>*01502>*020>*007; KIR3DL1*005 has a low expression level on the cell surface, but has a strong mediated inhibition ability. The most common KIR3DL1*004 allele in the Caucasian population is not expressed on the cell surface ^[5] . KIR2DS4*003, *004, *006, *007, *008, *009, *010, *012, and *013 have a 22bp deletion in the 5th exon CDS nt454~nt475 position, causing the reading frame shift, coding Truncated peptides are not normally expressed on the cell surface ^[6] . Given the different expression levels of different KIR genes, the expression levels and mediated activation/inhibition abilities of different alleles in the same KIR gene are also different. It is necessary to identify and screen common KIR alleles without activation/inhibition effects. . It is especially urgent to study and establish a high-throughput, high-resolution KIR gene simultaneous sequencing method, and to carry out KIR application research based on the allele level.

Currently, sequence specific primer-polymerase chain reaction (PCR-SSP) and sequence specific oligonucleotide probe-PCR (sequence specific oligonucleotide probe-PCR) The PCR-SSOP commercialization kit can only identify the presence or absence of the KIR gene, fail to achieve a fine allele level, and discriminate all KIR alleles and variants.

The sequence-based typing (SBT) method is the gold standard for genotyping. However, to date, there is no commercially available KIR sequencing typing kit and corresponding analysis software for determining KIR genotype; there is no internationally recognized KIR gene sequencing method. Since the KIR allele sequence is highly homologous and the sequence of the intron in the full length sequence of the genome is particularly long, it is very difficult to specifically PCR amplification of the KIR gene. At present, the KIR sequencing typing method has been reported to have the following problems: (1) only some exons of the KIR gene have been sequenced ^{[7, 8, 9]} , and the complete coding region sequence and analysis of the external display cannot be obtained. The polymorphism of the sub-segment is prone to ambiguous sequencing typing results. (2) The target gene fragment amplified by PCR during sequencing typing covers all the coding regions, but the amplified product fragments are too long, such as the amplification of KIR3DS1 exon 3 to 3'-UTR gene fragment up to 12.2 Kb, the 68 °C chain extension time in a single cycle is 13 min ^[10] , the time required for amplification is more than 10 hours, and the requirements for sample DNA quality and DNA polymerase are very high. (3) The sequence of the KIR gene is highly homologous, and the PCR primers for amplifying the target gene fragment sometimes have non-specific amplification/co-amplification, for example, the subject carries the KIR2DL1 and 2DS1 genes, and the 2DL1 is amplified. At the same time as the 1st to 5th exon sequences, the sequence of 2DS1 was also amplified. For non-specific amplification products, PCR primers are designed to be double-amplified by nested PCR, and the experimental operation is cumbersome ^[11] . (4) When performing PCR amplification on each gene fragment of KIR gene, the extension time is different due to the difference in annealing temperature of PCR primers and the difference in length of PCR gene amplification ^[10,11,12] . PCR simultaneous amplification and simultaneous sequencing under the same cycle parameters can not achieve high-pass quantification, and the experiment is time-consuming and laborious ^[13] . (5) Identification of alleles with one or more base deletions, and some need to be identified by molecular cloning methods, which cannot meet the needs of simple and rapid.

With the deepening of research on the biological functions of KIR molecules, KIR gene and disease association, and transplant rejection, KIR has become more and more widely used in the field of application, and it has established a high-efficiency, low-cost, functional KIR gene synchronization sequencing type suitable for Qualcomm quantification. The method and commercialization and industrialization are the problems that need to be solved urgently.

Summary of the invention

The present invention aims to solve the above problems, and for the first time provides a method for synchronizing sequencing of 14 functional killer cell immunoglobulin-like receptors (KIRs) genes, which can be widely applied to population genetics, bone marrow transplantation tissue matching. Research areas such as disease association, and lay the foundation for the commercialization of KIR gene sequencing and typing reagents, and change the current status of the international KIR-free sequencing and typing reagents.

In order to achieve the above object, the present invention adopts the following technical solutions:

First, according to the structure of the full-length sequence of each KIR gene, the distribution of single nucleotide polymorphisms in the coding region and the length of the introns of the two wings of the exon, a scientific PCR amplification strategy was developed, each functional. The entire coding region sequence of the KIR gene was amplified by PCR using 3 to 5 pairs of KIR gene-specific PCR primers with similar annealing temperatures. For each exon contained in the purified PCR amplification product, forward and reverse bidirectional sequencing were performed, as shown in FIG. 1 .

The coding region sequence of the 2DL1 gene is amplified by using 5 pairs of 2DL1 gene-specific PCR primers; wherein the first pair of PCR primers amplifies the first exon, the first intron, the second exon of the 2DL1 gene, and a portion of the 5'-promoter region and a portion of the second intron; the second pair of PCR primers amplify the 4th exon of 2DL1 and a partial sequence of its flanking; the third pair of PCR primers amplifies the 5th of 2DL1 a partial sequence of the exon and its flanks; a fourth pair of PCR primers amplifies the 6th exon of 2DL1 and a partial sequence of its flanking; the fifth pair of PCR primers amplifies the 7th exon of 2DL1, 7th Intron, 8th exon, 8th intron, ninth exon, and part 6 intron and part 3'UTR Sequence of regions;

The coding regions of the 2DL2, 2DL3, 2DS1, 2DS2, 2DS3, 2DS4 and 2DS5 genes were amplified by four pairs of KIR gene-specific PCR primers; the first pair of PCR-specific primers amplified the first exon, the first The intron and the second exon, and the sequence of the partial 5'-promoter region and part of the second intron; the second pair of PCR-specific primers amplify the fourth exon, the fourth intron, the first 5 exons, and some 2/3 introns (because the third exon is a pseudo exon, the sequence between the second exon and the fourth exon is collectively referred to as the 2/3 inclusive) And a part of the 5th intron; the third pair of PCR-specific primers amplify the partial sequence of the exon 6 and its flanking; the fourth pair of PCR-specific primers amplifies the exon 7 and the 7th Sequences of an intron, an 8th exon, an 8th intron, a 9th exon, and a portion of the 6th intron and a portion of the 3' UTR region;

The coding sequences of the 2DL4 and 2DL5 genes were amplified by four pairs of KIR gene-specific PCR primers; the first pair of PCR-specific primers amplified the first exon, the first intron, and the second exon. And a portion of the 5'-promoter region and a portion of the second intron; the second pair of PCR-specific primers amplify the third exon, the 3/4 intron (due to the deletion of the fourth exon, the first The sequence between exon 3 and exon 5 is collectively referred to as the 3/4 intron), the exon 5, and the sequence of part of the second intron and part of the 5th intron; The PCR-specific primers amplify the partial sequence of the exon 6 and its flanks; the fourth pair of PCR-specific primers amplify the 7th exon, the 7th intron, the 8th exon, and the 8th a sequence comprising a subunit, a ninth exon, and a portion of the sixth intron and a portion of the 3' UTR region;

The coding regions of the 3DL1, 3DL2 and 3DS1 genes were amplified by four pairs of KIR gene-specific PCR primers; the first pair of PCR-specific primers amplified the first exon, the first intron, and the second exon. a sub-and a partial 5'-promoter region and a portion of the second intron; the second pair of PCR-specific primers amplify the third exon, the third intron, the fourth exon, and the fourth a sequence containing a sub-operon, a fifth exon, and a portion of the second intron and a portion of the fifth intron; a third pair of PCR-specific primers amplifying a partial sequence of the exon 6 and its flanks; Amplification of the 7th exon, the 7th intron, the 8th exon, the 8th intron, the 9th exon, and some of the 6th intron and part of the 3'UTR region by PCR-specific primers the sequence of;

The coding region of the 3DL3 gene was amplified by the exon 6 and the exon was not amplified. Only 3 pairs of 3DL3 gene-specific PCR primers were used for amplification; the first pair of PCR-specific primers amplified the first The sequence of the exon, the first intron, the second exon, and the partial 5'-promoter region and part of the second intron; the second pair of PCR-specific primers amplifies the third exon, the third Sequence of intron, 4th exon, 4th intron, 5th exon, and part 2nd intron and part 5 intron; 3rd PCR specific primer amplification 7th Sequences of an exon, a 7th intron, an 8th exon, an 8th intron, a 9th exon, and a portion of the 6th intron and a portion of the 3' UTR region.

2. A total of 56 pairs of specific PCR amplification primers for 14 functional KIR genes (112). Except for KIR3DL3, only 3 pairs of PCR primers were used for deletion of exon 6 and 5 pairs of PCR primers for KIR2DL1. For each functional KIR gene, 4 pairs of KIR gene-specific PCR primers were used. The characteristics of the sequence of each PCR primer, the position in the full length sequence of the KIR genome, and the length of the amplification target fragment are as described in Table 1.

Table 1 Specific PCR amplification primers for 14 functional KIR genes

(1) The first pair of PCR primers that amplify the entire coding region of the KIR2DL1 gene include the upstream primer 2DL1_PCR_Ex12_F (sequence: 5'-GTTCGGGAGGTTGGATCTC-3', the position in the full length sequence of the genome: nt-268 to nt-250) and the downstream primer 2DL1_PCR_Ex12_R (sequence: 5'-CACACTGCAGCCCCTACCG-3', nt1332 to nt1350), specifically amplifying the first exon, the first intron, the second exon, and a part of the 5'-promoter region and part The sequence of the 2 introns, the length of the amplified fragment of interest is 1618 bp. The second pair of PCR primers include the upstream primer 2DL1_PCR_Ex4_F (sequence: 5'-TGATTCTCCTGAGTCTCCAGAGG-3', nt2501 to nt2523) and the downstream primer 2DL1_PCR_Ex4_R (sequence: 5'-TGGAAGGAGAAGAGGCAGTTTCC-3', nt5288 to nt5310), specific The 4th exon and its partial flanking sequences were amplified, and the amplified fragment of interest was 2810 bp. The third pair of PCR primers includes 2DL1_PCR_Ex5_F (sequence: 5'-CTGGCAGGGACCTACAGATGC-3', nt3692~nt3712) and downstream primer 2DL1_PCR_Ex5_R (sequence: 5'-GGACAGCCATGGGCTTTCCTC-3', nt5608~nt5628), specific amplification of the fifth explicit The partial sequence of the daughter and its flanks, the length of the amplified fragment of interest is 1937 bp. The fourth pair of PCR primers includes the upstream primer 2DL1_PCR_Ex6_F (sequence: 5'-TCCTGATTGTGAGTTCTTGGCAT-3', nt8082 to nt8104) and the downstream primer 2DL1_PCR_Ex6_R (sequence: 5'-TGAGTCAGTSAGTCGAARTGTGC-3', nt9279 to nt9301), specific amplification 6th The partial sequence of the exon and its flanks, the length of the amplified fragment of interest is 1220 bp. The fifth pair of PCR primers includes the upstream primer 2DL1_PCR_Ex789_F (sequence: 5'-CCTCAGCACGTTCTATGGTTACT-3', nt12880-nt12902) and the downstream primer 2DL1_PCR_Ex789_R (sequence: 5'-TGTGATTGCAGCCTCAAGTAGAC-3', nt14249-nt14271), specific amplification 7th Exon, 7th intron, 8th exon, 8th intron, ninth exon, and part of the 6th intron and part of the 3'-untranslated region, the amplified fragment of interest The length is 1392 bp.

(2) The first pair of PCR primers that amplify the entire coding region of the KIR2DL2 gene include the upstream primer 2DL2_PCR_Ex12_F (sequence: 5'-AGAGGTTGGATCTGAGACGTC-3', nt-263-nt-243) and the downstream primer 2DL2_PCR_Ex12_R (sequence: 5'-GGACCGATGGAGAAGTTGGCT -3', nt3590 to nt3610), specifically amplifying the sequence of the first exon, the first intron, the second exon, and a part of the 5'-promoter region and part of the second intron, The amplified fragment was 3873 bp in length. The second pair of PCR primers include the upstream primer 2DL2_PCR_Ex45_F (sequence: 5'-GAGGCTACTAGAGACAGAGGGAC-3', nt3207-nt3229) and the downstream primer 2DL2_PCR_Ex45_R (sequence: 5'-CCCAAGCTTCGTCTTCTCTCT-3', nt5617-nt5637), specific amplification 4 The sequence of the exon, the 4th intron, the 5th exon, and part of the 2/3 intron and part of the 5th intron, the amplified fragment of interest is 2431 bp. The third pair of PCR primers includes the upstream primer 2DL2_PCR_Ex6_F (sequence: 5'-CATGCCAACATCATGCTGTC-3', nt8530~nt8549) and downstream primer 2DL2_PCR_Ex6_R (sequence: 5'-TCCCTGTCCTAGCCTCCATAC-3', nt9879~nt9899), specifically amplifying the partial sequence of exon 6 and its flanks, The amplified fragment was 1370 bp in length. The fourth pair of PCR primers includes the upstream primer 2DL2_PCR_Ex789_F (sequence: 5'-GAAGTTCCACTTGCCAAGGAATG-3', nt9210~nt9232) and the downstream primer 2DL2_PCR_Ex789_R (sequence: 5'-CAGCTGCTGGTACATGGGAGC-3', nt14071-nt14091), specific amplification 7th Exon, 7th intron, 8th exon, 8th intron, ninth exon, and part of the 6th intron and part of the 3'-untranslated region, the amplified fragment of interest The length is 4882 bp.

