TWI479024B - Method for determining p1/p2 blood type and detection kit thereof - Google Patents
Method for determining p1/p2 blood type and detection kit thereof Download PDFInfo
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本發明係關於檢測P1 /P2 血型的方法及套組，尤係關於以單一核苷酸多型性來檢測P1 /P2 血型的方法及其檢測套組。The present invention is based on the detected P 1 / P 2 blood method and set, especially a method and detection kit based on single nucleotide polymorphism is detected P 1 / P 2 blood group.
1927年，奧地利生物學家卡爾蘭德斯泰納和美國免疫學家菲利普列文在研究新生兒溶血症期間，從輸入人類血液的家兔體內發現一種新抗體。這種抗體可以凝集一部分人的紅細胞(red cells)，而對另一部分人群的紅細胞則沒有效果。他們將這兩種血型分別稱為P1(+)型和P1(-)型。後來，將P1(+)型和P1(-)型分別稱為P1 型和P2 型，此種P1 /P2 血型系統在目前33種人類血型系統中屬於第三種。In 1927, the Austrian biologist Carl Landsteiner and the American immunologist Philip Levin discovered a new antibody from rabbits who had imported human blood during the study of neonatal hemolysis. This kind of antibody can agglutinate some people's red cells, but it has no effect on red blood cells in another part of the population. They refer to these two blood types as P1 (+) and P1 (-), respectively. Later, the P1 (+) type and P1 (-) are referred to as P-type and P-type 2 type 1, this P 1 / P 2 blood group system is present in a third of the 33 kinds of human blood group systems.
P1 /P2 血型系統是由醣脂質抗原組成的系統，然而其分子遺傳特性卻尚未釐清。P1 型和P2 型是與P1及Pk 血型抗原表現在紅細胞上有關，P1及Pk 係為醣類抗原(carbohydrate antigens)可經由不同的生物合成途徑(biosynthetic route)合成，自常見的醣脂(glycosphingolipid) 前驅物-乳糖苷(lactosylceramide)起始。P1及Pk 抗原係由α-(1,4)-半乳糖基轉移酶(galactosyltransferase，簡稱A4GALT)活性所測定。P1 /P2 是一種常見的表現型多型性，其分佈比例在不同的人種間而有不同，P1 表現型頻率在白種人(Caucasians)與非洲人種(Africans)間分別為80%及90%至95%，而在亞洲人種(Asians)間則相對較低，在台灣人種中P1 表現型頻率係介於30%至40%。The P 1 /P 2 blood group system is a system composed of glycolipid antigens, but its molecular genetic properties have not yet been clarified. P 1 and P 2 are related to the expression of P1 and P k blood group antigens on erythrocytes, and P1 and P k are carbohydrate antigens which can be synthesized via different biosynthetic routes. The glycosphingolipid precursor, lactosylceramide, begins. The P1 and Pk antigens are determined by the activity of α-(1,4)-galactosyltransferase (A4GALT). P 1 /P 2 is a common phenotypic polymorphism, and its distribution ratio varies from person to person. The frequency of P 1 phenotype is 80 between Caucasians and Africans. % and 90% to 95%, while relatively low among Asians (Asians), the P 1 phenotype frequency in Taiwanese population is between 30% and 40%.
以DNA基因型作為血型檢測為未來之趨勢。例如，Progenika Biopharma,S.A.(Spain)已開發血液晶片(BLOOD Chip)，而此血液晶片目前包含多種血型之基因型變異。但由於過去P1 /P2 血型之分子遺傳機制並未被證明，使得目前現有的DNA檢測，包括Progenika的血液晶片無法包括此於人群中常見的P1 /P2 血型多型性進行DNA檢測。惟，對於P1 /P2 型血型的檢測，仍有需求。The use of DNA genotypes as blood type tests is a future trend. For example, Progenika Biopharma, SA (Spain) has developed a BLOOD Chip, which currently contains genotype variations of various blood types. However, because the molecular genetic mechanism of the P 1 /P 2 blood type has not been proven in the past, the current DNA testing, including Progenika's blood wafer, cannot include the P 1 /P 2 blood group polymorphism commonly used in the human population for DNA detection. . However, there is still a need for detection of P 1 /P 2 blood types.
Thuresson等人(Thuresson B,Westman JS,Olsson ML.Identification of a novel A4GALT exon reveals the genetic basis of the P1/P2 histo-blood groups.Blood.2011；117(2)：678-687.)先前已報導SNP rs8138197與P1 /P2 表現型的關聯性，且提出了一種新穎之P1 /P2 血型形成的分子模式。Thuresson et al. (Thuresson B, Westman JS, Olsson ML. Identification of a novel A4GALT exon reveals the genetic basis of the P1/P2 histo-blood groups. Blood. 2011; 117(2): 678-687.) previously reported SNP rs8138197 is associated with the P 1 /P 2 phenotype and proposes a novel molecular pattern of P 1 /P 2 blood group formation.
