Patent Application
20180305744
The present application claims priority to Chinese Patent Application No. 201710284545.3 filed on Apr. 25, 2017, the entire contents of which are incorporated herein by reference.
The Sequence Listing is submitted concurrently with the specification as an ASCII formatted text file via EFS-Web, with a file name of “Sequence listing.txt”, a creation date of Sep. 4, 2017, and a size of 61,660 bytes. The Sequence Listing filed via EFS-Web is part of the specification and is incorporated in its entirety by reference herein.
The present disclosure is involved in biomedicine, and particularly gets involved in DNA sequence-based typing (SBT). The present disclosure provides a method for high-throughput simultaneous sequence-based typing of all the 14 functional killer cell immunoglobulin-like receptor (KIR) genes.
Killer cell immunoglobulin-like receptors (KIRs) belong to immunoglobulin superfamily and are expressed on both natural killer (NK) cells and a subset of T cells. Based on the number of extra-cellular domains, KIR genes are classified as KIR2D and KIR3D. Depending on the length of the cytoplasmic tail and the presence or absence of immunoreceptor tyrosine-based inhibitory motif (ITIM), KIRs can be functionally divided into inhibitory KIR (iKIR) and activating KIR (aKIR). KIR receptors regulate NK cell activities and convey activating or inhibitory signal through interaction with class I human leukocyte antigen (HLA) ligands, which play an important role in transplantation, elimination of tumor cells and resistance to viral infection through innate immune pathways.
The KIR gene cluster on human chromosome 19 consists of 14 functional KIR genes (KIR2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1) and two pseudogenes (KIR2DP1 and 3DP1) (1). The structure of KIR genes is extremely complicated. Except that KIR3DL3 lacks exon 6, other functional KIR3D genes including 3DL1, 3DL2, and 3DS1 possess 9 exons, 8 introns, a 5′-promoter region and a 3′-untranslated region (3′-UTR). Exons 1 and 2 encode leader peptide. Exons 3, 4 and 5 encode the extra-cellular domains D0, D1 and D2, respectively. Exon 6 encodes the stem. Exon 7 encodes the transmembrane region. Exons 8 and 9 encode the cytoplasmic region. Among the KIR2D genes, KIR2DL1-3 and 2DS1˜5 have an untranslated pseudoexon 3, which results in the absence of the corresponding extra-cellular domain D0. KIR2DL4 and 2DL5 are characterized by the complete absence of exon 4, and therefore their protein product has no extra-cellular domain D1.
The full genomic sequences for all the KIR alleles released in the IPD-KIR database varies from 9901 bp to 17009 bp in size (2). However, the coding sequences (CDS) of each functional KIR gene has a total length of only 915˜1368 bp (see Table 3), which imply that the non-coding sequence (8773˜15641 bp) accounts for the majority of full genomic sequences of KIR gene (see Table 4). Particularly, the length of introns 5 and 6 accounts for 44.0%˜61.2% of the full genomic sequence of corresponding KIR gene (also see Table 4).
Both exon 1 (either 34 or 40 bp) and exon 2 (36 bp) of each functional KIR gene are short in length and have limited single nucleotide polymorphism sites (SNPs). KIR2DL2, 2DL4 and 2DS4 lack SNPs in both exons 1 and 2, whereas other functional KIR genes possess 1-3 SNPs, respectively. Thus, routine sequence-based typing at exons 1 and 2 is not required for each KIR gene. In addition, intron 1, which is located between exon 1 and exon 2, is 199˜2280 bp in length. Polymerase chain reaction (PCR) amplificon covering the entire exon 1, exon 2 and the intervening intron 1 will be moderate in length for each KIR gene and can be amplified effectively.
Exon 3, exon 4 or exon 5 of each functional KIR gene is relatively long in length (282˜300 bp) and has much SNPs. Since pseudoexon 3 for KIR2DL1˜3 and 2DS1˜5 doesn't required to be detected, PCR amplification covering the entire exon 4, intron 4 and exon 5 can be achieved in a single amplicon using one pair of KIR gene-specific PCR primers, and then sequencing of exons 4 and 5 needs to be performed separately in both directions for these 8 functional KIR genes. Likewise, both KIR2DL4 and 2DL5 miss exon 4, PCR amplication covering the entire exon 3, intron 3/4 and exon 5 can be achieved in a single amplicon using one pair of KIR gene-specific PCR primers and then sequencing of exons 3 and 5 needs to be performed separately in both directions. As for the other four functional KIR genes (KIR3DS1, 3DL1˜3), which all possess exons 3, 4 and 5, the PCR amplication covering the entire exon 3, intron 3, exon 4, intron 4 and exon 5 can be achieved in a single amplicon using one pair of KIR gene-specific primers and then the sequencing of exons 3, 4 and 5 needs to be performed separately in both directions.
Apart from KIR3DL3 without exon 6, the exon 6 of all the other functional KIR genes is only 51 bp in length. According to the IPD-KIR Database (Release 2.6.0), KIR2DS4, 3DL1, 3DL2, and 3DS1 genes lack SNPs in exon 6, other functional KIR genes possess 1˜2 SNPs. The distribution of SNPs located in exon 6 of each KIR gene is limited. However, the flanking intronic sequences of exon 6 which include introns 5 and 6 have a total length of up to 4937˜9841 bp (see Table 4). To ensure the effective PCR amplication, the entire intronic sequences of intron 5 and/or intron 6 should be avoided in case of generating extreme long PCR amplicon. Thus, the target sequence of exon 6 for each KIR gene should be amplified separately in a single amplicon if necessary.
The length for exons 7, 8 and 9 of each functional KIR gene is 102˜105 bp, 51˜53 bp and 8˜270 bp, respectively. The length for introns 7 and 8 is 460˜462 bp and 98˜118 bp, respectively. Therefore, PCR amplification covering entire exon 7, intron 7, exon 8, intron 8, and exon 9 can be performed in a single amplicon and will not generate ultra-long PCR fragment.
The structural features of full genomic sequence for all functional KIR genes, the characteristic of SNPs distribution in coding sequence and the length of each exon and its flanking intronic sequence that we have mentioned above, are critical to develop a scientific and efficient PCR amplification strategy.
KIR genes exhibit extensive diversity in both haplotype content and allelic diversification. So far 698 KIR alleles including 7 null alleles have been released in the IPD-KIR Database (Release 2.6.0). Among the 14 functional KIR genes, KIR3DL2 exhibits the highest level of allelic diversity with 112 different identified alleles (see Table 5).
Identification of KIR alleles can carry functional significance. McErlean et al. (3) have found that mRNA expression level for the 14 functional KIR genes varies with the hierarchy KIR3DL2>KIR2DS2>KIR3DS1>KIR2DS5>KIR2DL5>KIR2DS3>KIR2DL1>KIR3DL1>KIR2DS1>KIR2DL2>KIR2DL4>KIR2DS4>KIR2DL3. Even within the same KIR gene, the expression level on NK cell surface, the affinity to cognate ligand and the capacity of medicated inhibition or activation can be influenced by different allele. It has been reported by Yawata et al. (4) that the expression levels of KIR3DL1 alleles were in the order of KIR3DL1*01502>*020>*001>*007>*005, whereas the levels of 3DL1-mediated NK cell inhibition were in the order of KIR3DL1*001>*005>*01502>*020>*007. KIR3DL1*005 combines low cell surface expression with a high inhibitory capacity. KIR3DL1*004, a most common KIR3DL1 allele in Caucasians, is poorly expressed at the cell surface (5). KIR2DS4*003, *004, *006, *007, *008, *009, *010, *012, and *013 alleles have a 22 bp deletion at coding sequence (CDS) nucleotide position nt454˜nt475 in exon 5, which causes a reading frame shift, yielding a truncated KIR2DS4 protein with loss of the transmembrane and cytoplasmic domains. These deleted variants of KIR2DS4 protein can't be anchored to the cell surface (6). Thus, it is critical important to identify allelic variation within the 14 functional KIR genes, especially those common null alleles. Since KIR allelic variation alters the level of protein expression and the affinity for cognate ligand as well as the mediated inhibitory/activating capacity, it is an urgent task to develop a low-cost, high-throughput, simultaneous sequence-based typing (SBT) method and apply the established SBT method in KIR-associated disease studies.
To date, the widely-used polymerase chain reaction-sequence specific primer (PCR-SSP) and PCR-sequence specific oligonucleotide probe (PCR-SSOP) commercial kits can only identify the presence or absence of KIR genes on low-resolution level, but can not identify all the KIR alleles at the allele level, especially for those null alleles.
Sequence-based typing is a powerful technique for KIR genotyping at allele level. However, there are no commercial KIR SBT kit and corresponding software for KIR allele assignment in worldwide until now. As KIR genes share extensive sequence homology, it is difficult to design KIR gene-specific primers for PCR amplification of target sequences. While summarizing the characteristic of the KIR SBT methods in the previously published literatures, several problems existed in: {circle around (1)} Only exons encoding extra-cellular domains and/or cytoplasmic region were sequenced for some KIR genes (7, 8, 9). Since the entire coding sequence was not sequenced and the diversity of each exon could not be allowed for analyzing, which led to being prone to generate ambiguous allele combination in SBT test. {circle around (2)} The PCR amplicons could cover the entire coding sequence of each KIR gene, however, the fragments size of PCR amplicon were extremely too long. For example, the KIR3DS1 amplicon covering exon 3 through 3′ untranslated region (3′-UTR) generated a fragment of approximate 12.2 kb in length, and PCR extension at 68V required up to 13 min in each cycle (10), as a result the total PCR amplification time exceeded 10 hours, more high requirement for DNA quality as well as the high-fidelity DNA polymerase were needed in PCR amplification. {circle around (3)} As KIR genes share extensive sequence homology, non-specific amplification or co-amplification occurred in PCR procedure. e.g., while amplifying the target sequence covering exon 1 through exon 5 of 2DL1 in a subject carrying both 2DL1 and 2DS1 genes, 2DS1 would also be co-amplified (11). To obtain 2DL1 specific PCR products, the secondary amplification needed to be carried out using nested PCR primers, which made the PCR procedure cumbersome. {circle around (4)} Due to the different annealing temperatures for PCR primers and varied extension time in each PCR cycling, PCR amplifications could not be carried out simultaneously under the same thermocycling parameters while amplifying the target sequences of 14 functional KIR genes (10, 11, 12, 13), which made PCR procedure more time-consuming and labour-consuming. {circle around (5)} Identification of the KIR alleles with one or more base pair insertion/deletion by traditional cloning and sequencing could not allow for the desired rapidity and simplicity in routine KIR genotyping.
With the elucidation of biological functions for KIR molecules, the clinical significance of the increasingly recognized KIR polymorphism and its role played in transplantation and disease associated studies have drawn extensive interest. Therefore, establishment of the method for high-throughput simultaneous sequence-based typing of 14 functional KIR genes, together with its commercialization and industrialization are currently urgent problems to be solved.
The present disclosure aims to solve the problems mentioned above and for the first time provide a simultaneous sequence-based typing (SBT) method for all the 14 functional KIR genes. The established KIR SBT method can be widely-used in population genetics, tissue typing for bone marrow transplantation, disease-associated studies, and also lays the foundation for the commercialization of KIR SBT reagents, which change the current status that no available commercial KIR SBT reagents meet the marketing.
In order to achieve the above objective, the present disclosure adopts the following technical strategy:
I. Based on the structural features of KIR full genomic sequences, the distribution of single nucleotide polymorphisms in their coding regions and the length of flanking intronic sequence of each exon, a scientific and reasonable PCR amplification strategy has been developed in the present disclosure. The complete coding sequence of each functional KIR gene is simultaneously amplified under the same thermocycling parameters using 3˜5 pairs of KIR gene-specific PCR primers that have similar annealing temperature. The nucleotide sequences of the exons carried by each PCR amplicon were determined in both directions using the specific forward and reverse sequencing primers, respectively, as shown in
The coding sequence of KIR2DL1 is amplified using 5 pairs of KIR2DL1 gene-specific PCR primers. In particular, the first pair of PCR primers is used to amplify the target sequence of KIR2DL1 covering exon 1, intron 1, exon 2, partial sequences of 5′-promoter region and intron 2/3; the second pair of PCR primers is used to amplify the target sequence of KIR2DL1 covering exon 4 and its partial flanking intronic sequences; the third pair of PCR primers is used to amplify the target sequence of KIR2DL1 covering exon 5 and its partial flanking intronic sequences; the fourth pair of PCR primers is used to amplify the target sequence of KIR2DL1 covering exon 6 and partial flanking intronic sequences; the fifth pair of PCR primers is used to amplify the target sequence of KIR2DL1 covering exon 7, intron 7, exon 8, intron 8, exon 9, partial sequences of intron 6 and 3′-UTR region.
The coding sequences of KIR2DL2, 2DL3, 2DS1, 2DS2, 2DS3, 2DS4 and 2DS5 are amplified using 4 pairs of KIR gene-specific PCR primers, respectively. The first pair of PCR primers is used to amplify the target sequence of each KIR gene mentioned above covering exon 1, intron 1, exon 2, partial sequences of 5′-promoter region and intron 2/3 (since exon 3 is a pseudoexon, the sequence between exon 2 and exon 4 is referred as intron 2/3); the second pair of PCR primers is used to amplify the target sequence of each KIR gene mentioned above covering exon 4, intron 4, exon 5, partial sequences of intron 2/3 and intron 5; the third pair of PCR primers is used to amplify the target sequence of each KIR gene mentioned above covering exon 6 and partial flanking intronic sequences; the fourth pair of PCR primers is used to amplify the target sequence of each KIR gene mentioned above covering exon 7, intron 7, exon 8, intron 8, exon 9, partial sequences of intron 6 and 3′-UTR region.