(3) The first pair of PCR primers that amplify the entire coding region of the KIR2DL3 gene include the upstream primer 2DL3_PCR_Ex12_F (sequence: 5'-GGCYGMCTGTCTGCACAGA-3', nt-26 to nt-8) and the downstream primer 2DL3_PCR_Ex12_R (sequence: 5'-GGTTTCCTGTTGCTGCTGTAG -3', nt2560 to nt2580), specific amplification of the first exon, the first intron, the second exon, and a part of the 5'-promoter region and part of the second intron, the purpose The amplified fragment was 2606 bp in length. The second pair of PCR primers includes the upstream primer 2DL3_PCR_Ex45_F (sequence: 5'-AGAGAAGAGGGAGGGAGACAGAT-3', nt3231 to nt3253) and the downstream primer 2DL3_PCR_Ex45_R (sequence: 5'-GCCATCCTGTGCCCTGATC-3', nt5651 to nt5669), specific amplification 4 The sequence of the exon, the 4th intron, the 5th exon, and part of the 2/3 intron and part of the 5th intron, the amplified fragment of interest is 2439 bp in length. The third pair of PCR primers include the upstream primer 2DL3_PCR_Ex6_F (sequence: 5'-CCCACCTCAGGCTCTCAAAGG-3', nt7497-nt7517) and the downstream primer 2DL3_PCR_Ex6_R (sequence: 5'-GGCGTACAATGTCAGAGCTGC-3', nt8908-nt8928), specific amplification 6th The partial sequence of the exon and its flanks, the length of the amplified fragment of interest is 1432 bp. The fourth pair of PCR primers includes the upstream primer 2DL3_PCR_Ex789_F (sequence: 5'-ACTGAGAAAGCAGGAGAAAGCTG-3', nt12934-nt12956) and the downstream primer 2DL3_PCR_Ex789_R (sequence: 5'-CCTTCAGATTCCAGCTGCTGG-3', nt14063-nt14083), specific amplification of exon 7, intron 7, exon 8, intron, ninth exon, and part The sequence of the 6 intron and part of the 3'-untranslated region, the length of the amplified fragment of interest is 1150 bp.

(4) The first pair of PCR primers for amplifying the entire coding region of the KIR2DL4 gene includes the upstream primer 2DL4_PCR_Ex12_F (sequence: 5'-GTGGTCAATGTGTCAACTGCACG-3', nt-99-nt-77) and the downstream primer 2DL4_PCR_Ex12_R (sequence: 5'-CACAGGCTCCAAGGATTACAATG) -3', nt1639 to nt1661), specific amplification of the first exon, the first intron, the second exon, and a part of the 5'-promoter region and part of the second intron, the purpose The amplified fragment was 1760 bp in length. The second pair of PCR primers include the upstream primer 2DL4_PCR_Ex35_F (sequence: 5'-CTTTCTTCCCCATGGCTGAGTTG-3', nt571-nt593) and the downstream primer 2DL4_PCR_Ex35_R (sequence: 5'-CTTGGGCAACAAGAGTGAAACGC-3', nt3848-nt3870), specific amplification 3 The sequence of the exon, the 3/4 intron, the 5th exon, and part of the 2nd intron and part of the 5th intron, the amplified fragment of interest is 3300 bp. The third pair of PCR primers includes the upstream primer 2DL4_PCR_Ex6_F (sequence: 5'-AACCTCTACCTCCAGGATTCAAG-3', nt3904-nt3926) and the downstream primer 2DL4_PCR_Ex6_R (sequence: 5'-GTAAGTGGAAGTGTCATGTGCAC-3', nt5738-nt5760), specific amplification 6th The partial sequence of the exon and its flanks, the length of the amplified fragment of interest is 1857 bp. The fourth pair of PCR primers includes the upstream primer 2DL4_PCR_Ex789_F (sequence: 5'-CCAAGAAATGAGAGACAATCCAC-3', nt9442 to nt9464) and the downstream primer 2DL4_PCR_Ex789_R (sequence: 5'-AGGCACCAGATTTGTGGTGTG-3', nt10540 to nt10560), specific amplification 7th Exon, 7th intron, 8th exon, 8th intron, ninth exon, and part of the 6th intron and part of the 3'-untranslated region, the amplified fragment of interest The length is 1119 bp.

(5) The first pair of PCR primers for amplifying the entire coding region of the KIR2DL5 gene includes the upstream primer 2DL5_PCR_Ex12_F (sequence: 5'-TCATAGTGAAGGACGYGAGGTGC-3', nt-230 to nt-208) and downstream introduction. 2DL5_PCR_Ex12_R (sequence: 5'-AGCCAATGTGTGAACCACAATAC-3', nt1238~nt1260), specifically amplifying the first exon, the first intron, the second exon, and a part of the 5'-promoter region and part The sequence of the second intron, the amplified fragment of interest is 1490 bp in length. The second pair of PCR primers include the upstream primer 2DL5_PCR_Ex35_F (sequence: 5'-CAGGACAAGCCCTTGCTGTCT-3', nt1571 to nt1591) and the downstream primer 2DL5_PCR_Ex35_R (sequence: 5'-GACAGAAACAAGCAGTGGGTCAC-3', nt2993 to nt3015), specific amplification 3 The sequence of the exon, the 3/4 intron, the 5th exon, and part of the 2nd intron and part of the 5th intron, the amplified fragment of interest is 1445 bp. The third pair of PCR primers include the upstream primer 2DL5_PCR_Ex6_F (sequence: 5'-CATTTCCTCACCTCTCTCCTGTCCT-3', nt5158-nt5182) and the downstream primer 2DL5_PCR_Ex6_R (sequence: 5'-AAGAGCAGAGGCCAAATGCATCG-3', nt6351 to nt6373), specific amplification 6th The partial sequence of the exon and its flanks, the length of the amplified fragment of interest is 1216 bp. The fourth pair of PCR primers includes the upstream primer 2DL5_PCR_Ex789_F (sequence: 5'-CAGATGTTGTATGTGCTTAGCTG-3', nt7907-nt7929) and the downstream primer 2DL5_PCR_Ex789_R (sequence: 5'-GGTTTTGAGACAGGGCTGTTGTC-3', nt8937-nt8959), specific amplification 7th Exon, 7th intron, 8th exon, 8th intron, ninth exon, and part of the 6th intron and part of the 3'-untranslated region, the amplified fragment of interest The length is 1053 bp.

(6) The first pair of PCR primers for amplifying the entire coding region of the KIR2DS1 gene includes the upstream primer 2DS1_PCR_Ex12_F (sequence: 5'-CATAGTGAAGGACGCTAGGTGTA-3', nt-229-nt-207) and the downstream primer 2DS1_PCR_Ex12_R (sequence: 5'-GAGCCCTCTGACCTGTGACCG -3', nt2035-nt2055), specific amplification of the first exon, the first intron, the second exon, and a part of the 5'-promoter region and part of the second intron, the purpose The amplified fragment was 2284 bp in length. The second pair of PCR primers includes the upstream primer 2DS1_PCR_Ex45_F (sequence: 5'-GTTCCTCTTCCACCCCCACAC-3', nt3175 to nt3195) and the downstream primer 2DS1_PCR_Ex45_R (sequence: 5'-GAGGGTTTGGAGGTGCCCTGTCG-3', nt5747-nt5769), specifically amplifying the 4th exon, the 4th intron, the 5th exon, and part of the 2/3 intron and The sequence of part of the 5th intron, the length of the amplified fragment of interest is 2595 bp. The third pair of PCR primers include the upstream primer 2DS1_PCR_Ex6_F (sequence: 5'-TCCTGATTGTGAGTTCTTGGCAT-3', nt8078-nt8100) and the downstream primer 2DS1_PCR_Ex6_R (sequence: 5'-GTCTCCTAGATTCCAGTTACGCC-3', nt10742-nt10764), specific amplification 6th The partial sequence of the exon and its flanks, the length of the amplified fragment of interest is 2687 bp. The fourth pair of PCR primers includes the upstream primer 2DS1_PCR_Ex789_F (sequence: 5'-CGTGGAAAAGGCAATTCCCGA-3', nt10765-nt10785) and the downstream primer 2DS1_PCR_Ex789_R (sequence: 5'-GGAGGTGGAACAGCACGTGTC-3', nt14330-nt14350), specific amplification 7th Exon, 7th intron, 8th exon, 8th intron, ninth exon, and part of the 6th intron and part of the 3'-untranslated region, the amplified fragment of interest The length is 3586 bp.

(7) The first pair of PCR primers for amplifying the entire coding region of the KIR2DS2 gene includes the upstream primer 2DS2_PCR_Ex12_F (sequence: 5'-TGAGAGGTTGGATCTGAGACGTC-3', nt-265-nt-243) and the downstream primer 2DS2_PCR_Ex12_R (sequence: 5'-ACATCCAGGCTCTTATCAGCCTT -3', nt2956-nt2978), specific amplification of the first exon, the first intron, the second exon, and a part of the 5'-promoter region and part of the second intron, the purpose The amplified fragment was 3243 bp in length. The second pair of PCR primers includes the upstream primer 2DS2_PCR_Ex45_F (sequence: 5'-GCTTCCATGCTTCTGATAATTTTG-3', nt2420~nt2443) and the downstream primer 2DS2_PCR_Ex45_R (sequence: 5'-CTCTGGGTCTCTCCTGACCGT-3', nt5639~nt5659), specific amplification 4 The sequence of the exon, the fourth intron, the exon 5, and part of the 2/3 intron and part of the 5th intron, the amplified fragment of interest is 3240 bp in length. The third pair of PCR primers includes the upstream primer 2DS2_PCR_Ex6_F (sequence: 5'-CATTCTGCTCCGTTGTTCTATGTC-3', nt8282 to nt8305) and the downstream primer 2DS2_PCR_Ex6_R (sequence: 5'-GCCAGGGTTGCTTCATGACCTAT-3', Nt9024~nt9046) specifically amplified the partial sequence of exon 6 and its flanks, and the amplified fragment of interest was 765 bp. The fourth pair of PCR primers includes the upstream primer 2DS2_PCR_Ex789_F (sequence: 5'-GATAGGCCATGGGGAGGTAAATT-3', nt11463-nt11485) and the downstream primer 2DS2_PCR_Ex789_R (sequence: 5'-GGGCAGACATGTTTATTTGAAGGC-3', nt14250-nt14273), specific amplification 7th Exon, 7th intron, 8th exon, 8th intron, ninth exon, and part of the 6th intron and part of the 3'-untranslated region, the amplified fragment of interest The length is 2811 bp.

(8) The first pair of PCR primers for amplifying the entire coding region of the KIR2DS3 gene includes the upstream primer 2DS3_PCR_Ex12_F (sequence: 5'-TGTAAACTGCATGGGCAGGGA-3', nt-90-nt-70) and the downstream primer 2DS3_PCR_Ex12_R (sequence: 5'-CTCTGACCTGTGACCATGATCAG -3', nt2368 to nt2390), specifically amplifying the sequence of the first exon, the first intron, the second exon, and a part of the 5'-promoter region and part of the second intron, The amplified fragment was 2480 bp in length. The second pair of PCR primers include the upstream primer 2DS3_PCR_Ex45_F (sequence: 5'-CTGAGCCCAGCGGCAAGGC-3', nt3586-nt3604) and the downstream primer 2DS3_PCR_Ex45_R (sequence: 5'-ATCCCTCCCTCACACCGAGGA-3', nt6039-nt6059), specific amplification 4 The sequence of the exon, the 4th intron, the 5th exon, and part of the 2/3 intron and part of the 5th intron, the amplified fragment of interest is 2474 bp. The third pair of PCR primers includes the upstream primer 2DS3_PCR_Ex6_F (sequence: 5'-TACCAGGGTTCTCCTTTCTCTAG-3', nt7491 to nt7513) and the downstream primer 2DS3_PCR_Ex6_R (sequence: 5'-AGGAAGGGGACCAGGAGCG-3', nt9878 to nt9896), specific amplification 6th The partial sequence of the exon and its flanks, the length of the amplified fragment of interest is 2406 bp. The fourth pair of PCR primers includes the upstream primer 2DS3_PCR_Ex789_F (sequence: 5'-TGATGTTGAAGGAAGAGGCTCTT-3', nt10853-nt10875) and the downstream primer 2DS3_PCR_Ex789_R (sequence: 5'-GATAGTCTGAGGGGAGGTGGAACT-3', nt14688-nt14711), specific amplification 7th Exon, 7th intron, 8th exon, 8th intron, ninth exon, and part of the 6th intron and part of the 3'-untranslated region, the amplified fragment of interest The length is 3859bp.