本發明利用不同族群遺傳連結研究(association study)，證明A4GALT基因上的單一核苷酸多型性(SNP)rs2143918及rs5751348和P1 /P2 血型具完全連結之現象。本發明結果提供P1 /P2 血型之分子遺傳機制，並進一步提供利用單一核苷酸多型性rs2143918及rs5751348之基因 型作為P1 /P2 血型DNA鑑定之標記。The present invention utilizes genetic coupling of different ethnic groups (association study), demonstrated the phenomenon is completely connected to the A4GALT gene single nucleotide polymorphism (SNP) rs2143918 and rs5751348 and P 1 / P 2 with blood. The results of the present invention provide a molecular genetic mechanism of the P 1 /P 2 blood type, and further provide genotypes using the single nucleotide polymorphisms rs2143918 and rs5751348 as markers for P 1 /P 2 blood group DNA identification.
本發明針對四個種族族群進行大規模的調查試驗，其結果顯示自具有P1 型非洲人以及標示為M3之台灣人家族之譜系分析所得之結果發現SNP rs8138197基因型並未與定義P1 /P2 血型呈現完全的關聯。The present invention conducted a large-scale investigation on four ethnic groups, and the results showed that the SNP rs8138197 genotype was not defined with the definition of P 1 / from the pedigree analysis of the P 1 type African and the Taiwanese family labeled M3. The P 2 blood type presents a complete association.
本發明提供一種鑑別P1 /P2 血型之方法，包括：提供個體之生物樣本；檢測該生物樣本之A4GALT基因中單一核苷酸多型性rs2143918或rs5751348位置之基因型；以及依據所檢測之該基因型，決定該個體之表現型。The invention provides a method for identifying a P 1 /P 2 blood type, comprising: providing a biological sample of an individual; detecting a genotype of a single nucleotide polymorphic rs2143918 or rs5751348 position in the A4GALT gene of the biological sample; This genotype determines the phenotype of the individual.
於本發明之一具體實施例中，該單一核苷酸多型性rs2143918位置之基因型為T/T、T/G或G/G。In a specific embodiment of the invention, the genotype of the single nucleotide polymorphic rs2143918 locus is T/T, T/G or G/G.
於本發明之一具體實施例中，該單一核苷酸多型性rs2143918位置之基因型為T/T或T/G係表示該個體具有P1 表現型。In a specific embodiment of the invention, the genotype of the single nucleotide polymorphic rs2143918 locus is T/T or T/G line indicating that the individual has a P 1 phenotype.
於本發明之一具體實施例中，該單一核苷酸多型性rs2143918位置之基因型為G/G係表示該個體具有P2 表現型。In a specific embodiment of the invention, the genotype of the single nucleotide polymorphic rs2143918 locus is G/G line indicating that the individual has a P 2 phenotype.
於本發明之一具體實施例中，該單一核苷酸多型性rs5751348位置之基因型為G/G、G/T或T/T。In a specific embodiment of the invention, the genotype of the single nucleotide polymorphic rs5751348 locus is G/G, G/T or T/T.
於本發明之一具體實施例中，該單一核苷酸多型性rs5751348位置之基因型為G/G或G/T係表示該個體具有P1 表現型。In a specific embodiment of the invention, the genotype of the single nucleotide polymorphic rs5751348 locus is G/G or G/T line indicating that the individual has a P 1 phenotype.
於本發明之一具體實施例中，該單一核苷酸多型性 rs5751348位置之基因型為T/T係表示該個體具有P2 表現型。In a specific embodiment of the invention, the genotype of the single nucleotide polymorphic rs5751348 locus is T/T line indicating that the individual has a P 2 phenotype.
於本發明之一具體實施例中，該生物樣本為該個體之血液或唾液。In a specific embodiment of the invention, the biological sample is blood or saliva of the individual.
於本發明之一具體實施例中，該生物樣本之檢測係以聚合酶連鎖反應(Polymerase Chain Reaction，PCR)檢測該生物樣本中之核酸。In a specific embodiment of the present invention, the biological sample is detected by a polymerase chain reaction (PCR) to detect nucleic acid in the biological sample.