The coding sequences of KIR2DL4 and KIR2DL5 are amplified using 4 pairs of corresponding KIR gene-specific PCR primers, respectively. The first pair of PCR primers is used to amplify the target sequence of each KIR gene mentioned above covering exon 1, intron 1, exon 2, partial sequences of 5′-promoter region and intron 2; the second pair of PCR primers is used to amplify the target sequence of covering exon 3, intron 3/4 (since exon 4 is deleted, the sequence between exon 3 and exon 5 is referred to as intron 3/4), exon 5, partial sequences of intron 2 and intron 5; the third pair of PCR primers is used to amplify the target sequence of each KIR gene mentioned above covering exon 6 and partial flanking intronic sequences; the fourth pair of PCR primers is used to amplify the target sequence of each KIR gene mentioned above covering exon 7, intron 7, exon 8, intron 8, exon 9, partial sequences of intron 6 and 3′-UTR region.
The coding sequences of KIR3DL1, 3DL2 and 3DS1 are amplified using 4 pairs of corresponding KIR gene-specific PCR primers, respectively. The first pair of PCR primers is used to amplify the target sequence of each KIR gene mentioned above covering exon 1, intron 1, exon 2, partial sequences of 5′-promoter region and intron 2; the second pair of PCR primers is used to amplify the target sequence of each KIR gene mentioned above covering exon 3, intron 3, exon 4, intron 4, exon 5, partial sequence of intron 2 and intron 5; the third pair of PCR primers is used to amplify the target sequence of each KIR gene mentioned above covering exon 6 and partial flanking intronic sequences; the fourth pair of PCR primers is used to amplify the target sequence of each KIR gene mentioned above covering exon 7, intron 7, exon 8, intron 8, exon 9, partial sequences of intron 6 and 3′-UTR region.
Since KIR3DL3 lacks exon 6, only 3 pairs of KIR3DL3 specific PCR primers are used to amplify the coding sequence of KIR3DL3. The first pair of PCR primers is used to amplify the target sequence of KIR3DL3 covering exon 1, intron 1, exon 2, partial sequences of 5′-promoter and intron 2; the second pair of PCR primers is used to amplify the target sequence of KIR3DL3 covering exon 3, intron 3, exon 4, intron 4, exon 5, partial sequences of intron 2 and intron 5/6; the third pair of PCR primers is used to amplify the target sequence of KIR3DL3 covering exon 7, intron 7, exon 8, intron 8, exon 9, partial sequences of intron 5/6 and 3′-UTR region.
II. The coding sequences of 14 functional KIR genes are amplified using a total of 56 pairs of KIR gene-specific PCR primers. Except that only three pairs of PCR primers are used for KIR3DL3 and five pairs of PCR primers are used for KIR2DL1, four pairs of KIR gene-specific PCR primers are used for each other functional KIR gene. All the PCR primers are designed using the Primer Premier 5.0 software and have been examined using NCBI BLAST to confirm homology with the expected KIR gene. The sequence of each PCR primer, its position in the full genomic sequence and the length of each PCR amplicon are illustrated in Table 1.
(1) The first pair of 2DL1 specific PCR primers includes a forward primer 2DL1_PCR_Ex12_F (sequence: 5′-GTTCGGGAGGTTGGATCTC-3′, its position in the full genomic sequence: nt-268˜nt-250, SEQ ID No: 1) and a reverse primer 2DL1_PCR_Ex12_R (5′-CACACTGCAGCCCCTACCG-3′, nt1332˜nt1350, SEQ ID No: 2), which is used for amplifying the target sequence of KIR2DL1 covering exon 1, intron 1, exon 2, partial sequences of 5′-promoter region and intron 2/3, the target amplicon is 1618 bp in length. The second pair of 2DL1 specific PCR primers includes a forward primer 2DL1_PCR_Ex4_F (5′-TGATTCTCCTGAGTCTCCAGAGG-3′, nt2501˜nt2523, SEQ ID No: 3) and a reverse primer 2DL1_PCR_Ex4_R (5′-TGGAAGGAGAAGAGGCAGTTTCC-3′, nt5288˜nt5310, SEQ ID No: 4), which is used for amplifying the target sequence of KIR2DL1 covering exon 4 and its partial flanking intronic sequences, the target amplicon is 2810 bp in length. The third pair of 2DL1 specific PCR primers includes a forward primer 2DL1_PCR_Ex5_F (5′-CTGGCAGGGACCTACAGATGC-3′, nt3692˜nt3712, SEQ ID No: 5) and a reverse primer 2DL1_PCR_Ex5_R (5′-GGACAGCCATGGGCTTTCCTC-3′, nt5608˜nt5628, SEQ ID No: 6), which is used for amplifying the target sequence of KIR2DL1 covering exon 5 and its partial flanking intronic sequences, the target amplicon is 1937 bp in length. The fourth pair of 2DL1 specific PCR primers includes a forward primer 2DL1_PCR_Ex6_F (5′-TCCTGATTGTGAGTTCTTGGCAT-3′, nt8082˜nt8104, SEQ ID No: 7) and a reverse primer 2DL1_PCR_Ex6_R (5′-TGAGTCAGTSAGTCGAARTGTGC-3′, nt9279˜nt9301, SEQ ID No: 8), which is used for amplifying the target sequence of KIR2DL1 covering exon 6 and its partial flanking intronic sequences, the target amplicon is 1220 bp in length. The fifth pair of 2DL1 specific PCR primers includes a forward primer 2DL1_PCR_Ex789_F (5′-CCTCAGCACGTTCTATGGTTACT-3′, nt12880˜nt12902, SEQ ID No: 9) and a reverse primer 2DL1_PCR_Ex789_R (5′-TGTGATTGCAGCCTCAAGTAGAC-3′, nt14249˜nt14271, SEQ ID No: 10), which is used for amplifying the target sequence of KIR2DL1 covering exon 7, intron 7, exon 8, intron 8, exon 9, partial sequences of intron 6 and 3′-UTR region, the target amplicon is 1392 bp in length.
(2) The first pair of 2DL2 specific PCR primers includes a forward primer 2DL2_PCR_Ex12_F (5′-AGAGGTTGGATCTGAGACGTC-3′, nt-263˜nt-243, SEQ ID No: 11) and a reverse primer 2DL2_PCR_Ex12_R (5′-GGACCGATGGAGAAGTTGGCT-3′, nt3590˜nt3610, SEQ ID No: 12), which is used for amplifying the target sequence of KIR2DL2 covering exon 1, intron 1, exon 2, intron 2/3, partial sequences of the 5′-promoter region and exon 4, the target amplicon is 3873 bp in length. The second pair of 2DL2 specific PCR primers includes a forward primer 2DL2_PCR_Ex45_F (5′-GAGGCTACTAGAGACAGAGGGAC-3′, nt3207˜nt3229, SEQ ID No: 13) and a reverse primer 2DL2_PCR_Ex45_R (5′-CCCAAGCTTCGTCTTCTCTCT-3′, nt5617˜nt5637, SEQ ID No: 14), which is used for amplifying the target sequence of KIR2DL2 covering exon 4, intron 4, exon 5, partial sequences of intron 2/3 and intron 5, the target amplicon is 2431 bp in length. The third pair of 2DL2 specific PCR primers includes a forward primer 2DL2_PCR_Ex6_F (5′-CATGCCAACATCATGCTGTC-3′, nt8530˜nt8549, SEQ ID No: 15) and a reverse primer 2DL2_PCR_Ex6_R (5′-TCCCTGTCCTAGCCTCCATAC-3′, nt9879˜nt9899, SEQ ID No: 16), which is used for amplifying the target sequence of KIR2DL2 covering exon 6 and its partial flanking intronic sequences, the target amplicon is 1370 bp in length. The fourth pair of 2DL2 specific PCR primers includes a forward primer 2DL2_PCR_Ex789_F (5′-GAAGTTCCACTTGCCAAGGAATG-3′, nt9210˜nt9232, SEQ ID No: 17) and a reverse primer 2DL2_PCR_Ex789_R (5′-CAGCTGCTGGTACATGGGAGC-3′, nt14071˜nt14091, SEQ ID No: 18), which is used for amplifying the target sequence of KIR2DL2 covering exon 7, intron 7, exon 8, intron 8, exon 9, partial sequences of intron 6 and 3′-UTR region, the target amplicon is 4882 bp in length.
(3) The first pair of 2DL3 specific PCR primers includes a forward primer 2DL3_PCR_Ex12_F (5′-GGCYGMCTGTCTGCACAGA-3′, nt-26˜nt-8, SEQ ID No: 19) and a reverse primer 2DL3_PCR_Ex12_R (5′-GGTTTCCTGTTGCTGCTGTAG-3′, nt2560˜nt2580, SEQ ID No: 20), which is used for amplifying the target sequence of KIR2DL3 covering exon 1, intron 1, exon 2, partial sequences of the 5′-promoter region and intron 2/3, the target amplicon is 2606 bp in length. The second pair of 2DL3 specific PCR primers includes a forward primer 2DL3_PCR_Ex45_F (5′-AGAGAAGAGGGAGGGAGACAGAT-3′, nt3231˜nt3253, SEQ ID No: 21) and a reverse primer 2DL3_PCR_Ex45_R (5′-GCCATCCTGTGCCCTGATC-3′, nt5651˜nt5669, SEQ ID No: 22), which is used for amplifying the target sequences of KIR2DL3 covering exon 4, intron 4, exon 5, partial sequences of intron 2/3 and intron 5, the target amplicon is 2439 bp in length. The third pair of 2DL3 specific PCR primers includes a forward primer 2DL3_PCR_Ex6_F (5′-CCCACCTCAGGCTCTCAAAGG-3′, nt7497˜nt7517, SEQ ID No: 23) and a reverse primer 2DL3_PCR_Ex6_R (5′-GGCGTACAATGTCAGAGCTGC-3′, nt8908˜nt8928, SEQ ID No: 24), which is used for amplifying the target sequence of KIR2DL3 covering exon 6 and its partial fanking sequences, the target amplicon is 1432 bp in length. The fourth pair of 2DL3 specific PCR primers includes a forward primer 2DL3_PCR_Ex789_F (5′-ACTGAGAAAGCAGGAGAAAGCTG-3′, nt12934˜nt12956, SEQ ID No: 25) and a reverse primer 2DL3_PCR_Ex789_R (5′-CCTTCAGATTCCAGCTGCTGG-3′, nt14063˜nt14083, SEQ ID No: 26), which is used for amplifying the target sequence of KIR2DL3 covering exon 7, intron 7, exon 8, intron 8, exon 9, partial sequences of intron 6 and 3′-UTR region, the target amplicon is 1150 bp in length.
(4) The first pair of 2DL4 specific PCR primers includes a forward primer 2DL4_PCR_Ex12_F (5′-GTGGTCAATGTGTCAACTGCACG-3′, nt-99˜nt-77, SEQ ID No: 27) and a reverse primer 2DL4_PCR_Ex12_R (5′-CACAGGCTCCAAGGATTACAATG-3′, nt1639˜nt1661, SEQ ID No: 28), which is used for amplifying the target sequence of KIR2DL4 covering exon 1, intron 1, exon 2, intron 2, exon 3, partial sequences of 5′-promoter region and intron 3/4, the target amplicon is 1760 bp in length. The second pair of 2DL4 specific PCR primers includes a forward primer 2DL4_PCR_Ex35_F (5′-CTTTCTTCCCCATGGCTGAGTTG-3′, nt571˜nt593, SEQ ID No: 29) and a reverse primer 2DL4_PCR_Ex35_R (5′-CTTGGGCAACAAGAGTGAAACGC-3′, nt3848˜nt3870, SEQ ID No: 30), which is used for amplifying the target sequence of KIR2DL4 covering exon 3, intron 3/4, exon 5, partial sequences of intron 2 and intron 5, the target amplicon is 3300 bp in length. The third pair of 2DL4 specific PCR primers includes a forward primer 2DL4_PCR_Ex6_F (5′-AACCTCTACCTCCAGGATTCAAG-3′, nt3904˜nt3926, SEQ ID No: 31) and a reverse primer 2DL4_PCR_Ex6_R (5′-GTAAGTGGAAGTGTCATGTGCAC-3′, nt5738˜nt5760, SEQ ID No: 32), which is used for amplifying the target sequence of KIR2DL4 covering exon 6 and its partial flanking sequences, the target amplicon is 1857 bp in length. The fourth pair of 2DL4 specific PCR primers includes a forward primer 2DL4_PCR_Ex789_F (5′-CCAAGAAATGAGAGACAATCCAC-3′, nt9442˜nt9464, SEQ ID No: 33) and a reverse primer 2DL4_PCR_Ex789_R (5′-AGGCACCAGATTTGTGGTGTG-3′, nt10540˜nt10560, SEQ ID No: 34), which is used for amplifying the target sequence of KIR2DL4 covering exon 7, intron 7, exon 8, intron 8, exon 9, partial sequences of intron 6 and 3′UTR region, the target amplicon is 1119 bp in length.