(9) The first pair of PCR primers that amplify the entire coding region of the KIR2DS4 gene include the upstream primer 2DS4_PCR_Ex12_F (sequence: 5'-ACCATGTCGCTCATGGTCATCAT-3', nt-3 to nt20) and the downstream primer 2DS4_PCR_Ex12_R (sequence: 5'-TTGTCCTGACCACCTTGGGGT-3 ', nt3070 to nt3090), specific amplification of the first exon, the first intron, the second exon, and a part of the 5'-promoter region and part of the second intron, the purpose of amplification The fragment length is 3093 bp. The second pair of PCR primers includes the upstream primer 2DS4_PCR_Ex45_F (sequence: 5'-TCAGTTCATACCTCCTGCCAAGG-3', nt4419~nt4441) and the downstream primer 2DS4_PCR_Ex45_R (sequence: 5'-CGTGGTCAGGAGTTCCAGAGC-3', nt7611 to nt7631), specific amplification 4 The sequence of the exon, the fourth intron, the exon 5, and part of the 2/3 intron and part of the 5th intron, the amplified fragment of interest is 3213 bp in length. The third pair of PCR primers includes the upstream primer 2DS4_PCR_Ex6_F (sequence: 5'-CTGGACTCCCAGGGCCCAATG-3', nt10004 to nt10024) and the downstream primer 2DS4_PCR_Ex6_R (sequence: 5'-AAGGTTTCCACCTCCCCAGGG-3', nt10212 to nt10232), specific amplification 6th The partial sequence of the exon and its flanks, the length of the amplified fragment of interest is 229 bp. The fourth pair of PCR primers includes the upstream primer 2DS4_PCR_Ex789_F (sequence: 5'-GAAAGCCCGCTGAATCCTC-3', nt12884-nt12902) and the downstream primer 2DS4_PCR_Ex789_R (sequence: 5'-GCAGAAGGCTGAAAGATAGTCTG-3', nt15726-nt15748), specific amplification 7th Exon, 7th intron, 8th exon, 8th intron, ninth exon, and part of the 6th intron and part of the 3'-untranslated region, the amplified fragment of interest The length is 2865 bp.

(10) The first pair of PCR primers for amplifying the entire coding region of the KIR2DS5 gene includes the upstream primer 2DS5_PCR_Ex12_F (sequence: 5'-TGAGAACAATTTCCAGGAAGCCG-3', nt-199-nt-177) and the downstream primer 2DS5_PCR_Ex12_R (sequence: 5'-CCTTTCCTGTGGACACTTGTC -3', nt2870~nt2890), specifically amplifying the sequence of the first exon, the first intron, the second exon, and a part of the 5'-promoter region and part of the second intron, The amplified fragment was 3089 bp in length. The second pair of PCR primers includes the upstream primer 2DS5_PCR_Ex45_F (preface Column: 5'-TCCTGCCAAGGATTCCAATTCGA-3', nt3609~nt3631) and downstream primer 2DS5_PCR_Ex45_R (sequence: 5'-TCTGTCCATGCTTCTCTCCATCC-3', nt6181~nt6203), specifically amplifying the fourth exon, the fourth intron, The 5th exon, and the sequence of part 2/3 intron and part 5 intron, the amplified fragment of interest is 2595 bp. The third pair of PCR primers includes the upstream primer 2DS5_PCR_Ex6_F (sequence: 5'-CTTGAAGTCTCAAGACAGTGGGT-3', nt9083 to nt9105) and the downstream primer 2DS5_PCR_Ex6_R (sequence: 5'-ATGCACTTCATACTTTGAGCTAG-3', nt9923 to nt9945), specific amplification 6th The partial sequence of the exon and its flanks, the length of the amplified fragment of interest is 863 bp. The fourth pair of PCR primers includes the upstream primer 2DS5_PCR_Ex789_F (sequence: 5'-TGATGTKGAAGGAAGAGGCTCTG-3', nt11029-nt11051) and the downstream primer 2DS5_PCR_Ex789_R (sequence: 5'-AGGGGAGGTGGAACTGCATGAGA-3', nt14857~nt14879), specific amplification 7th Exon, 7th intron, 8th exon, 8th intron, ninth exon, and part of the 6th intron and part of the 3'-untranslated region, the amplified fragment of interest The length is 3851 bp.

(11) The first pair of PCR primers that amplify the entire coding region of the KIR3DL1 gene include the upstream primer 3DL1_PCR_Ex12_F (sequence: 5'-CGAGGTGTCAATTCTAGTGAGAG-3', nt-215-nt-193) and the downstream primer 3DL1_PCR_Ex12_R (sequence: 5'-TACCACAAACATGGCAGCG -3', nt2689 to nt2707), specifically amplifying the sequence of the first exon, the first intron, the second exon, and a part of the 5'-promoter region and a part of the second intron, The amplified fragment was 2922 bp in length. The second pair of PCR primers include the upstream primer 3DL1_PCR_Ex345_F (sequence: 5'-CACCCAGGTGTGGTAGGAGCC-3', nt1700-nt1720) and the downstream primer 3DL1_PCR_Ex345_R (sequence: 5'-CTCTGTGTGGGTGAGAGGCCATG-3', nt5684-nt5706), specific amplification 3 The sequence of the exon, the third intron, the fourth exon, the fourth intron, the fifth exon, and some of the second intron and part of the fifth intron, the length of the amplified fragment of interest It is 4007bp. The third pair of PCR primers includes the upstream primer 3DL1_PCR_Ex6_F (sequence: 5'-GCCTGTAATACCACTACTCGGGT-3', nt8050~nt8072) and downstream primer 3DL1_PCR_Ex6_R (sequence: 5'-CTAAAACACCTCGCCCTCATC-3', nt8921~nt8941), specifically amplifying the partial sequence of exon 6 and its flanks, The amplified fragment was 892 bp in length. The fourth pair of PCR primers includes the upstream primer 3DL1_PCR_Ex789_F (sequence: 5'-GCTATAACTGAGAAAGCAGGAGG-3', nt12700-nt12722) and the downstream primer 3DL1_PCR_Ex789_R (sequence: 5'-CTGGAAAATAGTCCGAAGAAAGG-3', nt14173-nt14195), specific amplification 7th Exon, 7th intron, 8th exon, 8th intron, ninth exon, and part of the 6th intron and part of the 3'-untranslated region, the amplified fragment of interest The length is 1496 bp.

(12) The first pair of PCR primers for amplifying the entire coding region of the KIR3DL2 gene includes the upstream primer 3DL2_PCR_Ex12_F (sequence: 5'-TGCAAGGTGGCAATTGTAGTCAC-3', nt-217-nt-195) and the downstream primer 3DL2_PCR_Ex12_R (sequence: 5'-CGACGATAGTGACACTGAAGAGC -3', nt1588 to nt1610), specifically amplifying the sequence of the first exon, the first intron, the second exon, and a part of the 5'-promoter region and part of the second intron, The amplified fragment was 1827 bp in length. The second pair of PCR primers includes the upstream primer 3DL2_PCR_Ex345_F (sequence: 5'-CCTCCTCTCTAAGGCAGTGCCTC-3', nt1488~nt1510) and the downstream primer 3DL2_PCR_Ex345_R (sequence: 5'-CGGGTTTTCCTCACCTGTGACAG-3', nt5429~nt5451), specific amplification 3 The sequence of the exon, the third intron, the fourth exon, the fourth intron, the fifth exon, and some of the second intron and part of the fifth intron, the length of the amplified fragment of interest It is 3964bp. The third pair of PCR primers include the upstream primer 3DL2_PCR_Ex6_F (sequence: 5'-GACAGGGCACCTCCAAACCCTCT-3', nt5584-nt5606) and the downstream primer 3DL2_PCR_Ex6_R (sequence: 5'-ATTTTAGCCCAGTGACATGCACG-3', nt9282 to nt9304), specific amplification 6th The partial sequence of the exon and its flanks, the length of the amplified fragment of interest is 3721 bp. The fourth pair of PCR primers includes the upstream primer 3DL2_PCR_Ex789_F (sequence: 5'-GCAGGAGAAAGCTGGGTCTCC-3', nt15186 to nt15206) and the downstream primer 3DL2_PCR_Ex789_R (sequence: 5'-CTGGTTTTGAGACAGGGCTGTTG-3', nt16262-nt16284), specific amplification of exon 7, intron 7, exon 8, intron, ninth exon The sub-and a part of the 6th intron and part of the 3'-untranslated region are sequenced, and the amplified fragment of interest is 1099 bp in length.

(13) The first pair of PCR primers for amplifying the entire coding region of the KIR3DL3 gene includes the upstream primer 3DL3_PCR_Ex12_F (sequence: 5'-ACAACATCCTGTGTGCTGCTGAA-3', nt-63-nt-41) and the downstream primer 3DL3_PCR_Ex12_R (sequence: 5'-GTCAACCCCCTGTGTCGCCTG -3', nt815 to nt835), specifically amplifying the sequence of the first exon, the first intron, the second exon, and a part of the 5'-promoter region and part of the second intron, The amplified fragment was 898 bp in length. The second pair of PCR primers include the upstream primer 3DL3_PCR_Ex345_F (sequence: 5'-GGAACCACAGTCATGACCCTGAC-3', nt1156-nt1178) and the downstream primer 3DL3_PCR_Ex345_R (sequence: 5'-AAAGGGTGTAGGCGTTGCTGG-3', nt5608-nt5630), specific amplification third The sequence of the exon, the third intron, the fourth exon, the fourth intron, the fifth exon, and some of the second intron and part of the fifth intron, the length of the amplified fragment of interest It is 4475 bp. The third pair of PCR primers include the upstream primer 3DL3_PCR_Ex789_F (sequence: 5'-TGAGCCAGTCCCTCAAGGCTC-3', nt9865-nt9885) and the downstream primer 3DL3_PCR_Ex789_R (sequence: 5'-GTTTTACTGCTGACAGAAGGCTG-3', nt12007-nt12029), specific amplification 7th Exon, 7th intron, 8th exon, 8th intron, ninth exon, and part of the 6th intron and part of the 3'-untranslated region, the amplified fragment of interest The length is 2165 bp.

(14) The first pair of PCR primers for amplifying the entire coding region of the KIR3DS1 gene includes the upstream primer 3DS1_PCR_Ex12_F (sequence: 5'-CGAGGTGTCAATTCTAGTGAGAG-3', nt-215-nt-193) and the downstream primer 3DS1_PCR_Ex12_R (sequence: 5'-CCTGTGACCATGATCACCAT) -3', nt2080 to nt2099), specifically amplifying the sequence of the first exon, the first intron, the second exon, and a part of the 5'-promoter region and part of the second intron, Amplified fragment length It is 2314 bp. The second pair of PCR primers include the upstream primer 3DS1_PCR_Ex345_F (sequence: 5'-CAGCTGACACTTGTTGTAGGGAG-3', nt1634-nt1656) and the downstream primer 3DS1_PCR_Ex345_R (sequence: 5'-AGTGGCATGATCTCGGCTCAG-3', nt6472 to nt6492), specific amplification 3 The sequence of the exon, the third intron, the fourth exon, the fourth intron, the fifth exon, and some of the second intron and part of the fifth intron, the length of the amplified fragment of interest It is 4859bp. The third pair of PCR primers includes the upstream primer 3DS1_PCR_Ex6_F (sequence: 5'-TGATCCGCCCACCTCCGCT-3', nt7633~nt7651) and the downstream primer 3DS1_PCR_Ex6_R (sequence: 5'-GCTGGGAGGTTTGAGCCAACG-3', nt9048-nt9068), specific amplification 6th The partial sequence of the exon and its flanks, the length of the amplified fragment of interest is 1436 bp. The fourth pair of PCR primers includes the upstream primer 3DS1_PCR_Ex789_F (sequence: 5'-GCTATAACTGAGAAAGCAGGAGG-3', nt13101-nt13123) and the downstream primer 3DS1_PCR_Ex789_R (sequence: 5'-GAAGGCTGAAAGCTAGTCTGAGG-3', nt14562-nt14584), specific amplification 7th Exon, 7th intron, 8th exon, 8th intron, ninth exon, and part of the 6th intron and part of the 3'-untranslated region, the amplified fragment of interest The length is 1484 bp.