於本發明之一具體實施例中，該聚合酶連鎖反應係以SEQ ID NOS：1及2之引子對檢測該生物樣本中之該核酸。In a specific embodiment of the invention, the polymerase chain reaction detects the nucleic acid in the biological sample with a primer pair of SEQ ID NOS: 1 and 2.
於本發明之一具體實施例中，該聚合酶連鎖反應係以SEQ ID NOS：3及4之引子對檢測該生物樣本中之該核酸。In a specific embodiment of the invention, the polymerase chain reaction detects the nucleic acid in the biological sample with a primer pair of SEQ ID NOS: 3 and 4.
本發明提供一種用於鑑別P1 /P2 血型之套組，包括：一組引子對，用以檢測個體核酸樣本中A4GALT因基單一核苷酸多型性rs2143918或rs5751348位置之基因型。The invention provides a kit for identifying a P 1 /P 2 blood type, comprising: a set of primer pairs for detecting the genotype of the A4GALT dependent single nucleotide polymorphic rs2143918 or rs5751348 position in an individual nucleic acid sample.
於本發明之一具體實施例中，該引子對為分別具有SEQ ID NOS：1及2之核苷酸序列。In a specific embodiment of the invention, the primer pair is a nucleotide sequence having SEQ ID NOS: 1 and 2, respectively.
於本發明之一具體實施例中，該引子對為分別具有SEQ ID NOS：3及4之核苷酸序列。In a specific embodiment of the invention, the primer pair is a nucleotide sequence having SEQ ID NOS: 3 and 4, respectively.
於本發明之一具體實施例中，該單一核苷酸多型性rs2143918位置之基因型為T/T、T/G或G/G。In a specific embodiment of the invention, the genotype of the single nucleotide polymorphic rs2143918 locus is T/T, T/G or G/G.
於本發明之一具體實施例中，該單一核苷酸多型性rs2143918位置之基因型為T/T或T/G係表示該個體具有P1 表現型。In a specific embodiment of the invention, the genotype of the single nucleotide polymorphic rs2143918 locus is T/T or T/G line indicating that the individual has a P 1 phenotype.
於本發明之一具體實施例中，該單一核苷酸多型性 rs2143918位置之基因型為G/G係表示該個體具有P2 表現型。In a specific embodiment of the invention, the genotype of the single nucleotide polymorphic rs2143918 locus is G/G line indicating that the individual has a P 2 phenotype.
於本發明之一具體實施例中，該單一核苷酸多型性rs5751348位置之基因型為G/G、G/T或T/T。In a specific embodiment of the invention, the genotype of the single nucleotide polymorphic rs5751348 locus is G/G, G/T or T/T.
於本發明之一具體實施例中，該單一核苷酸多型性rs5751348位置之基因型為G/G或G/T係表示該個體具有P1 表現型。In a specific embodiment of the invention, the genotype of the single nucleotide polymorphic rs5751348 locus is G/G or G/T line indicating that the individual has a P 1 phenotype.
於本發明之一具體實施例中，該單一核苷酸多型性rs5751348位置之基因型為T/T係表示該個體具有P2 表現型。In a specific embodiment of the invention, the genotype of the single nucleotide polymorphic rs5751348 locus is T/T line indicating that the individual has a P 2 phenotype.
第1圖係顯示遍及四個種族族群之P1 及P2 個體中最有可能的單倍型對的分佈；以及第2圖係顯示P1 /P2 表現型以及M3家族中最有可能的單倍型對。Figure 1 shows the distribution of the most likely haplotype pairs among the P 1 and P 2 individuals across the four ethnic groups; and Figure 2 shows the P 1 /P 2 phenotype and the most likely of the M3 family. Haplotype pair.
以下係藉由特定的具體實施例說明本發明之實施方式，熟習此專業之人士可由本說明書所揭示之內容輕易地瞭解本發明之優點及功效。本發明亦可藉由其它不同之實施方式加以施行或應用，本說明書中的各項細節亦可基於不同觀點與應用，在不悖離本發明所揭示之精神下賦予不同之修飾與變更。The embodiments of the present invention are described by way of specific examples, and those skilled in the art can readily understand the advantages and effects of the present invention from the disclosure. The present invention may be embodied or applied by other different embodiments, and the various details of the present invention may be variously modified and changed without departing from the spirit and scope of the invention.