(5) The first pair of 2DL5 specific PCR primers includes a forward primer 2DL5_PCR_Ex12_F (5′-TCATAGTGAAGGACGYGAGGTGC-3′, nt-230˜nt-208, SEQ ID No: 35) and a reverse primer 2DL5_PCR_Ex12_R (5′-AGCCAATGTGTGAACCACAATAC-3′, nt1238˜nt1260, SEQ ID No: 36), which is used for amplifying the target sequence of KIR2DL5 covering exon 1, intron 1, exon 2, partial sequences of 5′-promoter region and intron 2, the target amplicon is 1490 bp in length. The second pair of 2DL5 specific PCR primers includes a forward primer 2DL5_PCR_Ex35_F (5′-CAGGACAAGCCCTTGCTGTCT-3′, nt1571˜nt1591, SEQ ID No: 37) and a reverse primer 2DL5_PCR_Ex35_R (5′-GACAGAAACAAGCAGTGGGTCAC-3′, nt2993˜nt3015, SEQ ID No: 38), which is used for amplifying the target sequence of KIR2DL5 covering exon 3, intron 3/4, exon 5, the target amplicon is 1445 bp in length. The third pair of 2DL5 specific PCR primers includes a forward primer 2DL5_PCR_Ex6_F (5′-CATTTCCTCACCTCTCTCCTGTCCT-3′, nt5158˜nt5182, SEQ ID No: 39) and a reverse primer 2DL5_PCR_Ex6_R (5′-AAGAGCAGAGGCCAAATGCATCG-3′, nt6351˜nt6373, SEQ ID No: 40), which is used for amplifying the target sequence of KIR2DL5 covering exon 6 and its partial flanking sequences, the target amplicon is 1216 bp in length. The fourth pair of 2DL5 specific PCR primers includes a forward primer 2DL5_PCR_Ex789_F (5′-CAGATGTTGTATGTGCTTAGCTG-3′, nt7907˜nt7929, SEQ ID No: 41) and a reverse primer 2DL5_PCR_Ex789_R (5′-GGTTTTGAGACAGGGCTGTTGTC-3′, nt8937˜nt8959, SEQ ID No: 42), which is used for amplifying the target sequence of KIR2DL5 covering exon 7, intron 7, exon 8, intron 8, exon 9 and partial sequences of intron 6, the target amplicon is 1053 bp in length.
(6) The first pair of 2DS1 specific PCR primers includes a forward primer 2DS1_PCR_Ex12_F (5′-CATAGTGAAGGACGCTAGGTGTA-3′, nt-229˜nt-207, SEQ ID No: 43) and a reverse primer 2DS1_PCR_Ex12_R (5′-GAGCCCTCTGACCTGTGACCG-3′, nt2035˜nt2055, SEQ ID No: 44), which is used for amplifying the target sequence of KIR2DS1 covering exon 1, intron 1, exon 2, partial sequences of 5′-promoter region and intron 2/3, the target amplicon is 2284 bp in length. The second pair of 2DS1 specific PCR primers includes a forward primer 2DS1_PCR_Ex45_F (5′-GTTCCTCTTCCACCCCCACAC-3′, nt3175˜nt3195, SEQ ID No: 45) and a reverse primer 2DS1_PCR_Ex45_R (5′-GAGGGTTTGGAGGTGCCCTGTCG-3′, nt5747˜nt5769, SEQ ID No: 46), which is used for amplifying the target sequence of KIR2DS1 covering exon 4, intron 4, exon 5, partial sequences of intron 2/3 and intron 5, the target amplicon is 2595 bp in length. The third pair of 2DS1 specific PCR primers includes a forward primer 2DS1_PCR_Ex6_F (5′-TCCTGATTGTGAGTTCTTGGCAT-3′, nt8078˜nt8100, SEQ ID No: 47) and a reverse primer 2DS1_PCR_Ex6_R (5′-GTCTCCTAGATTCCAGTTACGCC-3′, nt10742˜nt10764, SEQ ID No: 48), which is used for amplifying the target sequence of KIR2DS1 covering exon 6 and its partial flanking sequences, the target amplicon is 2687 bp in length. The fourth pair of 2DS1 specific PCR primers includes a forward primer 2DS1_PCR_Ex789_F (5′-CGTGGAAAAGGCAATTCCCGA-3′, nt10765˜nt10785, SEQ ID No: 49) and a reverse primer 2DS1_PCR_Ex789_R (5′-GGAGGTGGAACAGCACGTGTC-3′, nt14330˜nt14350, SEQ ID No: 50), which is used for amplifying the sequence of KIR2DS1 covering exon 7, intron 7, exon 8, intron 8, exon 9, partial sequences of intron 6 and 3′-UTR region, the target amplicon is 3586 bp in length.
(7) The first pair of 2DS2 specific PCR primers includes a forward primer 2DS2_PCR_Ex12_F (5′-TGAGAGGTTGGATCTGAGACGTC-3′, nt-265˜nt-243, SEQ ID No: 51) and a reverse primer 2DS2_PCR_Ex12_R (5′-ACATCCAGGCTCTTATCAGCCTT-3′, nt2956˜nt2978, SEQ ID No: 52), which is used for amplifying the target sequence of KIR2DS2 covering exon 1, intron 1, exon 2, partial sequences of 5′-promoter region and intron 2/3, the target amplicon is 3243 bp in length. The second pair of 2DS2 specific PCR primers includes a forward primer 2DS2_PCR_Ex45_F (5′-GCTTCCATGCTTCTGATAATTTTG-3′, nt2420˜nt2443, SEQ ID No: 53) and a reverse primer 2DS2_PCR_Ex45_R (5′-CTCTGGGTCTCTCCTGACCGT-3′, nt5639˜nt5659, SEQ ID No: 54), which is used for amplifying the sequence of KIR2DS2 covering exon 4, intron 4, exon 5, partial sequences of intron 2/3 and intron 5, the target amplicon is 3240 bp in length. The third pair of 2DS2 specific PCR primers includes a forward primer 2DS2_PCR_Ex6_F (5′-CATTCTGCTCCGTTGTTCTATGTC-3′, nt8282˜nt8305, SEQ ID No: 55) and a reverse primer 2DS2_PCR_Ex6_R (5′-GCCAGGGTTGCTTCATGACCTAT-3′, nt9024˜nt9046, SEQ ID No: 56), which is used for amplifying the sequence of KIR2DS2 covering exon 6 and its partial flanking sequences, the target amplicon is 765 bp in length. The fourth pair of 2DS2 specific PCR primers includes a forward primer 2DS2_PCR_Ex789_F (5′-GATAGGCCATGGGGAGGTAAATT-3′, nt11463˜nt11485, SEQ ID No: 57) and a reverse primer 2DS2_PCR_Ex789_R (5′-GGGCAGACATGTTTATTTGAAGGC-3′, nt14250˜nt14273, SEQ ID No: 58), which is used for amplifying the sequence of KIR2DS2 covering exon 7, intron 7, exon 8, intron 8, exon 9, partial sequences of intron 6 and 3′-UTR region, the target amplicon is 2811 bp in length.
(8) The first pair of 2DS3 specific PCR primers includes a forward primer 2DS3_PCR_Ex12_F (5′-TGTAAACTGCATGGGCAGGGA-3′, nt-90˜nt-70, SEQ ID No: 59) and a reverse primer 2DS3_PCR_Ex12_R (5′-CTCTGACCTGTGACCATGATCAG-3′, nt2368˜nt2390, SEQ ID No: 60), which is used for amplifying the target sequence of KIR2DS3 covering exon 1, intron 1, exon 2, partial sequences of 5′-promoter region and intron 2/3, the target amplicon is 2480 bp in length. The second pair of 2DS3 specific PCR primers includes a forward 2DS3_PCR_Ex45_F (5′-CTGAGCCCAGCGGCAAGGC-3′, nt3586˜nt3604, SEQ ID No: 61) and a reverse primer 2DS3_PCR_Ex45_R (5′-ATCCCTCCCTCACACCGAGGA-3′, nt6039˜nt6059, SEQ ID No: 62), which is used for amplifying the sequence of KIR2DS3 covering exon 4, intron 4, exon 5, partial sequences of intron 2/3 and intron 5, the target amplicon is 2474 bp in length. The third pair of 2DS3 specific PCR primers includes a forward primer 2DS3_PCR_Ex6_F (5′-TACCAGGGTTCTCCTTTCTCTAG-3′, nt7491˜nt7513, SEQ ID No: 63) and a reverse primer 2DS3_PCR_Ex6_R (5′-AGGAAGGGGACCAGGAGCG-3′, nt9878˜nt9896, SEQ ID No: 64), which is used for amplifying the sequence of KIR2DS3 covering exon 6 and its partial flanking sequences, the target amplicon is 2406 bp in length. The fourth pair of 2DS3 specific PCR primers includes a forward primer 2DS3_PCR_Ex789_F (5′-TGATGTTGAAGGAAGAGGCTCTT-3′, nt10853˜nt10875, SEQ ID No: 65) and a reverse primer 2DS3_PCR_Ex789_R (5′-GATAGTCTGAGGGGAGGTGGAACT-3′, nt14688˜nt14711, SEQ ID No: 66), which is used for amplifying the sequence of KIR2DS3 covering exon 7, intron 7, exon 8, intron 8, exon 9, partial sequences of intron 6 and 3′-UTR region, the target amplicon is 3859 bp in length.
(9) The first pair of 2DS4 specific PCR primers includes a forward primer 2DS4_PCR_Ex12_F (5′-ACCATGTCGCTCATGGTCATCAT-3′, nt-3˜nt20, SEQ ID No: 67) and a reverse primer 2DS4_PCR_Ex12_R (5′-TTGTCCTGACCACCTTGGGGT-3′, nt3070˜nt3090, SEQ ID No: 68), which is used for amplifying the target sequence of KIR2DS4 covering exon 1, intron 1, exon 2, partial sequences of 5′-promoter region and intron 2/3, the target amplicon is 3093 bp in length. The second pair of 2DS4 specific PCR primers includes a forward primer 2DS4_PCR_Ex45_F (5′-TCAGTTCATACCTCCTGCCAAGG-3′, nt4419˜nt4441, SEQ ID No: 69) and a reverse primer 2DS4_PCR_Ex45_R (5′-CGTGGTCAGGAGTTCCAGAGC-3′, nt7611˜nt7631, SEQ ID No: 70), which is used for amplifying the target sequence of KIR2DS4 covering exon 4, intron 4, exon 5, partial sequences of intron 2/3 and intron 5, the target amplicon is 3213 bp in length. The third pair of 2DS4 specific PCR primers includes a forward primer 2DS4_PCR_Ex6_F (5′-CTGGACTCCCAGGGCCCAATG-3′, nt10004˜nt10024, SEQ ID No: 71) and a reverse primer 2DS4_PCR_Ex6_R (5′-AAGGTTTCCACCTCCCCAGGG-3′, nt10212˜nt10232, SEQ ID No: 72), which is used for amplifying the target sequence of KIR2DS4 covering exon 6 and its partial flanking sequences, the target amplicon is 229 bp in length. The fourth pair of 2DS4 specific PCR primers includes a forward primer 2DS4_PCR_Ex789_F (5′-GAAAGCCCGCTGAATCCTC-3′, nt12884˜nt12902, SEQ ID No: 73) and a reverse primer 2DS4_PCR_Ex789_R (5′-GCAGAAGGCTGAAAGATAGTCTG-3′, nt15726˜nt15748, SEQ ID No: 74), which is used for amplifying the target sequence of KIR2DS4 covering exon 7, intron 7, exon 8, intron 8, exon 9, partial sequences of intron 6 and 3′-UTR region, the target amplicon is 2865 bp in length.
(10) The first pair of 2DS5 specific PCR primers includes a forward primer 2DS5_PCR_Ex12_F (5′-TGAGAACAATTTCCAGGAAGCCG-3′, nt-199˜nt-177, SEQ ID No: 75) and a reverse primer 2DS5_PCR_Ex12_R (5′-CCTTTCCTGTGGACACTTGTC-3′, nt2870˜nt2890, SEQ ID No: 76), which is used for amplifying the sequence of KIR2DS5 covering exon 1, intron 1, exon 2, partial sequences of 5′-promoter region and intron 2/3, the target amplicon is 3089 bp in length. The second pair of 2DS5 specific PCR primers includes a forward primer 2DS5_PCR_Ex45_F (5′-TCCTGCCAAGGATTCCAATTCGA-3′, nt3609˜nt3631, SEQ ID No: 77) and a reverse primer 2DS5_PCR_Ex45_R (5′-TCTGTCCATGCTTCTCTCCATCC-3′, nt6181˜nt6203, SEQ ID No: 78), which is used for amplifying the sequence of KIR2DS5 covering exon 4, intron 4, exon 5, partial of intron 2/3 and intron 5, the target amplicon is 2595 bp in length. The third pair of 2DS5 specific PCR primers includes a forward primer 2DS5_PCR_Ex6_F (5′-CTTGAAGTCTCAAGACAGTGGGT-3′, nt9083˜nt9105, SEQ ID No: 79) and a reverse primer 2DS5_PCR_Ex6_R (5′-ATGCACTTCATACTTTGAGCTAG-3′, nt9923˜nt9945, SEQ ID No: 80), which is used for amplifying the target sequence of KIR2DS5 covering exon 6 and its partial flanking sequences, the target amplicon is 863 bp in length. The fourth pair of 2DS5 specific PCR primers includes a forward primer 2DS5_PCR_Ex789_F (5′-TGATGTKGAAGGAAGAGGCTCTG-3′, nt11029˜nt11051, SEQ ID No: 81) and a reverse primer 2DS5_PCR_Ex789_R (5′-AGGGGAGGTGGAACTGCATGAGA-3′, nt14857˜nt14879, SEQ ID No: 82), which is used for amplifying the target sequence of KIR2DS5 covering exon 7, intron 7, exon 8, intron 8, exon 9, partial sequences of intron 6 and 3′-UTR region, the target amplicon is 3851 bp in length.