3. The system composition of the PCR amplification reaction is:

4. The PCR amplification reaction can be simultaneously amplified under the same cycle parameters, and the cycle parameters are:

5. The purification reaction system composition of the PCR amplification product is:

6. The purification reaction system of the PCR amplification product can be purified under the same cycle parameters, and the cycle parameters are:

37 ° C 45 min,

85 ° C 15 min,

4°C Infinite.

7. Purified KIR gene PCR-specific amplification products covered by exons, which are specific for sixteen (KIR2DL1~5, 2DS1~5 and KIR3DL3) or eighteen (KIR3DL1~2 and KIR3DS1) specific The purified PCR product was subjected to a bidirectional sequencing reaction by reverse and reverse sequencing primers. A total of 230 functional KIR gene-specific forward and reverse sequencing primers were shown in Table 2, as shown in Table 2:

Table 2 14 functional KIR gene-specific forward and reverse sequencing primers

(1) KIR2DL1 first exon forward sequencing primer 2DL1_SBT_Ex1_F (sequence: 5'-CGTGTTCCGCTCTTGAGCG-3', nt-177~nt-159), first exon reverse sequencing primer 2DL1_SBT_Ex1_R (sequence: 5'- TCACTCCCTCCCTCTATTG-3', nt50~nt68);

KIR2DL1 second exon forward sequencing primer 2DL1_SBT_Ex2_F (sequence: 5'-TTCTTGGGTGCAGGTAGGC-3', nt855~nt873), second exon reverse sequencing primer 2DL1_SBT_Ex2_R (sequence: 5'-ACCCTGGTCCCCACAGAAC-3', nt1210~nt1228);

KIR2DL1 fourth exon forward sequencing primer 2DL1_SBT_Ex4_F (sequence: 5'-AAGGGGAAGCCTGACTCAA-3', nt3400~nt3418), fourth exon reverse sequencing primer 2DL1_SBT_Ex4_R (sequence: 5'-CCAATTCCTGGATCATTCAC-3', nt3827~ Nt3846);

KIR2DL1 fifth exon forward sequencing primer 2DL1_SBT_Ex5_F (sequence: 5'-GTTCTCAGCTCAGGTGAAG-3', nt5240~nt5258), fifth exon reverse sequencing primer 2DL1_SBT_Ex5_R (sequence: 5'-AAACAAGCAGTGGGTCACTTGAC-3', nt5574~ Nt5596);

KIR2DL1 sixth exon forward sequencing primer 2DL1_SBT_Ex6_F (sequence: 5'-TTTCCACTGAGTGGAGGAC-3', nt8698~nt8716), sixth exon reverse sequencing primer 2DL1_SBT_Ex6_R (sequence: 5'-TGGAGTTCGGAGATGGTGG-3', nt8920~ Nt8938);

KIR2DL1 seventh exon forward sequencing primer 2DL1_SBT_Ex7_F (sequence: 5'-ATGTGGTTACCTGTCAATC-3', nt12979~nt12997), seventh exon reverse sequencing primer 2DL1_SBT_Ex7_R (sequence: 5'-TCCTGCTTCCCCACATGGC-3', nt13207~ Nt13225);

KIR2DL1 eighth exon forward sequencing primer 2DL1_SBT_Ex8_F (sequence: 5'-CTCAGCCACCTATGGTCTC-3', nt13533~nt13551), eighth exon reverse sequencing primer 2DL1_SBT_Ex8_R (sequence: 5'-TCTCTGTGTGAAAACGCAG-3', nt13835~ Nt13853);

KIR2DL1 ninth exon forward sequencing primer 2DL1_SBT_Ex9_F (sequence: 5'-ACAGAACAGCGAATAGCGA-3', nt13667~nt13685), ninth exon reverse sequencing primer 2DL1_SBT_Ex9_R (sequence: 5'-TAAGATGCAGACTCATGCC-3', nt14060~ Nt14078).

(2) KIR2DL2 first exon forward sequencing primer 2DL2_SBT_Ex1_F (sequence: 5'-AGAGGTTGGATCTGAGACGTC-3', nt-263~nt-243), first exon reverse sequencing primer 2DL2_SBT_Ex1_R (sequence: 5'- TCTCCAACTCTGGGCCCCG-3', nt81~nt99);

KIR2DL2 second exon forward sequencing primer 2DL2_SBT_Ex2_F (sequence: 5'-TTCTTGGGTGCAGGTAGGC-3', nt799 to nt817), second exon reverse sequencing primer 2DL2_SBT_Ex2_R (sequence: 5'-CCCAGTCTAACCCTGGTCC-3', nt1163 to nt1181);

KIR2DL2 fourth exon forward sequencing primer 2DL2_SBT_Ex4_F (sequence: 5'-AAGGGGAAGCCTCACTCAT-3', nt3332~nt3350), fourth exon reverse sequencing primer 2DL2_SBT_Ex4_R (sequence: 5'-GGCCCCTGTGTCTGTCCTC-3', nt3900~ Nt3918);

KIR2DL2 fifth exon forward sequencing primer 2DL2_SBT_Ex5_F (sequence: 5'-GCTGTGACAAGGAAGATCC-3', nt5179~nt5197), fifth exon reverse sequencing primer 2DL2_SBT_Ex5_R (sequence: 5'-AAGCTCCTCAGCTAAGGCT-3', nt5564~ Nt5582);

KIR2DL2 sixth exon forward sequencing primer 2DL2_SBT_Ex6_F (sequence: 5'-ATCCCAGGACTCCCAGGGC-3', nt8669~nt8687), sixth exon reverse sequencing primer 2DL2_SBT_Ex6_R (sequence: 5'-GGCGTACAATGTCAGAGCTGC-3', nt8928~ Nt8948);

KIR2DL2 seventh exon forward sequencing primer 2DL2_SBT_Ex7_F (sequence: 5'-ATCTGGGTGCTTGTCCTAA-3', nt12990~nt13008), seventh exon reverse sequencing primer 2DL2_SBT_Ex7_R (sequence: 5'-CCTCTGCTTCGTGAGACTTAC-3', nt13213~ Nt13233);

KIR2DL2 eighth exon forward sequencing primer 2DL2_SBT_Ex8_F (sequence: 5'-CCCAGAAGTGCCCTCCGAG-3', nt13628~nt13646), eighth exon reverse sequencing primer 2DL2_SBT_Ex8_R (sequence: 5'-TCTCTGTGTGAAAACGCAG-3', nt13876~ Nt13894);

KIR2DL2 ninth exon forward sequencing primer 2DL2_SBT_Ex9_F (sequence: 5'-ACAGAACAGCGAATAGCGA-3', nt13708~nt13726), ninth exon reverse sequencing primer 2DL2_SBT_Ex9_R (sequence: 5'-GGCTGTTGTCTCTAGAAGACG-3', nt14026~ Nt14048).

(3) KIR2DL3 first exon forward sequencing primer 2DL3_SBT_Ex1_F (sequence: 5'-CYGMCTGTCTGCACAGA-3', nt-24 to nt-8), first exon reverse sequencing primer 2DL3_SBT_Ex1_R (sequence: 5'- TCTCCAACTCTGGGCCCCG-3', Nt81~nt99);

KIR2DL3 second exon forward sequencing primer 2DL3_SBT_Ex2_F (sequence: 5'-TTCTTGGGTGCAGGTAGGC-3', nt799~nt817), second exon reverse sequencing primer 2DL3_SBT_Ex2_R (sequence: 5'-ACCCTGGTCCCCACAGAAC-3', nt1154~ Nt1172);

KIR2DL3 fourth exon forward sequencing primer 2DL3_SBT_Ex4_F (sequence: 5'-CAGCAAGGGGAAGCCTCA-3', nt3329~nt3346), fourth exon reverse sequencing primer 2DL3_SBT_Ex4_R (sequence: 5'-GGCCCCTGTGTCTGTCCTC-3', nt3901~ Nt3919);

KIR2DL3 fifth exon forward sequencing primer 2DL3_SBT_Ex5_F (sequence: 5'-GAGCATTAGGTCATAGAGC-3', nt5131 to nt5149), fifth exon reverse sequencing primer 2DL3_SBT_Ex5_R (sequence: 5'-CTCTCTGCATCTGTCCATGCTTC-3', nt5602~ Nt5624);

KIR2DL3 sixth exon forward sequencing primer 2DL3_SBT_Ex6_F (sequence: 5'-TACTCAGGAGTTTGAGGCC-3', nt8310~nt8328), sixth exon reverse sequencing primer 2DL3_SBT_Ex6_R (sequence: 5'-GGCGTACAATGTCAGAGCTGC-3', nt8908~ Nt8928);

KIR2DL3 seventh exon forward sequencing primer 2DL3_SBT_Ex7_F (sequence: 5'-TCTGGGTGCTTGTCCTAAAGG-3', nt12969~nt12989), seventh exon reverse sequencing primer 2DL3_SBT_Ex7_R (sequence: 5'-CAGGCAATGGTCTGTGAGC-3', nt13361~ Nt13379);

KIR2DL3 eighth exon forward sequencing primer 2DL3_SBT_Ex8_F (sequence: 5'-CTTCATCGCTGGTGCTG-3', nt13166~nt13182), eighth exon reverse sequencing primer 2DL3_SBT_Ex8_R (sequence: 5'-GCTGAGTGAGGGAGGGTGC-3', nt13772~ Nt13790);

KIR2DL3 ninth exon forward sequencing primer 2DL3_SBT_Ex9_F (sequence: 5'-CCCAGCCTCGTGGCTAG-3', nt13724~nt13740), ninth exon reverse sequencing primer 2DL3_SBT_Ex9_R (sequence: 5'-GGCAGGAGACAACTTTGGATCW-3', nt13957~ Nt13978).

(4) KIR2DL4 first exon forward sequencing primer 2DL4_SBT_Ex1_F (sequence: 5'-GTGGTCAATGTGTCAACTGCACG-3', nt-99 to nt-77), first exon reverse sequencing primer 2DL4_SBT_Ex1_R (sequence: 5'-CCTGAGCCACTGGGCGCCA-3', nt 166 to nt 184);

KIR2DL4 second exon forward sequencing primer 2DL4_SBT_Ex2_F (sequence: 5'-GAGCCATGTTCTGAAGCAAGT-3', nt111~nt131), second exon reverse sequencing primer 2DL4_SBT_Ex2_R (sequence: 5'-CACCCTCTGTGCTGCCTCC-3', nt345~ Nt363);

KIR2DL4 third exon forward sequencing primer 2DL4_SBT_Ex3_F (sequence: 5'-TACTCCTCTCTGAGGCGGC-3', nt1140~nt1158), third exon reverse sequencing primer 2DL4_SBT_Ex3_R (sequence: 5'-CCAGAAGCTCTGGGACTCA-3', nt1502~ Nt1520);

KIR2DL4 fifth exon forward sequencing primer 2DL4_SBT_Ex5_F (sequence: 5'-GGGAGGGGAGCTGTGACAA-3', nt2275~nt2293), fifth exon reverse sequencing primer 2DL4_SBT_Ex5_R (sequence: 5'-GCTTCTCTCCATCATCAGC-3', nt2691~ Nt2709);

KIR2DL4 sixth exon forward sequencing primer 2DL4_SBT_Ex6_F (sequence: 5'-CAGGCATCCTCATTGCCAC-3', nt5179~nt5197), sixth exon reverse sequencing primer 2DL4_SBT_Ex6_R (sequence: 5'-TGGCAGGTGCTGAGCCAAC-3', nt5341~ Nt5359);

KIR2DL4 seventh exon forward sequencing primer 2DL4_SBT_Ex7_F (sequence: 5'-TCGCCAGACACCTGCATGC-3', nt9519~nt9537), seventh exon reverse sequencing primer 2DL4_SBT_Ex7_R (sequence: 5'-TTTGGAGCACCAGC-3', nt9600~ Nt9613);

KIR2DL4 eighth exon forward sequencing primer 2DL4_SBT_Ex8_F (sequence: 5'-GAGGACCCAGAAGTGCCCT-3', nt10030~nt10048), eighth exon reverse sequencing primer 2DL4_SBT_Ex8_R (sequence: 5'-CTGGAGAGAGGGAAATCCT-3', nt10215~ Nt10233);

KIR2DL4 ninth exon forward sequencing primer 2DL4_SBT_Ex9_F (sequence: 5'-CCAGCCTCATGGATACAGTCT-3', nt10150~nt10170), ninth exon reverse sequencing primer 2DL4_SBT_Ex9_R (sequence: 5'-GGAAGAGTGATGCTCTAAGATGG-3', nt10516 to nt10538).