本發明提供一種鑑別P1 /P2 血型之方法，該方法包括：分析來自個體之生物樣本，以檢測該生物樣本中A4GALT 基因之單一核苷酸多型性(Single Nucleotide Polymorphism，SNP)之基因型，其中，該單一核苷酸多型性係位於該A4GALT基因中之SNP rs2143918或SNP rs5751348位置；以及依該SNP rs2143918或SNP rs5751348之基因型，鑑別該個體具有P1 表現型或P2 表現型。The present invention provides a method for identifying a P 1 /P 2 blood type, the method comprising: analyzing a biological sample from an individual to detect a gene of a single nucleotide polymorphism (SNP) of the A4GALT gene in the biological sample. Type, wherein the single nucleotide polymorphism is located at the SNP rs2143918 or SNP rs5751348 position in the A4GALT gene; and the genotype of the SNP rs2143918 or SNP rs5751348 is identified to have a P 1 phenotype or P 2 expression type.
本發明提供一種用於鑑別P1 /P2 血型之套組，其包括PCR引子對，其可用於以受檢測個體之核酸樣本為模版，擴增出帶有A4GALT因基之單一核苷酸多型性(SNP)之核酸片段，該單一核苷酸多型性係位於SNP rs2143918或SNP rs5751348位置，其中，該PCR引子對可進行引子延伸反應，藉此檢測SNP rs2143918或SNP rs5751348之基因型，以鑑別該個體具有P1 表現型或P2 表現型。The present invention provides a kit for identifying a P 1 /P 2 blood type, which comprises a PCR primer pair, which can be used to amplify a single nucleotide having an A4GALT factor base by using a nucleic acid sample of the test subject as a template. A nucleic acid fragment of the type (SNP), which is located at the position of SNP rs2143918 or SNP rs5751348, wherein the PCR primer pair can be subjected to primer extension reaction, thereby detecting the genotype of SNP rs2143918 or SNP rs5751348, To identify that the individual has a P 1 phenotype or a P 2 phenotype.
1.樣本製備及P1 /P2 表現型1. Sample preparation and P 1 /P 2 phenotype
將橫跨4個人種的338個無相關個體(包括227位台灣人、32位印度人、46位白種人及33位非洲人(包含2位非洲裔美國人))納入本發明的樣本中，自各個體收集周邊血液(peripheral blood)樣本，藉由使用抗P1單株抗體試劑(monoclonal anti-P1 reagent)(Immucor Inc.,Houston,TX)的標準血球凝集試驗(hemagglutination)測定各樣本的P1 /P2 表現型，使用QIA DNA血液小套組(QIA DNA Blood Mini Kit)(Qiagen GmbH,Germany)自該周邊血液細胞純化出基因組DNA。338 unrelated individuals (including 227 Taiwanese, 32 Indians, 46 Caucasians, and 33 Africans (including 2 African Americans) across 4 races were included in the samples of the present invention, Peripheral blood samples were collected from each body, and P 1 of each sample was determined by standard hemagglutination test using anti-P1 reagent (Immucor Inc., Houston, TX). /P 2 phenotype, genomic DNA was purified from the peripheral blood cells using the QIA DNA Blood Mini Kit (Qiagen GmbH, Germany).
2.單一核苷酸多型性的分析2. Analysis of single nucleotide polymorphism
藉由聚合酶連鎖反應(polymerase chain reaction，簡稱PCR)分別擴增57個DNA片段，此涵蓋所有的單一核苷酸多型性位點(SNP site)分布橫跨該A4GALT基因的-7.0-Kb至+17.3Kb區域。該PCR的引子序列及其位置係列示於表1中。藉由使用桑格法(Sanger’s method)將經擴增的DNA片段直接定序而測定在單一核苷酸多型性位點的核苷酸。Amplification of 57 DNA fragments by polymerase chain reaction (PCR), which covers all single nucleotide polymorphisms (SNP sites) distributed across the A4GALT gene -7.0-Kb To the +17.3Kb area. The primer sequence of this PCR and its position series are shown in Table 1. Nucleotides at a single nucleotide polymorphism site are determined by direct sequencing of the amplified DNA fragments using the Sanger's method.
3.用以擴增鑑定rs2143918及rs5751348基因型的引子對：用於擴增鑑定rs2143918基因型之正向引子具有如SEQ ID NO：1所示之序列，而反向引子則具有如SEQ ID NO：2所示之序列。3. A primer pair for amplifying and identifying the rs2143918 and rs5751348 genotypes: a forward primer for amplifying and identifying the rs2143918 genotype has the sequence shown in SEQ ID NO: 1, and a reverse primer having SEQ ID NO The sequence shown in :2.