(11) The first pair of 3DL1 specific PCR primers includes a forward primer 3DL1_PCR_Ex12_F (5′-CGAGGTGTCAATTCTAGTGAGAG-3′, nt-215˜nt-193, SEQ ID No: 83) and a reverse primer 3DL1_PCR_Ex12_R (5′-TACCACAAACATGGCAGCG-3′, nt2689˜nt2707, SEQ ID No: 84), which is used for amplifying the target sequence of KIR3DL1 covering exon 1, intron 1, exon 2, intron 2, exon 3, partial sequences of 5′-promoter region and intron 3, the target amplicon is 2922 bp in length. The second pair of 3DL1 specific PCR primers includes a forward primer 3DL1_PCR_Ex345_F (5′-CACCCAGGTGTGGTAGGAGCC-3′, nt1700˜nt1720, SEQ ID No: 85) and a reverse primer 3DL1_PCR_Ex345_R (5′-CTCTGTGTGGGTGAGAGGCCATG-3′, nt5684˜nt5706, SEQ ID No: 86), which is used for amplifying the target sequence of KIR3DL1 covering exon 3, intron 3, exon 4, intron 4, exon 5, partial sequences of intron 2 and intron 5, the target amplicon is 4007 bp in length. The third pair of 3DL1 specific PCR primers includes a forward primer 3DL1_PCR_Ex6_F (5′-GCCTGTAATACCACTACTCGGGT-3′, nt8050˜nt8072, SEQ ID No: 87) and a reverse primer 3DL1_PCR_Ex6_R (5′-CTAAAACACCTCGCCCTCATC-3′, nt8921˜nt8941, SEQ ID No: 88), which is used for amplifying the sequence of KIR3DL1 covering exon 6 and its partial flanking sequences, the target amplicon is 892 bp in length. The fourth pair of 3DL1 specific PCR primers includes a forward primer 3DL1_PCR_Ex789_F (5′-GCTATAACTGAGAAAGCAGGAGG-3′, nt12700˜nt12722, SEQ ID No: 89) and a reverse primer 3DL1_PCR_Ex789_R (5′-CTGGAAAATAGTCCGAAGAAAGG-3′, nt14173˜nt14195, SEQ ID No: 90), which is used for amplifying the target sequence of KIR3DL1 covering exon 7, intron 7, exon 8, intron 8, exon 9, partial sequences of intron 6 and 3′-UTR region, the target amplicon is 1496 bp in length.
(12) The first pair of 3DL2 specific PCR primers includes a forward primer 3DL2_PCR_Ex12_F (5′-TGCAAGGTGGCAATTGTAGTCAC-3′, nt-217˜nt-195, SEQ ID No: 91) and a reverse primer 3DL2_PCR_Ex12_R (5′-CGACGATAGTGACACTGAAGAGC-3′, nt1588˜nt1610, SEQ ID No: 92), which is used for amplifying the target sequence of KIR3DL2 covering exon 1, intron 1, exon 2, intron 2, partial sequences of 5′-promoter region and exon 3, the target amplicon is 1827 bp in length. The second pair of 3DL2 specific PCR primers includes a forward primer 3DL2_PCR_Ex345_F (5′-CCTCCTCTCTAAGGCAGTGCCTC-3′, nt1488˜nt1510, SEQ ID No: 93) and a reverse primer 3DL2_PCR_Ex345_R (5′-CGGGTTTTCCTCACCTGTGACAG-3′, nt5429˜nt5451, SEQ ID No: 94), which is used for amplifying the target sequence of KIR3DL2 covering exon 3, intron 3, exon 4, intron 4, exon 5, partial sequences of intron 2 and intron 5, the target amplicon is 3964 bp in length. The third pair of 3DL2 specific PCR primers includes a forward primer 3DL2_PCR_Ex6_F (5′-GACAGGGCACCTCCAAACCCTCT-3′, nt5584˜nt5606, SEQ ID No: 95) and a reverse primer 3DL2_PCR_Ex6_R (5′-ATTTTAGCCCAGTGACATGCACG-3′, nt9282˜nt9304, SEQ ID No: 96), which is used for amplifying the target sequence of KIR3DL2 covering exon 6 and its partial flanking sequences, the target amplicon is 3721 bp in length. The fourth pair of 3DL2 specific PCR primers includes a forward primer 3DL2_PCR_Ex789_F (5′-GCAGGAGAAAGCTGGGTCTCC-3′, nt15186˜nt15206, SEQ ID No: 97) and a reverse primer 3DL2_PCR_Ex789_R (5′-CTGGTTTTGAGACAGGGCTGTTG-3′, nt16262˜nt16284, SEQ ID No: 98), which is used for amplifying the target sequence of KIR3DL2 covering exon 7, intron 7, exon 8, intron 8, exon 9, partial sequences of intron 6 and 3′-UTR region, the target amplicon is 1099 bp in length.
(13) The first pair of 3DL3 specific PCR primers includes a forward primer 3DL3_PCR_Ex12_F (5′-ACAACATCCTGTGTGCTGCTGAA-3′, nt-63˜nt-41, SEQ ID No: 99) and a reverse primer 3DL3_PCR_Ex12_R (5′-GTCAACCCCCTGTGTCGCCTG-3′, nt815˜nt835, SEQ ID No: 100), which is used for amplifying the target sequence of KIR3DL3 covering exon 1, intron 1, exon 2, partial sequences of 5′-promoter region and intron 2, the target amplicon is 898 bp in length. The second pair of 3DL3 specific PCR primers includes a forward primer 3DL3_PCR_Ex345_F (5′-GGAACCACAGTCATGACCCTGAC-3′, nt1156˜nt1178, SEQ ID No: 101) and a reverse primer 3DL3_PCR_Ex345_R (5′-AAAGGGTGTAGGCGTTGCTGG-3′, nt5608˜nt5630, SEQ ID No: 102), which is used for amplifying the target sequence of KIR3DL3 covering exon 3, intron 3, exon 4, intron 4, exon 5, partial sequences of intron 2 and intron 5/6, the target amplicon is 4475 bp. The third pair of 3DL3 specific PCR primers includes a forward primer 3DL3_PCR_Ex789_F (5′-TGAGCCAGTCCCTCAAGGCTC-3′, nt9865˜nt9885, SEQ ID No: 103) and a reverse primer 3DL3_PCR_Ex789_R (5′-GTTTTACTGCTGACAGAAGGCTG-3′, nt12007˜nt12029, SEQ ID No: 104), which is used for amplifying the target sequence of KIR3DL3 covering exon 7, intron 7, exon 8, intron 8, exon 9, partial sequences of intron 5/6 and 3′-UTR region, the target amplicon is 2165 bp in length.
(14) The first pair of 3DS1 specific PCR primers includes a forward primer 3DS1_PCR_Ex12_F (5′-CGAGGTGTCAATTCTAGTGAGAG-3′, nt-215˜nt-193, SEQ ID No: 105) and a reverse primer 3DS1_PCR_Ex12_R (5′-CCTGTGACCATGATCACCAT-3′, nt2080˜nt2099, SEQ ID No: 106), which is used for amplifying the target sequence of KIR3DS1 covering exon 1, intron 1, exon 2, intron 2, partial sequences of 5′-promoter region and exon 3, the target amplicon is 2314 bp in length. The second pair of 3DS1 specific PCR primers includes a forward primer 3DS1_PCR_Ex345_F (5′-CAGCTGACACTTGTTGTAGGGAG-3′, nt1634˜nt1656, SEQ ID No: 107) and a reverse primer 3DS1_PCR_Ex345_R (5′-AGTGGCATGATCTCGGCTCAG-3′, nt6472˜nt6492, SEQ ID No: 108), which is used for amplifying the target sequence of KIR3DS1 covering exon 3, intron 3, exon 4, intron 4, exon 5, partial sequences of intron 2 and intron 5, the target amplicon is 4859 bp in length. The third pair of 3DS1 specific PCR primers includes a forward primer 3DS1_PCR_Ex6_F (5′-TGATCCGCCCACCTCCGCT-3′, nt7633˜nt7651, SEQ ID No: 109) and a reverse primer 3DS1_PCR_Ex6_R (5′-GCTGGGAGGTTTGAGCCAACG-3′, nt9048˜nt9068, SEQ ID No: 110), which is used for amplifying exon 6 and its partial flanking sequences, the target amplicon is 1436 bp in length. The fourth pair of 3DS1 specific PCR primers includes a forward primer 3DS1_PCR_Ex789_F (5′-GCTATAACTGAGAAAGCAGGAGG-3′, nt13101˜nt13123, SEQ ID No: 111) and a reverse primer 3DS1_PCR_Ex789_R (5′-GAAGGCTGAAAGCTAGTCTGAGG-3′, nt14562˜nt14584, SEQ ID No: 112), which is used for amplifying the target sequence of KIR3DS1 covering exon 7, intron 7, exon 8, intron 8, exon 9, partial sequences of intron 6 and 3′-UTR region, the target amplicon is 1484 bp in length.
III. All the PCR amplifications can be carried out in a volume of 10 μL containing:
IV. PCR amplifications can be conducted simultaneously under the same thermocycling parameters, and the thermocycling parameters are described below:
V. Purification of PCR products can be carried out using the purification system described below:
VI. Purification of PCR products can be carried out under the same thermocycling parameters, and the thermocycling parameters are described below:
VII. The nucleotide sequences of each exon carried by purified PCR amplicons are determined in both directions using the forward and reverse sequencing primers. As for KIR2DL1˜5, 2DS1˜5 and KIR3DL3 genes, each KIR gene is sequenced by sixteen specific sequencing primers, respectively. For KIR3DL1˜2 and KIR3DS1 genes, each KIR gene is sequenced by eighteen specific sequencing primers, respectively. A total number of 230 KIR gene-specific forward and reverse sequencing primers for all the 14 functional KIR genes are shown in the following Table 2:
(1) The forward and reverse sequencing primers for exon 1 of KIR2DL1 are 2DL1_SBT_Ex1_F (5′-CGTGTTCCGCTCTTGAGCG-3′, nt-177˜nt-159, SEQ ID No: 113) and 2DL1_SBT_Ex1_R (5′-TCACTCCCTCCCTCTATTG-3′, nt50˜nt68, SEQ ID No: 114), respectively;
The forward and reverse sequencing primers for exon 2 of KIR2DL1 are 2DL1_SBT_Ex2_F (5′-TTCTTGGGTGCAGGTAGGC-3′, nt855˜nt873, SEQ ID No: 115) and 2DL1_SBT_Ex2_R (5′-ACCCTGGTCCCCACAGAAC-3′, nt1210˜nt1228, SEQ ID No: 116), respectively;
The forward and reverse sequencing primers for exon 4 of KIR2DL1 are 2DL1_SBT_Ex4_F (5′-AAGGGGAAGCCTGACTCAA-3′, nt3400˜nt3418, SEQ ID No: 117) and 2DL1_SBT_Ex4_R (5′-CCAATTCCTGGATCATTCAC-3′, nt3827˜nt3846, SEQ ID No: 118), respectively;
The forward and reverse sequencing primers for exon 5 of KIR2DL1 are 2DL1_SBT_Ex5_F (5′-GTTCTCAGCTCAGGTGAAG-3′, nt5240˜nt5258, SEQ ID No: 119) and 2DL1_SBT_Ex5_R (5′-AAACAAGCAGTGGGTCACTTGAC-3′, nt5574˜nt5596, SEQ ID No: 120), respectively;
The forward and reverse sequencing primers for exon 6 of KIR2DL1 are 2DL1_SBT_Ex6_F (5′-TTTCCACTGAGTGGAGGAC-3′, nt8698˜nt8716, SEQ ID No: 121) and 2DL1_SBT_Ex6_R (5′-TGGAGTTCGGAGATGGTGG-3′, nt8920˜nt8938, SEQ ID No: 122), respectively;
The forward and reverse sequencing primers for exon 7 of KIR2DL1 are 2DL1_SBT_Ex7_F (5′-ATGTGGTTACCTGTCAATC-3′, nt12979˜nt12997, SEQ ID No: 123) and 2DL1_SBT_Ex7_R (5′-TCCTGCTTCCCCACATGGC-3′, nt13207˜nt13225, SEQ ID No: 124), respectively;
The forward and reverse sequencing primers for exon 8 of KIR2DL1 are 2DL1_SBT_Ex8_F (5′-CTCAGCCACCTATGGTCTC-3′, nt13533˜nt13551, SEQ ID No: 125) and 2DL1_SBT_Ex8_R (5′-TCTCTGTGTGAAAACGCAG-3′, nt13835˜nt13853, SEQ ID No: 126), respectively;
The forward and reverse sequencing primers for exon 9 of KIR2DL1 are 2DL1_SBT_Ex9_F (5′-ACAGAACAGCGAATAGCGA-3′, nt13667˜nt13685, SEQ ID No: 127) and 2DL1_SBT_Ex9_R (5′-TAAGATGCAGACTCATGCC-3′, nt14060˜nt14078, SEQ ID No: 128), respectively.