(5) KIR2DL5 first exon forward sequencing primer 2DL5_SBT_Ex1_F (sequence: 5'-CCAAATAACATCCTGTGCGCT-3', nt-67 to nt-47), first exon reverse sequencing primer 2DL5_SBT_Ex1_R (sequence: 5'- AGATCTCCATCCCCGCACT-3', nt64~nt82);

KIR2DL5 second exon forward sequencing primer 2DL5_SBT_Ex2_F (sequence: 5'-CAGCAAGGGCCTGGCTACC-3', nt668-nt686), second exon reverse sequencing primer 2DL5_SBT_Ex2_R (sequence: 5'-GAAAATCCCCCACCGGGCT-3', nt872~ Nt890);

KIR2DL5 third exon forward sequencing primer 2DL5_SBT_Ex3_F (sequence: 5'-ACAAGCCCTTGCTGTCTGCCT-3', nt1575~nt1595), third exon reverse sequencing primer 2DL5_SBT_Ex3_R (sequence: 5'-CAGATGCTCTGGGATTCAG-3', nt1891~ Nt1909);

KIR2DL5 fifth exon forward sequencing primer 2DL5_SBT_Ex5_F (sequence: 5'-CAGGTGTGAGGGGAGCTGT-3', nt2665~nt2683), fifth exon reverse sequencing primer 2DL5_SBT_Ex5_R (sequence: 5'-CGGGTCTGACCACTCATAGGGT-3', nt2970~ Nt2991);

KIR2DL5 sixth exon forward sequencing primer 2DL5_SBT_Ex6_F (sequence: 5'-TCACCTCTCTCCTGTCCTGTGT-3', nt5165~nt5186), sixth exon reverse sequencing primer 2DL5_SBT_Ex6_R (sequence: 5'-TGAGCCAATGCTTGAATCCAAGA-3', nt5295~ Nt5317);

KIR2DL5 seventh exon forward sequencing primer 2DL5_SBT_Ex7_F (sequence: 5'-ATCCATAAAGAGGAACTGCTATA-3', nt7951~nt7973), seventh exon reverse sequencing primer 2DL5_SBT_Ex7_R (sequence: 5'-CCTTGGTCCAGGGACCATC-3', nt8201~ Nt8219);

KIR2DL5 eighth exon forward sequencing primer 2DL5_SBT_Ex8_F (sequence: 5'-CACCTACGGCCTCCCGCTG-3', nt8480~nt8498), eighth exon reverse sequencing primer 2DL5_SBT_Ex8_R (sequence: 5'-GAGGGTGCTCACATTCTTCAA-3', nt8680~ Nt8700);

KIR2DL5 ninth exon forward sequencing primer 2DL5_SBT_Ex9_F (sequence: 5'-TGCCGGGGACAGAACAGTG-3', nt8600 to nt8618), ninth exon reverse sequencing primer 2DL5_SBT_Ex9_R (sequence: 5'-CTCAAGGCCTGACTGTGGTGCTT-3', nt8899 to nt8921).

(6) KIR2DS1 first exon forward sequencing primer 2DS1_SBT_Ex1_F (sequence: 5'-CTCCCATGATGTGGTCAAC-3', nt-109~nt-91), first exon reverse sequencing primer 2DS1_SBT_Ex1_R (sequence: 5'- TCTCCAACCCCACACTCCC-3', nt61~nt79);

KIR2DS1 second exon forward sequencing primer 2DS1_SBT_Ex2_F (sequence: 5'-TTCTTGGGTGCAGGTAGGC-3', nt855~nt873), second exon reverse sequencing primer 2DS1_SBT_Ex2_R (sequence: 5'-CTGCCAAGGGAATGAAAGG-3', nt1185~ Nt1203);

KIR2DS1 fourth exon forward sequencing primer 2DS1_SBT_Ex4_F (sequence: 5'-GGTGCCATGGATGGGATGA-3', nt3423~nt3441), fourth exon reverse sequencing primer 2DS1_SBT_Ex4_R (sequence: 5'-CAAGTCCTGGATCATTCAC-3', nt3827~ Nt3845);

KIR2DS1 fifth exon forward sequencing primer 2DS1_SBT_Ex5_F (sequence: 5'-AGAGCAGGGGAGTGAGTTC-3', nt5221~nt5239), fifth exon reverse sequencing primer 2DS1_SBT_Ex5_R (sequence: 5'-GGCTCTAGGATCATAGGAC-3', nt5628~ Nt5646);

KIR2DS1 sixth exon forward sequencing primer 2DS1_SBT_Ex6_F (sequence: 5'-TCCTCAAAGATTTCCACTGAGTG-3', nt8684~nt8706), sixth exon reverse sequencing primer 2DS1_SBT_Ex6_R (sequence: 5'-GTGAGATGCTGAGTCAACGC-3', nt8871~ Nt8890);

KIR2DS1 seventh exon forward sequencing primer 2DS1_SBT_Ex7_F (sequence: 5'-GTGGTTACCTGCCAATCAAG-3', nt12981~nt13000), seventh exon reverse sequencing primer 2DS1_SBT_Ex7_R (sequence: 5'-TGAGGAACACACATCCGCGT-3', nt13236~ Nt13255);

KIR2DS1 eighth exon forward sequencing primer 2DS1_SBT_Ex8_F (sequence: 5'-ATGGCCTCCCCCTGTTTGT-3', nt13547~nt13565), eighth exon reverse sequencing primer 2DS1_SBT_Ex8_R (sequence: 5'-GGGAATAAGACTAGCCACG-3', nt13713~nt13731);

KIR2DS1 ninth exon forward sequencing primer 2DS1_SBT_Ex9_F (sequence: 5'-CTCCTCGGCCCAGCCTCGT-3', nt13697~nt13715), ninth exon reverse sequencing primer 2DS1_SBT_Ex9_R (sequence: 5'-TCCCCTCAAGGCCTGACTG-3', nt13971~ Nt13989).

(7) KIR2DS2 first exon forward sequencing primer 2DS2_SBT_Ex1_F (sequence: 5'-ATAACATCCTGTGCGCTGC-3', nt-63~nt-45), first exon reverse sequencing primer 2DS2_SBT_Ex1_R (sequence: 5'- CCAACTCTGGGCCCCGATC-3', nt78~nt96);

KIR2DS2 second exon forward sequencing primer 2DS2_SBT_Ex2_F (sequence: 5'-AAGGGAGTCCTGGTTTGCC-3', nt772~nt790), second exon reverse sequencing primer 2DS2_SBT_Ex2_R (sequence: 5'-GTCAGAAATGTGGGCCGAG-3', nt981~ Nt999);

KIR2DS2 fourth exon forward sequencing primer 2DS2_SBT_Ex4_F (sequence: 5'-CACCTTCTAAACTCACAACC-3', nt3268~nt3287), fourth exon reverse sequencing primer 2DS2_SBT_Ex4_R (sequence: 5'-CACTCTGCAGCCCAATGAC-3', nt3624~ Nt3642);

KIR2DS2 fifth exon forward sequencing primer 2DS2_SBT_Ex5_F (sequence: 5'-AGAGCAGGGGAGTGAGTTC-3', nt5030~nt5048), fifth exon reverse sequencing primer 2DS2_SBT_Ex5_R (sequence: 5'-GAAGCTCCTCAGCTAAGGC-3', nt5453~ Nt5471);

KIR2DS2 sixth exon forward sequencing primer 2DS2_SBT_Ex6_F (sequence: 5'-CCAGGGCCCAATATTAGAT-3', nt8465~nt8483), sixth exon reverse sequencing primer 2DS2_SBT_Ex6_R (sequence: 5'-TGAGTCAACGCCTGAATCC-3', nt8686~ Nt8704);

KIR2DS2 seventh exon forward sequencing primer 2DS2_SBT_Ex7_F (sequence: 5'-GCCAATCAAGAAATGCGAG-3', nt12815~nt12833), seventh exon reverse sequencing primer 2DS2_SBT_Ex7_R (sequence: 5'-GTCCTGCCTCTGTGGCTCC-3', nt13108~ Nt13126);

KIR2DS2 eighth exon forward sequencing primer 2DS2_SBT_Ex8_F (sequence: 5'-ATGAGGACCCAGAAGTGCC-3', nt13407-nt13425), eighth exon reverse sequencing primer 2DS2_SBT_Ex8_R (sequence: 5'-CCTCCTGATGGTCTTGTTC-3', nt13621 to nt13639);

KIR2DS2 ninth exon forward sequencing primer 2DS2_SBT_Ex9_F (sequence: 5'-AGGTAGGTGCTCCTCGGCC-3', nt13512~nt13530), ninth exon reverse sequencing primer 2DS2_SBT_Ex9_R (sequence: 5'-AGAAGATCCCCTCAAGGCC-3', nt13801~ Nt13819).

(8) KIR2DS3 first exon forward sequencing primer 2DS3_SBT_Ex1_F (sequence: 5'-CAGGGAGCCAAATAACATC-3', nt-75~nt-57), first exon reverse sequencing primer 2DS3_SBT_Ex1_R (sequence: 5'- CGCTCCCTCCCTCTATTCC-3', nt49~nt67);

KIR2DS3 second exon forward sequencing primer 2DS3_SBT_Ex2_F (sequence: 5'-GCCGAGAGCCCTGTTCTTG-3', nt1182~nt1200), second exon reverse sequencing primer 2DS3_SBT_Ex2_R (sequence: 5'-ACAGGACTTCCCTCCCGTT-3', nt1432~ Nt1450);

KIR2DS3 fourth exon forward sequencing primer 2DS3_SBT_Ex4_F (sequence: 5'-AGAGAGACACCTTCTAAAT-3', nt3780~nt3798), fourth exon reverse sequencing primer 2DS3_SBT_Ex4_R (sequence: 5'-ATCATTCACTCTGTGTCCG-3', nt4152~ Nt4170);

KIR2DS3 fifth exon forward sequencing primer 2DS3_SBT_Ex5_F (sequence: 5'-AGGAAGATCCTCCATAAGG-3', nt5596~nt5614), fifth exon reverse sequencing primer 2DS3_SBT_Ex5_R (sequence: 5'-GGCTCTAGGATCATAGGAC-3', nt5957~ Nt5975);

KIR2DS3 sixth exon forward sequencing primer 2DS3_SBT_Ex6_F (sequence: 5'-TCCCAGGGCCCAATATTAG-3', nt8968~nt8986), sixth exon reverse sequencing primer 2DS3_SBT_Ex6_R (sequence: 5'-CACTGAGCCCTGTGTTGGG-3', nt9291~ Nt9309);

KIR2DS3 seventh exon forward sequencing primer 2DS3_SBT_Ex7_F (sequence: 5'-GTGCTTGTCCTAAAGAGACGT-3', nt13284~nt13304), seventh exon reverse sequencing primer 2DS3_SBT_Ex7_R (sequence: 5'-TGAGTGGCTGCAGGGGACG-3', nt13709~nt13727);

KIR2DS3 eighth exon forward sequencing primer 2DS3_SBT_Ex8_F (sequence: 5'-GACCTCAGGCACCTATGGC-3', nt13862~nt13880), eighth exon reverse sequencing primer 2DS3_SBT_Ex8_R (sequence: 5'-GCTGAGTGAGGGAGGGTGC-3', nt14082~ Nt14100);

KIR2DS3 ninth exon forward sequencing primer 2DS3_SBT_Ex9_F (sequence: 5'-CGGCCCAGCCTCGTGGCTA-3', nt14031~nt14049), ninth exon reverse sequencing primer 2DS3_SBT_Ex9_R (sequence: 5'-TGTCTTGGGCCTCTGAGAAGGGG-3', nt14196~ Nt14218).