(2) The forward and reverse sequencing primers for exon 1 of KIR2DL2 are 2DL2_SBT_Ex1_F (5′-AGAGGTTGGATCTGAGACGTC-3′, nt-263˜nt-243, SEQ ID No: 129) and 2DL2_SBT_Ex1_R (5′-TCTCCAACTCTGGGCCCCG-3′, nt81˜nt99, SEQ ID No: 130), respectively;
The forward and reverse sequencing primers for exon 2 of KIR2DL2 are 2DL2_SBT_Ex2_F (5′-TTCTTGGGTGCAGGTAGGC-3′, nt799˜nt817, SEQ ID No: 131) and 2DL2_SBT_Ex2_R (5′-CCCAGTCTAACCCTGGTCC-3′, nt1163˜nt1181, SEQ ID No: 132), respectively;
The forward and reverse sequencing primers for exon 4 of KIR2DL2 are 2DL2_SBT_Ex4_F (5′-AAGGGGAAGCCTCACTCAT-3′, nt3332˜nt3350, SEQ ID No: 133) and 2DL2_SBT_Ex4_R (5′-GGCCCCTGTGTCTGTCCTC-3′, nt3900˜nt3918, SEQ ID No: 134), respectively;
The forward and reverse sequencing primers for exon 5 of KIR2DL2 are 2DL2_SBT_Ex5_F (5′-GCTGTGACAAGGAAGATCC-3′, nt5179˜nt5197, SEQ ID No: 135) and 2DL2_SBT_Ex5_R (5′-AAGCTCCTCAGCTAAGGCT-3′, nt5564˜nt5582, SEQ ID No: 136), respectively;
The forward and reverse sequencing primers for exon 6 of KIR2DL2 are 2DL2_SBT_Ex6_F (5′-ATCCCAGGACTCCCAGGGC-3′, nt8669˜nt8687, SEQ ID No: 137) and 2DL2_SBT_Ex6_R (5′-GGCGTACAATGTCAGAGCTGC-3′, nt8928˜nt8948, SEQ ID No: 138), respectively;
The forward and reverse sequencing primers for exon 7 of KIR2DL2 are 2DL2_SBT_Ex7_F (5′-ATCTGGGTGCTTGTCCTAA-3′, nt12990˜nt13008, SEQ ID No: 139) and 2DL2_SBT_Ex7_R (5′-CCTCTGCTTCGTGAGACTTAC-3′, nt13213˜nt13233, SEQ ID No: 140), respectively;
The forward and reverse sequencing primers for exon 8 of KIR2DL2 are 2DL2_SBT_Ex8_F (5′-CCCAGAAGTGCCCTCCGAG-3′, nt13628˜nt13646, SEQ ID No: 141) and 2DL2_SBT_Ex8_R (5′-TCTCTGTGTGAAAACGCAG-3′, nt13876˜nt13894, SEQ ID No: 142), respectively;
The forward and reverse sequencing primers for exon 9 of KIR2DL2 are 2DL2_SBT_Ex9_F (5′-ACAGAACAGCGAATAGCGA-3′, nt13708˜nt13726, SEQ ID No: 143) and 2DL2_SBT_Ex9_R (5′-GGCTGTTGTCTCCCTAGAAGACG-3′, nt14026˜nt14048, SEQ ID No: 144), respectively.
(3) The forward and reverse sequencing primers for exon 1 of KIR2DL3 are 2DL3_SBT_Ex1_F (5′-CYGMCTGTCTGCACAGA-3′, nt-24˜nt-8, SEQ ID No: 145) and 2DL3_SBT_Ex1_R (5′-TCTCCAACTCTGGGCCCCG-3′, nt81˜nt99, SEQ ID No: 146), respectively;
The forward and reverse sequencing primers for exon 2 of KIR2DL3 are 2DL3_SBT_Ex2_F (5′-TTCTTGGGTGCAGGTAGGC-3′, nt799˜nt817, SEQ ID No: 147) and 2DL3_SBT_Ex2_R (5′-ACCCTGGTCCCCACAGAAC-3′, nt1154˜nt1172, SEQ ID No: 148), respectively;
The forward and reverse sequencing primers for exon 4 of KIR2DL3 are 2DL3_SBT_Ex4_F (5′-CAGCAAGGGGAAGCCTCA-3′, nt3329˜nt3346, SEQ ID No: 149) and 2DL3_SBT_Ex4_R (5′-GGCCCCTGTGTCTGTCCTC-3′, nt3901˜nt3919, SEQ ID No: 150), respectively;
The forward and reverse sequencing primers for exon 5 of KIR2DL3 are 2DL3_SBT_Ex5_F (5′-GAGCATTAGGTCATAGAGC-3′, nt5131˜nt5149, SEQ ID No: 151) and 2DL3_SBT_Ex5_R (5′-CTCTCTGCATCTGTCCATGCTTC-3′, nt5602˜nt5624, SEQ ID No: 152), respectively;
The forward and reverse sequencing primers for exon 6 of KIR2DL3 are 2DL3_SBT_Ex6_F (5′-TACTCAGGAGTTTGAGGCC-3′, nt8310˜nt8328, SEQ ID No: 153) and 2DL3_SBT_Ex6_R (5′-GGCGTACAATGTCAGAGCTGC-3′, nt8908˜nt8928, SEQ ID No: 154), respectively;
The forward and reverse sequencing primers for exon 7 of KIR2DL3 are 2DL3_SBT_Ex7_F (5′-TCTGGGTGCTTGTCCTAAAGG-3′, nt12969˜nt12989, SEQ ID No: 155) and 2DL3_SBT_Ex7_R (5′-CAGGCAATGGTCTGTGAGC-3′, nt13361˜nt13379, SEQ ID No: 156), respectively;
The forward and reverse sequencing primers for exon 8 of KIR2DL3 are 2DL3_SBT_Ex8_F (5′-CTTCATCGCTGGTGCTG-3′, nt13166˜nt13182, SEQ ID No: 157) and 2DL3_SBT_Ex8_R (5′-GCTGAGTGAGGGAGGGTGC-3′, nt13772˜nt13790, SEQ ID No: 158), respectively;
The forward and reverse sequencing primers for exon 9 of KIR2DL3 are 2DL3_SBT_Ex9_F (5′-CCCAGCCTCGTGGCTAG-3′, nt13724˜nt13740, SEQ ID No: 159) and 2DL3_SBT_Ex9_R (5′-GGCAGGAGACAACTTTGGATCW-3′, nt13957˜nt13978, SEQ ID No: 160), respectively.
(4) The forward and reverse sequencing primers for exon 1 of KIR2DL4 are 2DL4_SBT_Ex1_F (5′-GTGGTCAATGTGTCAACTGCACG-3′, nt-99˜nt-77, SEQ ID No: 161) and 2DL4_SBT_Ex1_R (5′-CCTGAGCCACTGGGCGCCA-3′, nt166˜nt184, SEQ ID No: 162), respectively;
The forward and reverse sequencing primers for exon 2 of KIR2DL4 are 2DL4_SBT_Ex2_F (5′-GAGCCATGTTCTGAAGCAAGT-3′, nt111˜nt131, SEQ ID No: 163) and 2DL4_SBT_Ex2_R (5′-CACCCTCTGTGCTGCCTCC-3′, nt345˜nt363, SEQ ID No: 164), respectively;
The forward and reverse sequencing primers for exon 3 of KIR2DL4 are 2DL4_SBT_Ex3_F (5′-TACTCCTCTCTGAGGCGGC-3′, nt1140˜nt1158, SEQ ID No: 165) and 2DL4_SBT_Ex3_R (5′-CCAGAAGCTCTGGGACTCA-3′, nt1502˜nt1520, SEQ ID No: 166), respectively;
The forward and reverse sequencing primers for exon 5 of KIR2DL4 are 2DL4_SBT_Ex5_F (5′-GGGAGGGGAGCTGTGACAA-3′, nt2275˜nt2293, SEQ ID No: 167) and 2DL4_SBT_Ex5_R (5′-GCTTCTCTCCATCATCAGC-3′, nt2691˜nt2709, SEQ ID No: 168), respectively;
The forward and reverse sequencing primers for exon 6 of KIR2DL4 are 2DL4_SBT_Ex6_F (5′-CAGGCATCCTCATTGCCAC-3′, nt5179˜nt5197, SEQ ID No: 169) and 2DL4_SBT_Ex6_R (5′-TGGCAGGTGCTGAGCCAAC-3′, nt5341˜nt5359, SEQ ID No: 170), respectively;
The forward and reverse sequencing primers for exon 7 of KIR2DL4 are 2DL4_SBT_Ex7_F (5′-TCGCCAGACACCTGCATGC-3′, nt9519˜nt9537, SEQ ID No: 171) and 2DL4_SBT_Ex7_R (5′-TTTGGAGCACCAGC-3′, nt9600˜nt9613, SEQ ID No: 172), respectively;
The forward and reverse sequencing primers for exon 8 of KIR2DL4 are 2DL4_SBT_Ex8_F (5′-GAGGACCCAGAAGTGCCCT-3′, nt10030˜nt10048, SEQ ID No: 173) and 2DL4_SBT_Ex8_R (5′-CTGGAGAGAGGGAAATCCT-3′, nt10215˜nt10233, SEQ ID No: 174), respectively;
The forward and reverse sequencing primers for exon 9 of KIR2DL4 are 2DL4_SBT_Ex9_F (5′-CCAGCCTCATGGATACAGTCT-3′, nt10150˜nt10170, SEQ ID No: 175) and 2DL4_SBT_Ex9_R (5′-GGAAGAGTGATGCTCTAAGATGG-3′, nt10516˜nt10538, SEQ ID No: 176), respectively.
(5) The forward and reverse sequencing primers for exon 1 of KIR2DL5 are 2DL5_SBT_Ex1_F (5′-CCAAATAACATCCTGTGCGCT-3′, nt-67˜nt-47, SEQ ID No: 177) and 2DL5_SBT_Ex1_R (5′-AGATCTCCATCCCCGCACT-3′, nt64˜nt82, SEQ ID No: 178), respectively;
The forward and reverse sequencing primers for exon 2 of KIR2DL5 are 2DL5_SBT_Ex2_F (5′-CAGCAAGGGCCTGGCTACC-3′, nt668˜nt686, SEQ ID No: 179) and 2DL5_SBT_Ex2_R (5′-GAAAATCCCCCACCGGGCT-3′, nt872˜nt890, SEQ ID No: 180), respectively;
The forward and reverse sequencing primers for exon 3 of KIR2DL5 are 2DL5_SBT_Ex3_F (5′-ACAAGCCCTTGCTGTCTGCCT-3′, nt1575˜nt1595, SEQ ID No: 181) and 2DL5_SBT_Ex3_R (5′-CAGATGCTCTGGGATTCAG-3′, nt1891˜nt1909, SEQ ID No: 182), respectively;
The forward and reverse sequencing primers for exon 5 of KIR2DL5 are 2DL5_SBT_Ex5_F (5′-CAGGTGTGAGGGGAGCTGT-3′, nt2665˜nt2683, SEQ ID No: 183) and 2DL5_SBT_Ex5_R (5′-CGGGTCTGACCACTCATAGGGT-3′, nt2970˜nt2991, SEQ ID No: 184), respectively;
The forward and reverse sequencing primers for exon 6 of KIR2DL5 are 2DL5_SBT_Ex6_F (5′-TCACCTCTCTCCTGTCCTGTGT-3′, nt5165˜nt5186, SEQ ID No: 185) and 2DL5_SBT_Ex6_R (5′-TGAGCCAATGCTTGAATCCAAGA-3′, nt5295˜nt5317, SEQ ID No: 186), respectively;
The forward and reverse sequencing primers for exon 7 of KIR2DL5 are 2DL5_SBT_Ex7_F (5′-ATCCATAAAGAGGAACTGCTATA-3′, nt7951˜nt7973, SEQ ID No: 187) and 2DL5_SBT_Ex7_R (5′-CCTTGGTCCAGGGACCATC-3′, nt8201˜nt8219, SEQ ID No: 188), respectively;
The forward and reverse sequencing primers for exon 8 of KIR2DL5 are 2DL5_SBT_Ex8_F (5′-CACCTACGGCCTCCCGCTG-3′, nt8480˜nt8498, SEQ ID No: 189) and 2DL5_SBT_Ex8_R (5′-GAGGGTGCTCACATTCTTCAA-3′, nt8680˜nt8700, SEQ ID No: 190), respectively;
The forward and reverse sequencing primers for exon 9 of KIR2DL5 are 2DL5_SBT_Ex9_F (5′-TGCCGGGGACAGAACAGTG-3′, nt8600˜nt8618, SEQ ID No: 191) and 2DL5_SBT_Ex9_R (5′-CTCAAGGCCTGACTGTGGTGCTT-3′, nt8899˜nt8921, SEQ ID No: 192), respectively.
(6) The forward and reverse sequencing primers for exon 1 of KIR2DS1 are 2DS1_SBT_Ex1_F (5′-CTCCCATGATGTGGTCAAC-3′, nt-109˜nt-91, SEQ ID No: 193) and 2DS1_SBT_Ex1_R (5′-TCTCCAACCCCACACTCCC-3′, nt61˜nt79, SEQ ID No: 194), respectively;
The forward and reverse sequencing primers for exon 2 of KIR2DS1 are 2DS1_SBT_Ex2_F (5′-TTCTTGGGTGCAGGTAGGC-3′, nt855˜nt873, SEQ ID No: 195) and 2DS1_SBT_Ex2_R (5′-CTGCCAAGGGAATGAAAGG-3′, nt1185˜nt1203, SEQ ID No: 196), respectively;
The forward and reverse sequencing primers for exon 4 of KIR2DS1 are 2DS1_SBT_Ex4_F (5′-GGTGCCATGGATGGGATGA-3′, nt3423˜nt3441, SEQ ID No: 197) and 2DS1_SBT_Ex4_R (5′-CAAGTCCTGGATCATTCAC-3′, nt3827˜nt3845, SEQ ID No: 198), respectively;
The forward and reverse sequencing primers for exon 5 of KIR2DS1 are 2DS1_SBT_Ex5_F (5′-AGAGCAGGGGAGTGAGTTC-3′, nt5221˜nt5239, SEQ ID No: 199) and 2DS1_SBT_Ex5_R (5′-GGCTCTAGGATCATAGGAC-3′, nt5628˜nt5646, SEQ ID No: 200), respectively;
The forward and reverse sequencing primers for exon 6 of KIR2DS1 are 2DS1_SBT_Ex6_F (5′-TCCTCAAAGATTTCCACTGAGTG-3′, nt8684˜nt8706, SEQ ID No: 201) and 2DS1_SBT_Ex6_R (5′-GTGAGATGCTGAGTCAACGC-3′, nt8871˜nt8890, SEQ ID No: 202), respectively;
The forward and reverse sequencing primers for exon 7 of KIR2DS1 are 2DS1_SBT_Ex7_F (5′-GTGGTTACCTGCCAATCAAG-3′, nt12981˜nt13000, SEQ ID No: 203) and 2DS1_SBT_Ex7_R (5′-TGAGGAACACACATCCGCGT-3′, nt13236˜nt13255, SEQ ID No: 204), respectively;
The forward and reverse sequencing primers for exon 8 of KIR2DS1 are 2D51_SBT_Ex8_F (5′-ATGGCCTCCCCCTGTTTGT-3′, nt13547˜nt13565, SEQ ID No: 205) and 2DS1_SBT_Ex8_R (5′-GGGAATAAGACTAGCCACG-3′, nt13713˜nt13731, SEQ ID No: 206), respectively;
The forward and reverse sequencing primers for exon 9 of KIR2DS1 are 2DS1_SBT_Ex9_F (5′-CTCCTCGGCCCAGCCTCGT-3′, nt13697˜nt13715, SEQ ID No: 207) and 2DS1_SBT_Ex9_R (5′-TCCCCTCAAGGCCTGACTG-3′, nt13971˜nt13989, SEQ ID No: 208), respectively.
(7) The forward and reverse sequencing primers for exon 1 of KIR2DS2 are 2DS2_SBT_Ex1_F (5′-ATAACATCCTGTGCGCTGC-3′, nt-63˜nt-45, SEQ ID No: 209) and 2DS2_SBT_Ex1_R (5′-CCAACTCTGGGCCCCGATC-3′, nt78˜nt96, SEQ ID No: 210), respectively;
The forward and reverse sequencing primers for exon 2 of KIR2DS2 are 2DS2_SBT_Ex2_F (5′-AAGGGAGTCCTGGTTTGCC-3′, nt772˜nt790, SEQ ID No: 211) and 2DS2_SBT_Ex2_R (5′-GTCAGAAATGTGGGCCGAG-3′, nt981˜nt999, SEQ ID No: 212), respectively;
The forward and reverse sequencing primers for exon 4 of KIR2DS2 are 2DS2_SBT_Ex4_F (5′-CACCTTCTAAACTCACAACC-3′, nt3268˜nt3287, SEQ ID No: 213) and 2DS2_SBT_Ex4_R (5′-CACTCTGCAGCCCAATGAC-3′, nt3624˜nt3642, SEQ ID No: 214), respectively;
The forward and reverse sequencing primers for exon 5 of KIR2DS2 are 2DS2_SBT_Ex5_F (5′-AGAGCAGGGGAGTGAGTTC-3′, nt5030˜nt5048, SEQ ID No: 215) and 2DS2_SBT_Ex5_R (5′-GAAGCTCCTCAGCTAAGGC-3′, nt5453˜nt5471, SEQ ID No: 216), respectively;
The forward and reverse sequencing primers for exon 6 of KIR2DS2 are 2DS2_SBT_Ex6_F (5′-CCAGGGCCCAATATTAGAT-3′, nt8465˜nt8483, SEQ ID No: 217) and 2DS2_SBT_Ex6_R (5′-TGAGTCAACGCCTGAATCC-3′, nt8686˜nt8704, SEQ ID No: 218), respectively;
The forward and reverse sequencing primers for exon 7 of KIR2DS2 are 2DS2_SBT_Ex7_F (5′-GCCAATCAAGAAATGCGAG-3′, nt12815˜nt12833, SEQ ID No: 219) and 2DS2_SBT_Ex7_R (5′-GTCCTGCCTCTGTGGCTCC-3′, nt13108˜nt13126, SEQ ID No: 220), respectively;
The forward and reverse sequencing primers for exon 8 of KIR2DS2 are 2DS2_SBT_Ex8_F (5′-ATGAGGACCCAGAAGTGCC-3′, nt13407˜nt13425, SEQ ID No: 221) and 2DS2_SBT_Ex8_R (5′-CCTCCTGATGGTCTTGTTC-3′, nt13621˜nt13639, SEQ ID No: 222), respectively;
The forward and reverse sequencing primers for exon 9 of KIR2DS2 are 2DS2_SBT_Ex9_F (5′-AGGTAGGTGCTCCTCGGCC-3′, nt13512˜nt13530, SEQ ID No: 223) and 2DS2_SBT_Ex9_R (5′-AGAAGATCCCCTCAAGGCC-3′, nt13801˜nt13819, SEQ ID No: 224), respectively.
(8) The forward and reverse sequencing primers for exon 1 of KIR2DS3 are 2DS3_SBT_Ex1_F (5′-CAGGGAGCCAAATAACATC-3′, nt-75˜nt-57, SEQ ID No: 225) and 2DS3_SBT_Ex1_R (5′-CGCTCCCTCCCTCTATTCC-3′, nt49˜nt67, SEQ ID No: 226), respectively;
The forward and reverse sequencing primers for exon 2 of KIR2DS3 are 2DS3_SBT_Ex2_F (5′-GCCGAGAGCCCTGTTCTTG-3′, nt1182˜nt1200, SEQ ID No: 227) and 2DS3_SBT_Ex2_R (5′-ACAGGACTTCCCTCCCGTT-3′, nt1432˜nt1450, SEQ ID No: 228), respectively;
The forward and reverse sequencing primers for exon 4 of KIR2DS3 are 2DS3_SBT_Ex4_F (5′-AGAGAGACACCTTCTAAAT-3′, nt3780˜nt3798, SEQ ID No: 229) and 2DS3_SBT_Ex4_R (5′-ATCATTCACTCTGTGTCCG-3′, nt4152˜nt4170, SEQ ID No: 230), respectively;
The forward and reverse sequencing primers for exon 5 of KIR2DS3 are 2DS3_SBT_Ex5_F (5′-AGGAAGATCCTCCATAAGG-3′, nt5596˜nt5614, SEQ ID No: 231) and 2DS3_SBT_Ex5_R (5′-GGCTCTAGGATCATAGGAC-3′, nt5957˜nt5975, SEQ ID No: 232), respectively;
The forward and reverse sequencing primers for exon 6 of KIR2DS3 are 2DS3_SBT_Ex6_F (5′-TCCCAGGGCCCAATATTAG-3′, nt8968˜nt8986, SEQ ID No: 233) and 2DS3_SBT_Ex6_R (5′-CACTGAGCCCTGTGTTGGG-3′, nt9291˜nt9309, SEQ ID No: 234), respectively;
The forward and reverse sequencing primers for exon 7 of KIR2DS3 are 2DS3_SBT_Ex7_F (5′-GTGCTTGTCCTAAAGAGACGT-3′, nt13284˜nt13304, SEQ ID No: 235) and 2DS3_SBT_Ex7_R (5′-TGAGTGGCTGCAGGGGACG-3′, nt13709˜nt13727, SEQ ID No: 236), respectively;
The forward and reverse sequencing primers for exon 8 of KIR2DS3 are 2DS3_SBT_Ex8_F (5′-GACCTCAGGCACCTATGGC-3′, nt13862˜nt13880, SEQ ID No: 237) and 2DS3_SBT_Ex8_R (5′-GCTGAGTGAGGGAGGGTGC-3′, nt14082˜nt14100, SEQ ID No: 238), respectively;
The forward and reverse sequencing primers for exon 9 of KIR2DS3 are 2DS3_SBT_Ex9_F (5′-CGGCCCAGCCTCGTGGCTA-3′, nt14031˜nt14049, SEQ ID No: 239) and 2DS3_SBT_Ex9_R (5′-TGTCTTGGGCCTCTGAGAAGGGG-3′, nt14196˜nt14218, SEQ ID No: 240), respectively.
(9) The forward and reverse sequencing primers for exon 1 of KIR2DS4 are 2DS4_SBT_Ex1_F (5′-ACCATGTCGCTCATGGTC-3′, nt-3˜nt15, SEQ ID No: 241) and 2DS4_SBT_Ex1_R (5′-GGCTCATCACTCCATCTCT-3′, nt148˜nt166, SEQ ID No: 242), respectively;
The forward and reverse sequencing primers for exon 2 of KIR2DS4 are 2DS4_SBT_Ex2_F (5′-GAAGGGGCTGGCTATCAAG-3′, nt2218˜nt2236, SEQ ID No: 243) and 2DS4_SBT_Ex2_R (5′-GACTTCCCTCCCGTTTCAG-3′, nt2404˜nt2422, SEQ ID No: 244), respectively;
The forward and reverse sequencing primers for exon 4 of KIR2DS4 are 2DS4_SBT_Ex4_F (5′-AGAGAGACACCTTCTAAAC-3′, nt4774˜nt4792, SEQ ID No: 245) and 2DS4_SBT_Ex4_R (5′-CACCTGGGTCTCCAAGTCC-3′, nt5168˜nt5186, SEQ ID No: 246), respectively;
The forward and reverse sequencing primers for exon 5 of KIR2DS4 are 2DS4_SBT_Ex5_F (5′-AGTTCTCAGGTCAGGTGTG-3′, nt6589˜nt6607, SEQ ID No: 247) and 2DS4_SBT_Ex5_R (5′-GGAAGCTCCTCAGCTAAGG-3′, nt7001˜nt7019, SEQ ID No: 248), respectively;
The forward and reverse sequencing primers for exon 6 of KIR2DS4 are 2DS4_SBT_Ex6_F (5′-CTGGACTCCCAGGGCCCAATG-3′, nt10004˜nt10024, SEQ ID No: 249) and 2DS4_SBT_Ex6_R (5′-TTCCACCTCCCCAGGGTTC-3′, nt10209˜nt10227, SEQ ID No: 250), respectively;
The forward and reverse sequencing primers for exon 7 of KIR2DS4 are 2DS4_SBT_Ex7_F (5′-CGCCATTTGGGTGCTTGTC-3′, nt14317˜nt14335, SEQ ID No: 251) and 2DS4_SBT_Ex7_R (5′-GGTGAGGAACACACATCCG-3′, nt14611˜nt14629, SEQ ID No: 252), respectively;
The forward and reverse sequencing primers for exon 8 of KIR2DS4 are 2DS4_SBT_Ex8_F (5′-AGTCTGCTGTTGGCAACTG-3′, nt14883˜nt14901, SEQ ID No: 253) and 2DS4_SBT_Ex8_R (5′-CCTCCTGATGGTCTTGTTC-3′, nt15169˜nt15187, SEQ ID No: 254), respectively;
The forward and reverse sequencing primers for exon 9 of KIR2DS4 are 2DS4_SBT_Ex9_F (5′-CTCGGCCCAGCCTCGTGGC-3′, nt15072˜nt15090, SEQ ID No: 255) and 2DS4_SBT_Ex9_R (5′-CAACTTTGGATCTGGGCTC-3′, nt15304˜nt15322, SEQ ID No: 256), respectively.