(9) KIR2DS4 first exon forward sequencing primer 2DS4_SBT_Ex1_F (sequence: 5'-ACCATGTCGCTCATGGTC-3', nt-3 to nt15), first exon reverse sequencing primer 2DS4_SBT_Ex1_R (sequence: 5'-GGCTCATCACTCCATCTCT- 3', nt148~nt166);

KIR2DS4 second exon forward sequencing primer 2DS4_SBT_Ex2_F (sequence: 5'-GAAGGGGCTGGCTATCAAG-3', nt2218~nt2236), second exon reverse sequencing primer 2DS4_SBT_Ex2_R (sequence: 5'-GACTTCCCTCCCGTTTCAG-3', nt2404~ Nt2422);

KIR2DS4 fourth exon forward sequencing primer 2DS4_SBT_Ex4_F (sequence: 5'-AGAGAGACACCTTCTAAAC-3', nt4774~nt4792), fourth exon reverse sequencing primer 2DS4_SBT_Ex4_R (sequence: 5'-CACCTGGGTCTCCAAGTCC-3', nt5168~ Nt5186);

KIR2DS4 fifth exon forward sequencing primer 2DS4_SBT_Ex5_F (sequence: 5'-AGTTCTCAGGTCAGGTGTG-3', nt6589~nt6607), fifth exon reverse sequencing primer 2DS4_SBT_Ex5_R (sequence: 5'-GGAAGCTCCTCAGCTAAGG-3', nt7001~ Nt7019);

KIR2DS4 sixth exon forward sequencing primer 2DS4_SBT_Ex6_F (sequence: 5'-CTGGACTCCCAGGGCCCAATG-3', nt10004~nt10024), sixth exon reverse sequencing primer 2DS4_SBT_Ex6_R (sequence: 5'-TTCCACCTCCCCAGGGTTC-3', nt10209~ Nt10227);

KIR2DS4 seventh exon forward sequencing primer 2DS4_SBT_Ex7_F (sequence: 5'-CGCCATTTGGGTGCTTGTC-3', nt14317 to nt14335), seventh exon reverse sequencing primer 2DS4_SBT_Ex7_R (sequence: 5'-GGTGAGGAACACACATCCG-3', nt14611 to nt14629);

KIR2DS4 eighth exon forward sequencing primer 2DS4_SBT_Ex8_F (sequence: 5'-AGTCTGCTGTTGGCAACTG-3', nt14883~nt14901), eighth exon reverse sequencing primer 2DS4_SBT_Ex8_R (sequence: 5'-CCTCCTGATGGTCTTGTTC-3', nt15169~ Nt15187);

KIR2DS4 ninth exon forward sequencing primer 2DS4_SBT_Ex9_F (sequence: 5'-CTCGGCCCAGCCTCGTGGC-3', nt15072~nt15090), ninth exon reverse sequencing primer 2DS4_SBT_Ex9_R (sequence: 5'-CAACTTTGGATCTGGGCTC-3', nt15304~ Nt15322).

(10) KIR2DS5 first exon forward sequencing primer 2DS5_SBT_Ex1_F (sequence: 5'-GGCGCCAAATAACATCCTG-3', nt-72-nt-54), first exon reverse sequencing primer 2DS5_SBT_Ex1_R (sequence: 5'- GCCCAGATCTCCATCCCCG-3', nt68~nt86);

KIR2DS5 second exon forward sequencing primer 2DS5_SBT_Ex2_F (sequence: 5'-GGCACTGAGKGTGAGTTTC-3', nt1383~nt1401), second exon reverse sequencing primer 2DS5_SBT_Ex2_R (sequence: 5'-TGACAGGACTTCCCTCCCG-3', nt1606~ Nt1624);

KIR2DS5 fourth exon forward sequencing primer 2DS5_SBT_Ex4_F (sequence: 5'-GACACCTTCTAAATTCACAAAC-3', nt3958~nt3979), fourth exon reverse sequencing primer 2DS5_SBT_Ex4_R (sequence: 5'-CTCTGCATCCCAATGACAATG-3', nt4315~ Nt4335);

KIR2DS5 fifth exon forward sequencing primer 2DS5_SBT_Ex5_F (sequence: 5'-CCTCCCTGAGGAAAATGCC-3', nt5786~nt5804), fifth exon reverse sequencing primer 2DS5_SBT_Ex5_R (sequence: 5'-TCATAGGACATGGGACAGC-3', nt6129~ Nt6147);

KIR2DS5 sixth exon forward sequencing primer 2DS5_SBT_Ex6_F (sequence: 5'-CAGGGCCCAATATTAGATAAC-3', nt9147~nt9167), sixth exon reverse sequencing primer 2DS5_SBT_Ex6_R (sequence: 5'-GGAGTATCTGGAGTTCGGAGA-3', nt9426 to nt9446);

KIR2DS5 seventh exon forward sequencing primer 2DS5_SBT_Ex7_F (sequence: 5'-CTGTCAATCAAGAAATGCGAG-3', nt13495~nt13515), seventh exon reverse sequencing primer 2DS5_SBT_Ex7_R (sequence: 5'-GGAACACACCCCCGTGC-3', nt13740~ Nt13758);

KIR2DS5 eighth exon forward sequencing primer 2DS5_SBT_Ex8_F (sequence: 5'-AGATAGAATGTCTGAGTCTGC-3', nt14003~nt14023), eighth exon reverse sequencing primer 2DS5_SBT_Ex8_R (sequence: 5'-ACACAGTGATCCAATTATGCG-3', nt14329~ Nt14349);

KIR2DS5 ninth exon forward sequencing primer 2DS5_SBT_Ex9_F (sequence: 5'-GGTAGGTGCTCCTCGGCCC-3', nt14195~nt14213), ninth exon reverse sequencing primer 2DS5_SBT_Ex9_R (sequence: 5'-ATGGGAGCTGGCAACCCGG-3', nt14528~ Nt14546).

(11) KIR3DL1 first exon forward sequencing primer 3DL1_SBT_Ex1_F (sequence: 5'-CAGGGCGCCAAATAACATC-3', nt-74 to nt-56), first exon reverse sequencing primer 3DL1_SBT_Ex1_R (sequence: 5'- CAGATCTCCATCCCCGCAC-3', nt65~nt83);

KIR3DL1 second exon forward sequencing primer 3DL1_SBT_Ex2_F (sequence: 5'-AGGGCCTGGCTGCCAAGAC-3', nt940~nt958), second exon reverse sequencing primer 3DL1_SBT_Ex2_R (sequence: 5'-AATGTGGGCCGAGCATCCG-3', nt1182~ Nt1200);

KIR3DL1 third exon forward sequencing primer 3DL1_SBT_Ex3_F (sequence: 5'-GGGGAGAATCTTCTGGGCACT-3', nt1736~nt1756), third exon reverse sequencing primer 3DL1_SBT_Ex3_R (sequence: 5'-TGATGGGACCCTGACGGAC-3', nt2167~ Nt2185);

KIR3DL1 fourth exon forward sequencing primer 3DL1_SBT_Ex4_F (sequence: 5'-TGGAGGCACCTGCACCAGG-3', nt3052~nt3070), fourth exon reverse sequencing primer 3DL1_SBT_Ex4_R (sequence: 5'-TGGTACAGACCTCACCAAG-3', nt3633~ Nt3651);

KIR3DL1 fifth exon forward sequencing primer 3DL1_SBT_Ex5_F (sequence: 5'-CAGGTATGAGGGGAGCTATG-3', nt5001 to nt5020), fifth exon reverse sequencing primer 3DL1_SBT_Ex5_R (sequence: 5'-CCTGTCTGCCATCCTGCGC-3', nt5490 to nt5508);

KIR3DL1 sixth exon forward sequencing primer 3DL1_SBT_Ex6_F (sequence: 5'-AAGCACCCTCATTTCCTCAC-3', nt8485~nt8504), sixth exon reverse sequencing primer 3DL1_SBT_Ex6_R (sequence: 5'-CAACACTTGCATCCAAGGC-3', nt8631~ Nt8649);

KIR3DL1 seventh exon forward sequencing primer 3DL1_SBT_Ex7_F (sequence: 5'-CCCGCCATCTGGGTGCTTG-3', nt12734~nt12752), seventh exon reverse sequencing primer 3DL1_SBT_Ex7_R (sequence: 5'-TCCTGCTTCCCCACATGGC-3', nt13001~ Nt13019);

KIR3DL1 eighth exon forward sequencing primer 3DL1_SBT_Ex8_F (sequence: 5'-CCAGAAGTGCCCTCCGAGC-3', nt13382~nt13400), eighth exon reverse sequencing primer 3DL1_SBT_Ex8_R (sequence: 5'-TGTTTGGGAATAACACTAGCC-3', nt13507~ Nt13527);

KIR3DL1 ninth exon forward sequencing primer 3DL1_SBT_Ex9_F (sequence: 5'-CGTGGCTAGTGTTATTCCC-3', nt13504~nt13522), ninth exon reverse sequencing primer 3DL1_SBT_Ex9_R (sequence: 5'-ATGGGAGCTGGCAACTCGG-3', nt13833~ Nt13851).

(12) KIR3DL2 first exon forward sequencing primer 3DL2_SBT_Ex1_F (sequence: 5'-GCCAAATAACATCCTGTGCGC-3', nt-68-nt-48), first exon reverse sequencing primer 3DL2_SBT_Ex1_R (sequence: 5'- TAGGCCGAGATCTCCATCC-3', nt71~nt89);

KIR3DL2 second exon forward sequencing primer 3DL2_SBT_Ex2_F (sequence: 5'-GAGGCTAAGTTTACCTTCAGC-3', nt624~nt644), second exon reverse sequencing primer 3DL2_SBT_Ex2_R (sequence: 5'-GACTTCCCTCCTGTTTCAG-3', nt834~ Nt852);

KIR3DL2 third exon forward sequencing primer 3DL2_SBT_Ex3_F (sequence: 5'-GGCCCAGCACTGTGGTGCC-3', nt1553~nt1571), third exon reverse sequencing primer 3DL2_SBT_Ex3_R (sequence: 5'-GCCCATTTCCCCTGTATTC-3', nt1930~nt1948);

KIR3DL2 fourth exon forward sequencing primer 3DL2_SBT_Ex4_F (sequence: 5'-GAGAGATGCCTTCTAAACT-3', nt3235~nt3253), fourth exon reverse sequencing primer 3DL2_SBT_Ex4_R (sequence: 5'-TCTCCATAAGAATCCCACGCT-3', nt3663~ Nt3683);

KIR3DL2 fifth exon forward sequencing primer 3DL2_SBT_Ex5_F (sequence: 5'-CCTCCCTGAGGAAACTGCC-3', nt5111~nt5129), fifth exon reverse sequencing primer 3DL2_SBT_Ex5_R (sequence: 5'-GAAAGAGCCGAAGCATCTG-3', nt5361~ Nt5379);

KIR3DL2 sixth exon forward sequencing primer 3DL2_SBT_Ex6_F (sequence: 5'-CAACCTCAAAGATTTCCATTG-3', nt8530~nt8550), sixth exon reverse sequencing primer 3DL2_SBT_Ex6_R (sequence: 5'-CAACACTTGCATCCAAGGC-3', nt8707~ Nt8725);

KIR3DL2 seventh exon forward sequencing primer 3DL2_SBT_Ex7_F (sequence: 5'-GAGATGTTCCATGTGGTTACC-3', nt15231~nt15251), seventh exon reverse sequencing primer 3DL2_SBT_Ex7_R (sequence: 5'-GGAACACACCCCCGTGC-3', nt15494~ Nt15512);

KIR3DL2 eighth exon forward sequencing primer 3DL2_SBT_Ex8_F (sequence: 5'-TCTGAGTCTGGATGTTGGC-3', nt15764~nt15782), eighth exon reverse sequencing primer 3DL2_SBT_Ex8_R (sequence: 5'-GGGTCTTGTTCATCAGAGTCC-3', nt16046~ Nt16066);

KIR3DL2 ninth exon forward sequencing primer 3DL2_SBT_Ex9_F (sequence: 5'-CCTCGGCCCAGCCTCACGG-3', nt15957-nt15975), ninth exon reverse sequencing primer 3DL2_SBT_Ex9_R (sequence: 5'-GACTGTGGTGCTCGTGGGC-3', nt16216~ Nt16234).