(10) The forward and reverse sequencing primers for exon 1 of KIR2DS5 are 2DS5_SBT_Ex1_F (5′-GGCGCCAAATAACATCCTG-3′, nt-72˜nt-54, SEQ ID No: 257) and 2DS5_SBT_Ex1_R (5′-GCCCAGATCTCCATCCCCG-3′, nt68˜nt86, SEQ ID No: 258), respectively;
The forward and reverse sequencing primers for exon 2 of KIR2DS5 are 2DS5_SBT_Ex2_F (5′-GGCACTGAGKGTGAGTTTC-3′, nt1383˜nt1401, SEQ ID No: 259) and 2DS5_SBT_Ex2_R (5′-TGACAGGACTTCCCTCCCG-3′, nt1606˜nt1624, SEQ ID No: 260), respectively;
The forward and reverse sequencing primers for exon 4 of KIR2DS5 are 2DS5_SBT_Ex4_F (5′-GACACCTTCTAAATTCACAAAC-3′, nt3958˜nt3979, SEQ ID No: 261) and 2DS5_SBT_Ex4_R (5′-CTCTGCATCCCAATGACAATG-3′, nt4315˜nt4335, SEQ ID No: 262), respectively;
The forward and reverse sequencing primers for exon 5 of KIR2DS5 are 2DS5_SBT_Ex5_F (5′-CCTCCCTGAGGAAAATGCC-3′, nt5786˜nt5804, SEQ ID No: 263) and 2DS5_SBT_Ex5_R (5′-TCATAGGACATGGGACAGC-3′, nt6129˜nt6147, SEQ ID No: 264), respectively;
The forward and reverse sequencing primers for exon 6 of KIR2DS5 are 2DS5_SBT_Ex6_F (5′-CAGGGCCCAATATTAGATAAC-3′, nt9147˜nt9167, SEQ ID No: 265) and 2DS5_SBT_Ex6_R (5′-GGAGTATCTGGAGTTCGGAGA-3′, nt9426˜nt9446, SEQ ID No: 266), respectively;
The forward and reverse sequencing primers for exon 7 of KIR2DS5 are 2DS5_SBT_Ex7_F (5′-CTGTCAATCAAGAAATGCGAG-3′, nt13495˜nt13515, SEQ ID No: 267) and 2DS5_SBT_Ex7_R (5′-GGAACACACACCCGCGTGC-3′, nt13740˜nt13758, SEQ ID No: 268), respectively;
The forward and reverse sequencing primers for exon 8 of KIR2DS5 are 2DS5_SBT_Ex8_F (5′-AGATAGAATGTCTGAGTCTGC-3′, nt14003˜nt14023, SEQ ID No: 269) and 2DS5_SBT_Ex8_R (5′-ACACAGTGATCCAATTATGCG-3′, nt14329˜nt14349, SEQ ID No: 270), respectively;
The forward and reverse sequencing primers for exon 9 of KIR2DS5 are 2DS5_SBT_Ex9_F (5′-GGTAGGTGCTCCTCGGCCC-3′, nt14195˜nt14213, SEQ ID No: 271) and 2DS5_SBT_Ex9_R (5′-ATGGGAGCTGGCAACCCGG-3′, nt14528˜nt14546, SEQ ID No: 272), respectively.
(11) The forward and reverse sequencing primers for exon 1 of KIR3DL1 are 3DL1_SBT_Ex1_F (5′-CAGGGCGCCAAATAACATC-3′, nt-74˜nt-56, SEQ ID No: 273) and 3DL1_SBT_Ex1_R (5′-CAGATCTCCATCCCCGCAC-3′, nt65˜nt83, SEQ ID No: 274), respectively;
The forward and reverse sequencing primers for exon 2 of KIR3DL1 are 3DL1_SBT_Ex2_F (5′-AGGGCCTGGCTGCCAAGAC-3′, nt940˜nt958, SEQ ID No: 275) and 3DL1_SBT_Ex2_R (5′-AATGTGGGCCGAGCATCCG-3′, nt1182˜nt1200, SEQ ID No: 276), respectively;
The forward and reverse sequencing primers for exon 3 of KIR3DL1 are 3DL1_SBT_Ex3_F (5′-GGGGAGAATCTTCTGGGCACT-3′, nt1736˜nt1756, SEQ ID No: 277) and 3DL1_SBT_Ex3_R (5′-TGATGGGACCCTGACGGAC-3′, nt2167˜nt2185, SEQ ID No: 278), respectively;
The forward and reverse sequencing primers for exon 4 of KIR3DL1 are 3DL1_SBT_Ex4_F (5′-TGGAGGCACCTGCACCAGG-3′, nt3052˜nt3070, SEQ ID No: 278) and 3DL1_SBT_Ex4_R (5′-TGGTACAGACCTCACCAAG-3′, nt3633˜nt3651, SEQ ID No: 280), respectively;
The forward and reverse sequencing primers for exon 5 of KIR3DL1 are 3DL1_SBT_Ex5_F (5′-CAGGTATGAGGGGAGCTATG-3′, nt5001˜nt5020, SEQ ID No: 281) and 3DL1_SBT_Ex5_R (5′-CCTGTCTGCCATCCTGCGC-3′, nt5490˜nt5508, SEQ ID No: 282), respectively;
The forward and reverse sequencing primers for exon 6 of KIR3DL1 are 3DL1_SBT_Ex6_F (5′-AAGCACCCTCATTTCCTCAC-3′, nt8485˜nt8504, SEQ ID No: 283) and 3DL1_SBT_Ex6_R (5′-CAACACTTGCATCCAAGGC-3′, nt8631˜nt8649, SEQ ID No: 284), respectively;
The forward and reverse sequencing primers for exon 7 of KIR3DL1 are 3DL1_SBT_Ex7_F (5′-CCCGCCATCTGGGTGCTTG-3′, nt12734˜nt12752, SEQ ID No: 285) and 3DL1_SBT_Ex7_R (5′-TCCTGCTTCCCCACATGGC-3′, nt13001˜nt13019, SEQ ID No: 286), respectively;
The forward and reverse sequencing primers for exon 8 of KIR3DL1 are 3DL1_SBT_Ex8_F (5′-CCAGAAGTGCCCTCCGAGC-3′, nt13382˜nt13400, SEQ ID No: 287) and 3DL1_SBT_Ex8_R (5′-TGTTTGGGAATAACACTAGCC-3′, nt13507˜nt13527, SEQ ID No: 288), respectively;
The forward and reverse sequencing primers for exon 9 of KIR3DL1 are 3DL1_SBT_Ex9_F (5′-CGTGGCTAGTGTTATTCCC-3′, nt13504˜nt13522, SEQ ID No: 289) and 3DL1_SBT_Ex9_R (5′-ATGGGAGCTGGCAACTCGG-3′, nt13833˜nt13851, SEQ ID No: 290), respectively.
(12) The forward and reverse sequencing primers for exon 1 of KIR3DL2 are 3DL2_SBT_Ex1_F (5′-GCCAAATAACATCCTGTGCGC-3′, nt-68˜nt-48, SEQ ID No: 291) and 3DL2_SBT_Ex1_R (5′-TAGGCCGAGATCTCCATCC-3′, nt71˜nt89, SEQ ID No: 292), respectively;
The forward and reverse sequencing primers for exon 2 of KIR3DL2 are 3DL2_SBT_Ex2_F (5′-GAGGCTAAGTTTACCTTCAGC-3′, nt624˜nt644, SEQ ID No: 293) and 3DL2_SBT_Ex2_R (5′-GACTTCCCTCCTGTTTCAG-3′, nt834˜nt852, SEQ ID No: 294), respectively;
The forward and reverse sequencing primers for exon 3 of KIR3DL2 are 3DL2_SBT_Ex3_F (5′-GGCCCAGCACTGTGGTGCC-3′, nt1553˜nt1571, SEQ ID No: 295) and 3DL2_SBT_Ex3_R (5′-GCCCATTTCCCCTGTATTC-3′, nt1930˜nt1948, SEQ ID No: 296), respectively;
The forward and reverse sequencing primers for exon 4 of KIR3DL2 are 3DL2_SBT_Ex4_F (5′-GAGAGATGCCTTCTAAACT-3′, nt3235˜nt3253, SEQ ID No: 297) and 3DL2_SBT_Ex4_R (5′-TCTCCATAAGAATCCCACGCT-3′, nt3663˜nt3683, SEQ ID No: 298), respectively;
The forward and reverse sequencing primers for exon 5 of KIR3DL2 are 3DL2_SBT_Ex5_F (5′-CCTCCCTGAGGAAACTGCC-3′, nt5111˜nt5129, SEQ ID No: 299) and 3DL2_SBT_Ex5_R (5′-GAAAGAGCCGAAGCATCTG-3′, nt5361˜nt5379, SEQ ID No: 300), respectively;
The forward and reverse sequencing primers for exon 6 of KIR3DL2 are 3DL2_SBT_Ex6_F (5′-CAACCTCAAAGATTTCCATTG-3′, nt8530˜nt8550, SEQ ID No: 301) and 3DL2_SBT_Ex6_R (5′-CAACACTTGCATCCAAGGC-3′, nt8707˜nt8725, SEQ ID No: 302), respectively;
The forward and reverse sequencing primers for exon 7 of KIR3DL2 are 3DL2_SBT_Ex7_F (5′-GAGATGTTCCATGTGGTTACC-3′, nt15231˜nt15251, SEQ ID No: 303) and 3DL2_SBT_Ex7_R (5′-GGAACACACACCCGCGTGC-3′, nt15494˜nt15512, SEQ ID No: 304), respectively;
The forward and reverse sequencing primers for exon 8 of KIR3DL2 are 3DL2_SBT_Ex8_F (5′-TCTGAGTCTGGATGTTGGC-3′, nt15764˜nt15782, SEQ ID No: 305) and 3DL2_SBT_Ex8_R (5′-GGGTCTTGTTCATCAGAGTCC-3′, nt16046˜nt16066, SEQ ID No: 306), respectively;
The forward and reverse sequencing primers for exon 9 of KIR3DL2 are 3DL2_SBT_Ex9_F (5′-CCTCGGCCCAGCCTCACGG-3′, nt15957˜nt15975, SEQ ID No: 307) and 3DL2_SBT_Ex9_R (5′-GACTGTGGTGCTCGTGGGC-3′, nt16216˜nt16234, SEQ ID No: 308), respectively.
(13) The forward and reverse sequencing primers for exon 1 of KIR3DL3 are 3DL3_SBT_Ex1_F (5′-ACAACATCCTGTGTGCTGCTGAA-3′, nt-63˜nt-41, SEQ ID No: 309) and 3DL3_SBT_Ex1_R (5′-TCCCTCCCTCGATTCCCTT-3′, nt46˜nt64, SEQ ID No: 310), respectively;
The forward and reverse sequencing primers for exon 2 of KIR3DL3 are 3DL3_SBT_Ex2_F (5′-GATGTACAGATGGATCATC-3′, nt672˜nt690, SEQ ID No: 311) and 3DL3_SBT_Ex2_R (5′-GTCAACCCCCTGTGTCGCCTG-3′, nt815˜nt835, SEQ ID No: 312), respectively;
The forward and reverse sequencing primers for exon 3 of KIR3DL3 are 3DL3_SBT_Ex3_F (5′-GCTCCACATCCTCCTCTCT-3′, nt1474˜nt1492, SEQ ID No: 313) and 3DL3_SBT_Ex3_R (5′-ATCCCCCTTTACCCCAAAT-3′, nt1905˜nt1923, SEQ ID No: 314), respectively;
The forward and reverse sequencing primers for exon 4 of KIR3DL3 are 3DL3_SBT_Ex4_F (5′-GGGAAGCCTCACTTATTTCAG-3′, nt2996˜nt3016, SEQ ID No: 315) and 3DL3_SBT_Ex4_R (5′-ACCTGGGGCTTCCAGTCCT-3′, nt3431˜nt3449, SEQ ID No: 316), respectively;
The forward and reverse sequencing primers for exon 5 of KIR3DL3 are 3DL3_SBT_Ex5_F (5′-GAGAGCTGTGACAASGAAG-3′, nt4900˜nt4918, SEQ ID No: 317) and 3DL3_SBT_Ex5_R (5′-GCAGGAAGCTCCTCAGCTA-3′, nt5294˜nt5312, SEQ ID No: 318), respectively;
The forward and reverse sequencing primers for exon 7 of KIR3DL3 are 3DL3_SBT_Ex7_F (5′-GTGAGACAATTCATATAGA-3′, nt10650˜nt10668, SEQ ID No: 319) and 3DL3_SBT_Ex7_R (5′-TGCTTCCCCACATGGCCCT-3′, nt10852˜nt10870, SEQ ID No: 320), respectively;
The forward and reverse sequencing primers for exon 8 of KIR3DL3 are 3DL3_SBT_Ex8_F (5′-GACCTCAGGCACCTATGGC-3′, nt11178˜nt11196, SEQ ID No: 321) and 3DL3_SBT_Ex8_R (5′-GAGTGAGGGAGGGTGCTCA-3′, nt11395˜nt11413, SEQ ID No: 322), respectively;
The forward and reverse sequencing primers for exon 9 of KIR3DL3 are 3DL3_SBT_Ex9_F (5′-CRTGGCTAGTCTTATTCCC-3′, nt11358˜nt11376, SEQ ID No: 323) and 3DL3_SBT_Ex9_R (5′-CCCTAGAAGATCCCATCAA-3′, nt11627˜nt11645, SEQ ID No: 324), respectively.
(14) The forward and reverse sequencing primers for exon 1 of KIR3DS1 are 3DS1_SBT_Ex1_F (5′-AAGCCATGCTCCGCTCTTG-3′, nt-181˜nt-163, SEQ ID No: 325) and 3DS1_SBT_Ex1_R (5′-CAGATCTCCATCCCCGCAC-3′, nt65˜nt83, SEQ ID No: 326), respectively;
The forward and reverse sequencing primers for exon 2 of KIR3DS1 are 3D51_SBT_Ex2_F (5′-AGTGGGGGCAGCAGGGTG-3′, nt968˜nt985, SEQ ID No: 327) and 3DS1_SBT_Ex2_R (5′-AATGTGGGCCGAGCATCCG-3′, nt1182˜nt1200, SEQ ID No: 328), respectively;
The forward and reverse sequencing primers for exon 3 of KIR3DS1 are 3D51_SBT_Ex3_F (5′-GGGGAGAATCTTCTGGGCACT-3′, nt1735˜nt1755, SEQ ID No: 329) and 3DS1_SBT_Ex3_R (5′-TGATGGGACCCTGACGGAC-3′, nt2166˜nt2184, SEQ ID No: 330), respectively;
The forward and reverse sequencing primers for exon 4 of KIR3DS1 are 3DS1_SBT_Ex4_F (5′-GGAGAGAGACAGACACGGG-3′, nt3485˜nt3503, SEQ ID No: 331) and 3DS1_SBT_Ex4_R (5′-TGGTACAGACCTCACCAAG-3′, nt4007˜nt4025, SEQ ID No: 332), respectively;
The forward and reverse sequencing primers for exon 5 of KIR3DS1 are 3D51_SBT_Ex5_F (5′-CAGGTGTGAGGGGAGCTGT-3′, nt5403˜nt5421, SEQ ID No: 333) and 3DS1_SBT_Ex5_R (5′-CCTGTCTGCCATCCTGCGC-3′, nt5892˜nt5910, SEQ ID No: 334), respectively;
The forward and reverse sequencing primers for exon 6 of KIR3DS1 are 3DS1_SBT_Ex6_F (5′-TCAAGACAGTGGGCATCGCAC-3′, nt8763˜nt8783, SEQ ID No: 335) and 3D51_SBT_Ex6_R (5′-GGGAGGTTTGAGCCAACGCTT-3′, nt9045˜nt9065, SEQ ID No: 336), respectively;
The forward and reverse sequencing primers for exon 7 of KIR3DS1 are 3DS1_SBT_Ex7_F (5′-CGCTGTATGTGGTTACCTGTG-3′, nt13165˜nt13185, SEQ ID No: 337) and 3DS1_SBT_Ex7_R (5′-GGTGAGGAACACACACCCG-3′, nt13432˜nt13450, SEQ ID No: 338), respectively;
The forward and reverse sequencing primers for exon 8 of KIR3DS1 are 3D51_SBT_Ex8_F (5′-CCAGAAGTGCCCTCCGAGC-3′, nt13784˜nt13802, SEQ ID No: 339) and 3DS1_SBT_Ex8_R (5′-GCTGAGTGAGGGAGGGTGC-3′, nt13944˜nt13962, SEQ ID No: 340), respectively;
The forward and reverse sequencing primers for exon 9 of KIR3DS1 are 3D51_SBT_Ex9_F (5′-CGTGGCTAGTGTTATTCCC-3′, nt13904˜nt13922, SEQ ID No: 341) and 3DS1_SBT_Ex9_R (5′-GGCCTCTGAGAAGGGCGAG-3′, nt14055˜nt14073, SEQ ID No: 342), respectively.
VIII. All the sequencing reactions can be carried out in a volume of 10 μL containing:
IX. The thermocycling parameters for the sequencing reaction are described below:
Based on the structural features of KIR full genomic sequences, the distribution of single nucleotide polymorphisms in their coding regions and the length of flanking intronic sequence of each exon, the present disclosure has established a scientific and efficient PCR amplification strategy for all the 14 functional KIR genes. We design KIR gene-specific PCR and sequencing primers, and explore the optimal PCR amplification and sequencing conditions. This disclosure allows for simultaneous genotyping of 14 functional KIR genes by SBT, which is suitable for high-resolution level KIR genotyping, population genetics, tissue typing for bone marrow transplant and disease-associated studies.
The contributions of the present disclosure include: for the first time this disclosure has established the method for high-throughput simultaneous sequence-based typing of 14 functional KIR genes at high-resolution level. KIR gene-specific PCR primers with similar annealing temperatures have been designed, which allow for simultaneous PCR amplification of 14 functional KIR genes under the same PCR conditions and make the PCR procedure less time-consuming and labor-consuming. The occurrence of non-specific amplification or co-amplification events has been solved via designing KIR gene-specific PCR primers. Without using the extra commercial KIR-SSP kit, the presence or absence of 14 functional KIR genes can also be identified by agarose gel electrophoresis of PCR products. Based on our KIR SBT method, the entire coding sequence for all the 14 functional KIR genes are sequenced in both direction, the deficiencies existed in previous literatures can be overcome. Moreover, no noise and artifacts are observed in the obtained sequences, which can greatly facilitate the process of KIR allele assignment.
51% (7432/14577)
In order to more clearly illustrate the embodiments of the present disclosure or the prior art solutions, a brief description of the accompanying drawings for use with the illustration of the embodiments or the prior art are provided below. It is obvious that the drawings described below depict merely some embodiments of the disclosure and those of ordinary skill in the art can obtain other drawings based on the arrangements shown in these drawings without making inventive efforts.
3˜5 pairs of KIR gene-specific PCR primers (except that three pairs of PCR primers are used for KIR3DL3 and five pairs of PCR primers are used for KIR2DL1, four pairs of KIR gene-specific PCR primers are used for each of other functional KIR genes) are used to amplify the complete coding sequence of each functional KIR gene. The nucleotide sequences of the exons carried by each amplicon were determined in both directions using the specific forward and reverse sequencing primers. As for KIR2DL1˜5, 2DS1˜5 and KIR3DL3 genes, each KIR gene is sequenced by sixteen specific sequencing primers, respectively. For KIR3DL1˜2 and KIR3DS1 genes, each KIR gene is sequenced by eighteen specific sequencing primers, respectively.
The foregoing objects, features and advantages of the present disclosure will be described in greater detail with reference to the accompanying drawings.
In the present embodiment, a randomly selected DNA sample which had been previously identified as KIRAA1 profile using a commercial KIR-SSP kit was subjected to sequence-based typing according to the present disclosure with an aim to confirm the effect of this disclosure. Firstly, the complete coding region of each functional KIR gene was separately amplified using 3˜5 pairs of KIR gene-specific PCR primers in an ABI 9700 PCR cycler. All the PCR amplifications were carried out in a volume of 10 μL containing:
All the PCR amplifications were simultaneously amplified under the same thermocycling parameters described below:
To confirm successful PCR amplification, 2 μL PCR products mixed with 1 μL nucleic acid dye as well as 3 μL 5×loading buffer were electrophoresed on a 2% agarose gel. The expected sizes of PCR products were in comparison with Takara DL2000 DNA markers. As a result, specific and clear PCR product bands for 7 KIR genes (KIR2DL1, 2DL3, 2DL4, 2DS4, 3DL1, 3DL2 and 3DL3) were observed on the gel, indicating the tested sample carried the above KIR genes. However, the remaining 7 functional KIR genes including KIR2DL2, 2DL5, 2DS1, 2DS2, 2DS3, 2DS5 and 3DS1 were absent since no specific PCR product band was observed on the gel. Thus, the PCR products amplified by using the PCR primers of this disclosure were subjected to agarose gel electrophoresis, the result was completely consistent with the known KIRAA1 profile. The electrophoresis images of the tested sample are shown in
Now that the 7 functional KIR genes (KIR2DL1, 2DL3, 2DL4, 2DS4, 3DL1, 3DL2 and 3DL3) were present for the tested sample, the specific PCR products of these KIR genes were then purified using the same purification system described below:
Purification of PCR products were carried out under the same thermocycling parameters described below:
Upon completion, dilute the purified product 1:3 with sterile deionized water. Mix gently by vortexing and centrifuge briefly.
The nucleotide sequences of each exon carried by purified PCR amplicons were determined in both directions. As for KIR2DL1, 2DL3, 2DL4, 2DS4 and 3DL3 genes, each KIR gene was sequenced using sixteen specific sequencing primers, respectively. For KIR3DL1 and 3DL2 genes, each KIR gene was sequenced using eighteen specific sequencing primers, respectively. All the sequencing reactions were carried out in a volume of 10 μL containing:
The thermocycling parameters for all the sequencing reactions were carried out as follows:
When the sequencing reaction was completed, purification of sequencing reaction products was carried out by ethanol/NaOAc/EDTA precipitation method, and finally added 15 μL of Hi-Di formamide solution to each well and then denatured at 95° C. for 2.5 min in a PCR cycler. The purified sequencing reaction products were detected by capillary electrophoresis in an ABI 3730 DNA sequencer. All the obtained sequences were then imported into Assign 3.5 or 4.7 software (Conexio Genomics, Western Australia) (see
In the present embodiment, a randomly selected DNA sample which had been previously identified as KIRAB6 profile using a commercial KIR-SSP kit was subjected to sequence-based typing according to the present disclosure with an aim to confirm the effect of this disclosure. Firstly, the complete coding region of each functional KIR gene was separately amplified using 3˜5 pairs of KIR gene-specific PCR primers in an ABI 9700 PCR cycler. All the PCR amplifications were carried out in a volume of 10 μL containing:
All the PCR amplifications were simultaneously amplified under the same thermocycling parameters described below:
To confirm successful PCR amplification, 2 μL PCR products mixed with 1 μL nucleic acid dye as well as 3 μL loading buffer were electrophoresed on a 2% agarose gel. The expected sizes of PCR products were in comparison with Takara DL2000 DNA markers. As a result, specific and clear PCR product bands for 14 KIR genes (KIR2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3 and 3DS1) were observed on gel, indicating the tested sample carried the above KIR genes. Thus, the PCR products amplified by using the PCR primers of this disclosure subjected to agarose gel electrophoresis, the result is completely consistent with the known KIRAB6 profile. The electrophoresis images of the tested sample are shown in
Now that the 14 functional KIR genes (KIR2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3 and 3DS1) were present for this tested sample, the specific PCR products of these KIR genes were then purified using the same purification system described below:
Purification of PCR products were carried out under the same thermocycling parameters described below:
Upon completion, diluted the purified PCR product 1:3 with sterile deionized water. Mixed gently by vortexing and centrifuge briefly.
The nucleotide sequences of each exon carried by the purified PCR amplicons were determined in both directions. As for KIR2DL1˜5, 2DS1˜5 and KIR3DL3 genes, each KIR gene was sequenced using sixteen specific sequencing primers, respectively. For KIR3DL1˜2 and KIR3DS1 genes, each KIR gene was sequenced using eighteen specific sequencing primers, respectively. All the sequencing reactions were carried out in a volume of 10 μL containing:
The thermocycling parameters for all the sequencing reactions were carried out as follows:
When the sequencing reactions were completed, purification of sequencing reaction products was carried out by ethanol/NaOAc/EDTA precipitation method, and finally added 15 μL, of Hi-Di formamide solution to each well and then denatured at 95V for 2.5 min in a PCR cycler. The purified sequencing reaction products were detected by capillary electrophoresis in an ABI 3730 DNA sequencer. All the obtained sequences were then imported into Assign 3.5 or 4.7 software (Conexio Genomics, Western Australia) (
In the present embodiment, a total number of 306 randomly selected DNA samples which had been previously detected using a commercial KIR-SSP kit were subjected to sequence-based typing according to the present disclosure with an aim to confirm the effect of this disclosure. Firstly, the complete coding region of each functional KIR gene was separately amplified using 3˜5 pairs of KIR gene-specific PCR primers in an ABI 9700 PCR cycler. All the PCR amplifications were carried out in a volume of 10 μL containing:
All the PCR amplifications were simultaneously amplified under the same thermocycling parameters described below:
To confirm successful PCR amplification, 2 μL PCR products mixed with 1 μL nucleic acid dye as well as 3 μL loading buffer were electrophoresed on a 2% agarose gel. The expected sizes of PCR products were in comparison with Takara DL2000 DNA markers. As a result, the PCR products amplified by using the PCR primers of this disclosure were subjected to agarose gel electrophoresis, the results were completely consistent with the known KIR profiles for all the 306 DNA samples.
PCR products for KIR genes that were present in each tested DNA sample, were then purified using the same purification system described below:
Purification of PCR products were carried out under the same thermocycling parameters described below:
Upon completion, dilute the purified PCR products 1:3 with sterile deionized water. Mix gently by vortexing and centrifuge briefly.
The nucleotide sequences of each exon carried by purified PCR amplicons were determined in both directions. As for KIR2DL1˜5, 2DS1˜5, and KIR3DL3 genes, each KIR gene was sequenced using sixteen specific sequencing primers, respectively. For KIR3DL1˜2 and KIR3DS1 genes, each KIR gene was sequenced using eighteen specific sequencing primers, respectively. All the sequencing reactions were carried out in a volume of 10 μL containing:
The thermocycling parameters for all the sequencing reactions were carried out as follows:
When the sequencing reactions were completed, purification of sequencing reaction products was carried out by ethanol/NaOAc/EDTA precipitation method, and finally added 15 μL of Hi-Di formamide solution to each well and then denatured at 95° C. for 2.5 min in a PCR cycler. The purified sequencing reaction products were detected by capillary electrophoresis in an ABI 3730 DNA sequencer. All the obtained sequences were then imported into Assign 3.5 or 4.7 software (Conexio Genomics, Western Australia), the allele level KIR genotype for each sample was identified (see Table 6). The identified alleles and their frequencies of 14 functional KIR genes in southern Chinese Han population (n=306) were provided in Table 7.
indicates data missing or illegible when filed
*01003
*02602
*04802
*062
*063
*064
*065
*009
2*013
3*00109
3*00110
3*025
3*026
3*027
3*028
3*029
3*031
*00304
*00305
*030
*031
*033
*034
*00503
*00504
L1*01505
L1*079
S1*082
S1*083
S1*084
S1*085
*022
*00105
*017
*018
*00706
*00707
*083
*084
*091
*093
*099
The foregoing description merely depicts some exemplary embodiments of the present disclosure and therefore is not intended as limiting the scope of the present disclosure. Any equivalent structural transformations made to the disclosure, or any direct or indirect applications of the disclosure on any other related fields based on concepts of the present disclosure, shall all fall in the scope of the present disclosure.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201710284545.3 | Apr 2017 | CN | national |