(13) KIR3DL3 first exon forward sequencing primer 3DL3_SBT_Ex1_F (sequence: 5'-ACAACATCCTGTGTGCTGCTGAA-3', nt-63-nt-41), first exon reverse sequencing primer 3DL3_SBT_Ex1_R (sequence: 5'- TCCCTCCCTCGATTCCCTT-3', nt46~nt64);

KIR3DL3 second exon forward sequencing primer 3DL3_SBT_Ex2_F (sequence: 5'-GATGTACAGATGGATCATC-3', nt672 to nt690), second exon reverse sequencing primer 3DL3_SBT_Ex2_R (sequence: 5'-GTCAACCCCCTGTGTCGCCTG-3', nt815 to nt835);

KIR3DL3 third exon forward sequencing primer 3DL3_SBT_Ex3_F (sequence: 5'-GCTCCACATCCTCCTCTCT-3', nt1474~nt1492), third exon reverse sequencing primer 3DL3_SBT_Ex3_R (sequence: 5'-ATCCCCCTTTACCCCAAAT-3', nt1905~ Nt1923);

KIR3DL3 fourth exon forward sequencing primer 3DL3_SBT_Ex4_F (sequence: 5'-GGGAAGCCTCACTTATTTCAG-3', nt2996~nt3016), fourth exon reverse sequencing primer 3DL3_SBT_Ex4_R (sequence: 5'-ACCTGGGGCTTCCAGTCCT-3', nt3431~ Nt3449);

KIR3DL3 fifth exon forward sequencing primer 3DL3_SBT_Ex5_F (sequence: 5'-GAGAGCTGTGACAASGAAG-3', nt4900~nt4918), fifth exon reverse sequencing primer 3DL3_SBT_Ex5_R (sequence: 5'-GCAGGAAGCTCCTCAGCTA-3', nt5294~ Nt5312);

KIR3DL3 seventh exon forward sequencing primer 3DL3_SBT_Ex7_F (sequence: 5'-GTGAGACAATTCATATAGA-3', nt10650~nt10668), seventh exon reverse sequencing primer 3DL3_SBT_Ex7_R (sequence: 5'-TGCTTCCCCACATGGCCCT-3', nt10852~ Nt10870);

KIR3DL3 eighth exon forward sequencing primer 3DL3_SBT_Ex8_F (sequence: 5'-GACCTCAGGCACCTATGGC-3', nt11178~nt11196), eighth exon reverse sequencing primer 3DL3_SBT_Ex8_R (sequence: 5'-GAGTGAGGGAGGGTGCTCA-3', nt11395~ Nt11413);

KIR3DL3 ninth exon forward sequencing primer 3DL3_SBT_Ex9_F (sequence: 5'-CRTGGCTAGTCTTATTCCC-3', nt11358~nt11376), ninth exon reverse sequencing primer 3DL3_SBT_Ex9_R (sequence: 5'-CCCTAGAAGATCCCATCAA-3', nt11627~ Nt11645).

(14) KIR3DS1 first exon forward sequencing primer 3DS1_SBT_Ex1_F (sequence: 5'-AAGCCATGCTCCGCTCTTG-3', nt-181 to nt-163), first exon reverse sequencing primer 3DS1_SBT_Ex1_R (sequence: 5'-CAGATCTCCATCCCCGCAC-3', nt65 to nt83);

KIR3DS1 second exon forward sequencing primer 3DS1_SBT_Ex2_F (sequence: 5'-AGTGGGGGCAGCAGGGTG-3', nt968~nt985), second exon reverse sequencing primer 3DS1_SBT_Ex2_R (sequence: 5'-AATGTGGGCCGAGCATCCG-3', nt1182~ Nt1200);

KIR3DS1 third exon forward sequencing primer 3DS1_SBT_Ex3_F (sequence: 5'-GGGGAGAATCTTCTGGGCACT-3', nt1735~nt1755), third exon reverse sequencing primer 3DS1_SBT_Ex3_R (sequence: 5'-TGATGGGACCCTGACGGAC-3', nt2166~ Nt2184);

KIR3DS1 fourth exon forward sequencing primer 3DS1_SBT_Ex4_F (sequence: 5'-GGAGAGAGACAGACACGGG-3', nt3485~nt3503), fourth exon reverse sequencing primer 3DS1_SBT_Ex4_R (sequence: 5'-TGGTACAGACCTCACCAAG-3', nt4007~ Nt4025);

KIR3DS1 fifth exon forward sequencing primer 3DS1_SBT_Ex5_F (sequence: 5'-CAGGTGTGAGGGGAGCTGT-3', nt5403~nt5421), fifth exon reverse sequencing primer 3DS1_SBT_Ex5_R (sequence: 5'-CCTGTCTGCCATCCTGCGC-3', nt5892~ Nt5910);

KIR3DS1 sixth exon forward sequencing primer 3DS1_SBT_Ex6_F (sequence: 5'-TCAAGACAGTGGGCATCGCAC-3', nt8763~nt8783), sixth exon reverse sequencing primer 3DS1_SBT_Ex6_R (sequence: 5'-GGGAGGTTTGAGCCAACGCTT-3', nt9045~ Nt9065);

KIR3DS1 seventh exon forward sequencing primer 3DS1_SBT_Ex7_F (sequence: 5'-CGCTGTATGTGGTTACCTGTG-3', nt13165~nt13185), seventh exon reverse sequencing primer 3DS1_SBT_Ex7_R (sequence: 5'-GGTGAGGAACACACCCG-3', nt13432~ Nt13450);

KIR3DS1 eighth exon forward sequencing primer 3DS1_SBT_Ex8_F (sequence: 5'-CCAGAAGTGCCCTCCGAGC-3', nt13784~nt13802), eighth exon reverse sequencing primer 3DS1_SBT_Ex8_R (sequence: 5'-GCTGAGTGAGGGAGGGTGC-3', nt13944~ Nt13962);

KIR3DS1 ninth exon forward sequencing primer 3DS1_SBT_Ex9_F (sequence: 5'-CGTGGCTAGTGTTATTCCC-3', nt13904 to nt13922), ninth exon reverse sequencing primer 3DS1_SBT_Ex9_R (sequence: 5'-GGCCTCTGAGAAGGGCGAG-3', nt14055 to nt14073).

8. The sequencing reaction composition is:

9. The cycle parameters of the sequencing reaction are:

The invention develops a scientific and efficient KIR gene PCR amplification strategy based on the structural characteristics of the full-length sequence of the KIR gene, the polymorphism distribution of SNPs and the length of the intron of the exon. By designing KIR gene-specific PCR primers and sequencing primers, exploring optimal PCR amplification and sequencing reaction conditions, and researching and establishing all 14 functional KIR genes in the KIR gene family, simultaneous sequencing and typing techniques are suitable for KIR high resolution. Horizontal genotyping, population genetics, bone marrow transplantation tissue matching, disease association research and other fields.

The contribution of the present invention is that for the first time, a method for simultaneous sequencing of 14 functional KIR genes suitable for high-pass quantification and high resolution levels has been established. According to the structural characteristics of KIR gene, a scientific PCR amplification strategy was developed, and KIR gene-specific PCR primers with similar annealing temperatures were designed, which can be simultaneously amplified under the same PCR amplification conditions, and the time required is short. Time and effort; due to the specificity of PCR primers, the cause was solved Non-specific amplification problems with highly homologous KIR sequences. The amplification product can directly identify the presence or absence of the KIR gene by electrophoresis, and does not need to be detected by a commercial KIR-SSP kit. This method can sequence all the exons of each functional KIR gene, solve the deficiencies in the KIR sequencing typing method reported by previous researchers, and the obtained sequence has no background signal and peak, which is easy. The identification and interpretation of the results laid a good foundation for the next commercialization and industrialization.

Table 3 Structure and exon length of 14 functional KIR gene coding regions

Del: missing exon; Pseudoexon: false exon.

DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the description of the prior art will be briefly described below. Obviously, the drawings in the following description are only It is a certain embodiment of the present invention, and those skilled in the art can obtain other drawings according to the structures shown in the drawings without any creative work.

Figure 1 is a strategic map of 14 functional KIR gene simultaneous sequencing typing techniques;

The design uses 3 to 5 pairs of KIR gene-specific PCR primers (except KIR3DL3, which uses only 3 pairs of PCR primers for deletion of exon 6 and 5 pairs of PCR primers for KIR2DL1, and each of the functional KIR genes uses 4 pairs of PCR. Primers), specific PCR amplification of the entire coding region of each functional KIR gene. Purified PCR amplification products were passed through sixteen (KIR2DL1~5, 2DS1~5 and KIR3DL3) or eighteen (KIR3DL1~2 and KIR3DS1) specific forward and reverse sequencing primers for each exon. The sequence was subjected to a two-way sequencing reaction.

Fig. 2 is a diagram showing the effect of agarose gel electrophoresis of PCR amplification products of the KIRAA1 gene combination type sample of Example 1. M: DL2000 Marker; A1: Amplicon 1, amplifying the first exon to the second exon gene fragment; A2: Amplicon 2, amplifying the third exon of 3DL1~3, 3DS1, 2DL4~5 to A gene fragment of the exon 5 and a gene fragment of the exon 5 to exon 5 of KIR2DL1 to 3 and KIR2DS1 to 5. In particular, KIR2DL1 uses two pairs of PCR primers to amplify the fourth exon (Amplicon 2-1) and the fifth exon (Amplicon 2-2); A3: Amplicon 3, amplify the gene of exon 6 Fragment; A4: Amplicon 4, amplifying a gene fragment from exon 7 to exon 9;

Figures 3-1 to 3-7 respectively correspond to the KIR2DL1, 2DL3, 2DL4, 2DS4, 3DL1, 3DL2, and 3DL3 full coding region sequencing sequences of the KIRAA1 gene combination sample of Example 1 and are introduced into Assign 3.5 or 4.7 analysis software renderings. The KIR genotype of the sample is KIR2DL1*00302-2DL3*00101-2DL4*00102, 011-2DS4*00101, 010-3DL1*00501, 01502-3DL2*00201, 010-3DL3*00901, 010;

Fig. 4 is a graph showing the effect of agarose gel electrophoresis of PCR amplification products of the KIRAB6 gene combination type sample of Example 2. M: DL2000 Marker; A1: Amplicon 1, amplifying the first exon to the second Gene fragment of exon; A2: Amplicon 2, amplifying the 3rd exon to 3rd exon of 3DL1~3, 3DS1, 2DL4~5 and the 4th exon of KIR2DL1~3 and KIR2DS1~5 Gene fragment of the target to 5 exons. In particular, KIR2DL1 uses two pairs of PCR primers to amplify the fourth exon (Amplicon 2-1) and the fifth exon (Amplicon 2-2); A3: Amplicon 3, amplify the gene of exon 6 Fragment; A4: Amplicon 4, amplifying a gene fragment from exon 7 to exon 9;

Figure 5-1 to Figure 5-14 show the effect of the sequence of 14 functional KIR gene coding regions of the KIRAB6 sample of Example 2 into the analysis software of Assign 3.5 or 4.7. The KIR genotype of the sample is "KIR2DL1*00302,00401 -2DL2*00301-2DL3*00101-2DL4*00102,00501-2DL5A*00101,B*010-2DS1*00201-2DS2*00101-2DS3*00101-2DS4*00101-2DS5*00201-3DL1*01502-3DL2*00201 , 00701-3DL3*01002-3DS1*01301". The KIR2DS4*001 homozygous sample was sequenced and the results showed that the 5th exon of the 2DS4*001 allele lacked 22bp. After the sequence was introduced, the 5th exon was forward sequenced in the coding region nt454 and downstream. Base misalignment (Figure 5-9A), reverse sequencing also has no base misalignment at nt475 and upstream (Figure 5-9B).

The implementation, functional features, and advantages of the present invention will be further described in conjunction with the embodiments.

detailed description

Example 1

In this example, a sample of a KIRAA1 gene combination (KIR2DL1-2DL3-2DL4-2DS4-3DL1-3DL2-3DL3) detected by a commercial KIR-SSP kit was randomly selected, and 14 functional KIRs were performed on the sample using the present invention. The genes were simultaneously sequenced to verify the effects of the present invention.

First, KIR gene-specific PCR amplification primers were used for PCR amplification of all coding regions of each functional KIR gene. The amplification reaction was carried out on an ABI 9700 PCR machine. The amplification reaction system consisted of:

The PCR amplification reaction can be synchronously amplified under the same cycle parameters, and the cycle parameters are:

Take 2 μL of PCR product, stain with nucleic acid dye, electrophoresis on 2% agarose gel, compare the label of Takara DL2000 DNA, observe the specific PCR amplification product band in the gel imaging system, and clear the PCR product The specificity of the gene can be clearly interpreted as "KIR2DL1-2DL3-2DL4-2DS4-3DL1-3DL2-3DL3", and its electrophoresis effect is shown in Fig. 2.

The electrophoresis results showed that specific bands appeared in each of the seven functional KIR genes, such as KIR2DL1, 2DL3, 2DL4, 2DS4, 3DL1, 3DL2 and 3DL3, and the remaining KIR2DL2, 2DL5, 2DS1, 2DS2, 2DS3, 2DS5, 3DS1, etc. 7 Each of the functional KIR genes has no bands in each lane and conforms to the KIRAA1 gene combination.

The above seven KIR genes of the specific PCR amplification bands were detected by electrophoresis, and the PCR amplification products were further purified. The purification reaction system consisted of:

The purification reaction of the PCR amplification product can be purified under the same cycle parameter conditions, The loop parameters are:

37 ° C 45 min,

85 ° C 15 min,

4°C Infinite.

After the end of the purification reaction, the PCR product was diluted with 3 volumes of sterile deionized water and mixed well.

The PCR products were electrophoretically detected for each KIR gene of the specific amplified band, and were specifically forward and reverse sequenced by sixteen (KIR2DL1, 2DL3, 2DL4, 2DS4, and KIR3DL3) or eighteen (KIR3DL1~2). Primers, two-way sequencing reactions of purified and diluted amplification products, each sequencing reaction system is as follows:

The cycle parameters of the sequencing reaction are:

After the sequencing reaction, the sequencing product was purified by ethanol/NaOAc/EDTA precipitation method, and finally 15 μL of ultra-pure formamide solution (Hi-Di Formamide) was added and denatured at 95 ° C for 2.5 min on a PCR instrument. The purified sequencing product was detected by capillary electrophoresis on an ABI 3730 gene sequencer and the sequence data information after electrophoresis was collected. All the sequences measured in this sample were introduced into Assign 3.5 or 4.7 (Conexio Genomics, Western Australia) analysis software (Fig. 3), and the KIR genotype of the subject was clearly determined as "KIR2DL1*00302-KIR2DL3*00101-KIR2DL4 *00102,011-KIR2DS4*00101,0 10-KIR3DL1*00501, 01502-KIR3DL2*00201, 010-KIR3DL3*00901, 010".

Figure 3-4 shows the KIR2DS4*00101, 010 heterozygous sample sequencing results: 22 bp deletion in exon 5 of 2DS4*010 allele, and 22 bp deletion in 2DS4*00101, result 5 exon Forward sequencing occurred base misalignment at the nt454 and downstream positions of the coding region (Fig. 3-4A), and reverse sequencing revealed base misalignment at nt475 and upstream positions (Fig. 3-4B).

Example 2

In this example, a sample of a KIRAA6 gene combination (ie, carrying all 14 functional KIR genes) detected by a commercial KIR-SSP kit was randomly selected, and 14 functional KIRs were performed on the sample using the present invention. The genes were simultaneously sequenced to verify the effects of the present invention.

First, KIR gene-specific PCR amplification primers were used for PCR amplification of all coding regions of each functional KIR gene. The amplification reaction was carried out on an ABI 9700 PCR machine. The amplification reaction system consisted of:

The PCR amplification reaction can be synchronously amplified under the same cycle parameters, and the cycle parameters are:

Take 2 μL of PCR product, stain with nucleic acid dye, electrophoresis on 2% agarose gel, compare the label of Takara DL2000 DNA, observe the specific PCR amplification product band in the gel imaging system, and clear the PCR product The specificity of the gene can be clearly interpreted as "KIR2DL1-2DL2-2DL3-2DL4-2DL5-2DS1-2DS2-2DS3-2DS4-2DS5-3DL1-3DL2-3DL3-3DS1", and its electrophoresis effect is shown in Fig. 4.

Electrophoresis results showed that specific bands were detected in each of the 14 functional KIR genes including KIR2DL1, KIR2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, KIR2DS5, 3DL1, 3DL2, 3DL3 and 3DS1, in line with KIRAB6. Gene combination type.

All 14 functional KIR genes detected by electrophoresis were further purified by PCR amplification products. The purification reaction system consisted of:

The purification reaction of the PCR amplification product can be purified under the same cycle parameters, and the cycle parameters are:

37 ° C 45 min,

85 ° C 15 min,

4°C Infinite.

After the end of the purification reaction, the PCR product was diluted with 3 volumes of sterile deionized water and mixed well.

The PCR products were electrophoretically detected for each KIR gene of the specific amplified band, and were specifically forward and reverse by sixteen (KIR2DL1~5, 2DS1~5 and KIR3DL3) or eighteen (KIR3DL1~2 and KIR3DS1). The sequencing primers are subjected to a two-way sequencing reaction on the purified and diluted amplification products, and each sequencing reaction system is as follows:

The cycle parameters of the sequencing reaction are:

After the sequencing reaction, the sequencing product was purified by ethanol/NaOAc/EDTA precipitation method, and finally 15 μL of ultra-pure formamide solution (Hi-Di Formamide) was added and denatured at 95 ° C for 2.5 min on a PCR instrument. The purified sequencing product was detected by capillary electrophoresis on an ABI 3730 gene sequencer and the sequence data information after electrophoresis was collected.

All the sequences measured in this sample were introduced into the Assign 4.7 SBT (Conexio Genomics, Western Australia) software (Figs. 5-1 to 5-14), and the KIR genotype of the subject was clearly determined as "KIR2DL1*00302, 00401". -KIR2DL2*00301-KIR2DL3*00101-KIR2DL4*00102,00501-KIR2DL5A*00101,B*010-KIR2DS1*00201-KIR2DS2*00101-KIR2DS3*00101-KIR2DS4*00101-KIR2DS5*00201-KIR3DL1*01502-KIR3DL2*00201 , 00701-KIR3DL3*01002-KIR3DS1*01301".

Figure 5-9 shows the KIR2DS4*00101 homozygous sample sequencing results: the 5th exon of the 2DS4*00101 allele has no 22bp deletion. After the sequence is introduced, the 5th exon can be forward sequenced in the coding region nt454 and There was no base mislocation at the downstream position (Fig. 5-9A), and reverse sequencing also showed no base misalignment at nt475 and upstream (Fig. 5-9B).

Example 3

This example gives an example of a population genetic survey using 14 functional KIR genes for simultaneous sequencing of 306 southern Han nationality health-independent individuals using the present invention.

For each sample, KIR gene-specific PCR amplification primers were used for PCR amplification of all coding regions of each functional KIR gene. The amplification reaction was carried out on an ABI 9700 PCR machine. The amplification reaction system consisted of:

The PCR amplification reaction can be synchronously amplified under the same cycle parameters, and the cycle parameters are:

Take 2 μL of PCR product, stain with nucleic acid dye, electrophoresis on 2% agarose gel, compare the label of Takara DL2000 DNA, observe the specific PCR amplification product band in the gel imaging system, and clear the PCR product Specific, can clearly determine the presence or absence of each sample KIR gene.

Each KIR gene of the specific band was detected by agarose gel electrophoresis, and the PCR amplification product was further purified. The purification reaction system consisted of:

The purification reaction of the PCR amplification product can be purified under the same cycle parameters, and the cycle parameters are:

37 ° C 45 min,

85 ° C 15 min,

4°C Infinite.

After the end of the purification reaction, the PCR product was diluted with 3 volumes of sterile deionized water and mixed well.

The PCR products were electrophoretically detected for each KIR gene of the specific amplified band, and were specifically forward and reverse by sixteen (KIR2DL1~5, 2DS1~5 and KIR3DL3) or eighteen (KIR3DL1~2 and KIR3DS1). The sequencing primers are subjected to a two-way sequencing reaction on the purified and diluted amplification products, and each sequencing reaction system is as follows:

The cycle parameters of the sequencing reaction are:

After the sequencing reaction, the sequencing product was purified by ethanol/NaOAc/EDTA precipitation method, and finally 15 μL of ultra-pure formamide solution (Hi-Di Formamide) was added and denatured at 95 ° C for 2.5 min on a PCR instrument. The purified sequencing product was detected by capillary electrophoresis on an ABI 3730 gene sequencer and the sequence data information after electrophoresis was collected.

All the sequences measured in each sample were imported into Assign 3.5 or 4.7 (Conexio Genomics, Western Australia) analysis software to clearly identify alleles detected in all 14 functional KIR genes (see Table 6). The frequency of detection of each allele was shown (see Table 7).

The above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the invention, and the equivalent structural transformation, or direct/indirect use, of the present invention and the contents of the drawings are used in the inventive concept of the present invention. It is included in the scope of the patent protection of the present invention in other related technical fields.

references

[1] Vierra-Green C, Roe D, Hou L, et al. Allele-Level Haplotype Frequencies and Pairwise Linkage Disequilibrium for 14 KIR Loci in 506 European-American Individuals. PLoS One, 2012, 7(11): e47491.

[2] Robinson J, Mistry K, McWilliam H, Lopez R, Marsh SGE. IPD-the immuno polymorphism database. Nucleic Acids Res. 2010, 38: D863-869.

[3] McErlean C, Gonzalez AA, Cunningham R, et al. Differential RNA expression of KIR alleles. Immunogenetics, 2010, 62(7): 431-440.

[4] Yawata M, Yawata N, Draghi M, et al. Roles for HLA and KIR polymorphisms in natural killer cell repertoire selection and modulation of effector function. J Exp Med, 2006, 203(3): 633-645.

[5] Pando MJ, Gardiner CM, Gleimer M, et al. The protein made from a common allele of KIR3DL1(3DL1*004) is poorly expressed at cell surfaces due to substitution at positions 86 in Ig domain 0 and 182 in Ig domain 1. J Immunol, 2003, 171 (12): 6640-6649.

[6] Bao X, Hou L, Sun A, et al. Distribution of killer cell immunoglobulin-like receptor genes and 2DS4 alleles in the Chinese Han population. Hum Immunol, 2010, 71(3): 289-292.

[7] Lebedeva TV, Ohashi M, Zannelli G, et al. Comprehensive approach to high-resolution KIR typing. Hum Immunol, 2007, 68(9): 789-796.

[8]See comment in PubMed Commons below Yan LX,Zhu FM,Jiang K,et al.Differential of the killer cell immunoglobulin-like receptor gene KIR2DS4 in the Chinese population.Tissue Antigens,2007,69(2):133-138 .

[9] Buhler S, Di Cristofaro J, Frassati C, et al.igh levels of molecular polymorphism at the KIR2DL4 locus in French and Congolese populations: impact for anthropology and clinical studies. Hum Immunol. 2009, 70(11): 953- 959.

[10] Belle I, Hou L, Chen M, et al. Investigation of killer cell immunogloublin-like receptor gene diversity in KIR3DL1 and KIR3DS1 in a transplant population, Tissue Antigens. 2008, 71(5): 434-439.

[11] Hou L, Chen M, Steiner N, et al. Killer cell immunoglobulin-like receptors (KIR) typing by DNA sequencing. Methods Mol Biol, 2012, 882: 431-468.

[12] Meenagh A, Gonzalez A, Sleator C, et al. Investigation of killer cell immunoglobulin-like receptor gene diversity, KIR2DL1 and KIR2DS1. Tissue Antigens. 2008, 72(4): 383-391.

[13] Research progress of molecular genetic polymorphism and sequencing typing technology of KIR. Chinese Journal of Medical Genetics. 2016, 33(6): 867-870.

Claims (8)

A method for simultaneous sequencing of 14 functional killer cell immunoglobulin-like receptor KIRs genes, characterized in that the structure of the full-length sequence of the genome of each KIR gene, the distribution of single nucleotide polymorphisms in the coding region, and the The length of the introns of the two wings of the phenotype is determined by a scientific PCR amplification strategy. The entire coding region of each functional KIR gene is subjected to PCR synchronization using 3 to 5 pairs of KIR gene-specific PCR primers with similar annealing temperatures. Amplification; for each exon contained in the purified PCR amplification product, forward and reverse bidirectional sequencing, respectively, as shown in FIG. The method according to claim 1, wherein said KIR gene-specific PCR amplification primer, except KIR3DL3, which uses only 3 pairs of PCR primers for deletion of exon 6 and 5 pairs of PCR primers for KIR2DL1, Four pairs of PCR primers were used for the functional KIR gene; a total of 56 pairs of PCR primers, 112; the sequence of each PCR primer, the position in the full length sequence of the KIR genome, and the length of the amplified fragment, such as Table 1 is described. The method of claim 1 wherein the system composition of the PCR amplification reaction is:

The method of claim 1 wherein the PCR amplification conditions are:

The method of claim 1 wherein the system composition used for purification of each 10 μL of PCR amplification product is:

The method according to claim 1, wherein the purification system of the PCR amplification product is subjected to a purification reaction under the same cycle parameter, and the cycle parameters are: 37 ° C 45 min, 85 ° C 15 min, 4°C Infinite. The method according to claim 1, wherein each of the exons contained in the purified PCR product is subjected to a sequencing reaction, and a total of 230 functional KIR gene specific forward and reverse sequencing primers are used. The sequence of each sequencing primer and its position in the full length sequence of the KIR genome are as described in Table 2. The method of claim 7 wherein the sequencing reaction components are:

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