AAGTGCACCTCCTCTCACTC(SEQ ID NO：1)AAGTGCACCTCCTCTCACTC (SEQ ID NO: 1)
TCTAGCTTTCCCATCAGC(SEQ ID NO：2)TCTAGCTTTCCCATCAGC (SEQ ID NO: 2)
用於擴增鑑定rs5751348基因型之正向引子具有如SEQ ID NO：3所示之序列，而反向引子則具有如SEQ ID NO：4所示之序列。The forward primer for amplification identification of the rs5751348 genotype has the sequence shown in SEQ ID NO: 3, and the reverse primer has the sequence shown as SEQ ID NO: 4.
TCACGAGCATTCCTCATC(SEQ ID NO：3)TCACGAGCATTCCTCATC (SEQ ID NO: 3)
CTCCTCTCTATCTCTCTGTC(SEQ ID NO：4)CTCCTCTCTATCTCTCTGTC (SEQ ID NO: 4)
4.結果4. Results
(1).識別與P1 /P2 表現型關聯之A4GALT基因中的SNP之試驗調查(1). A pilot survey to identify SNPs in the A4GALT gene associated with the P 1 /P 2 phenotype
為了探究P1 /P2 血型系統的分子遺傳基礎，本發明進行了試驗性調查，其涉及自四位具有P1 表現型的台灣人和四位具有P2 表現型的台灣人進行詳細和逐步篩選A4GALT基因的SNP。該SNP的篩選起始自該基因的5’啟動子區域，且逐步延長至該基因的5’及3’區域，用以識別任何與P1 /P2 表現型有關之多型性核苷酸位置。將PCR用於擴增涵蓋A4GALT基因中各SNP的DNA片段，其係已記錄於美 國國家生物技術信息中心(National Center for Biotechnology Information，簡稱NCBI)的SNP資料庫中(詳見http：//www.ncbi.nlm.nih.gov/snp)。最終，57個DNA片段各別以PCR擴增，且藉由此經擴增的DNA片段包含共計有416個不同SNP位點，該SNP位點係分佈於A4GALT基因的24.3-kb區域，包括7.0Kb之5’啟動子區域、外顯子1(74bp)以及17.3kb之5’部分的內含子1。測定遍及八個樣品的各SNP之核苷酸，其結果總結於表2，其顯示八個個體之11個SNP位點表現與P1 /P2 表現型之關聯性，此11個SNP分佈於A4GALT基因內含子1之+1.3-kb至+11.5-kb區域間，其在本發明中標示為自5’至3’之SNP1至SNP11。In order to explore the molecular genetic basis of the P 1 /P 2 blood group system, the present invention conducted a pilot investigation involving detailed and stepwise implementation from four Taiwanese with P 1 phenotype and four Taiwanese with P 2 phenotype. Screening for SNPs of the A4GALT gene. This SNP screening starting from the 5 'promoter region, and to gradually extend the gene 5' of the gene and the 3 'region, as much as any type to identify related nucleotide P 1 / P 2 phenotype position. PCR was used to amplify DNA fragments covering each SNP in the A4GALT gene, which has been recorded in the SNP database of the National Center for Biotechnology Information (NCBI) (see http://www for details). .ncbi.nlm.nih.gov/snp). Finally, 57 DNA fragments were each amplified by PCR, and the amplified DNA fragment contained a total of 416 different SNP sites distributed in the 24.3-kb region of the A4GALT gene, including 7.0. Intron 1 of the 5' promoter region of Kb, exon 1 (74 bp) and intron 1 of the 5' portion of 17.3 kb. The nucleotides of each SNP were measured over eight samples. The results are summarized in Table 2, which shows the association of 11 SNP loci in eight individuals with the P 1 /P 2 phenotype, and the 11 SNPs are distributed in The A4GALT gene contains the +1.3-kb to +11.5-kb region of intron 1, which is indicated in the present invention as SNP1 to SNP11 from 5' to 3'.
(2).SNP rs2143918及rs5751348顯示在不同種族人群間具有與P1 /P2 表現型明確的關聯(2).SNP rs2143918 and rs5751348 show a clear association with P 1 /P 2 phenotype among different ethnic groups
為了驗證這11個SNP(SNP1至SNP11)與P1 /P2 表現型的關聯性，進行不同種族人群的大規模關聯性試驗，此試驗包含227位台灣人(包含上述試驗性調查中所分析的八個個體)、32位印度人、46位白種人及33位非洲人(黑人)，測定此338個體之P1 /P2 表現型及SNP1至SNP11的基因型，各種族之P1 及P2 個體中之11個SNP的基因型分佈係顯示於表3。重建該四族群中11個SNP的單倍型(haplotype)，使用PHASE程式(第2.1版)將最有可能的單倍型對(haplotype pair)分配各個體，第1圖顯示各族群之P1 及P2 個體中最有可能之單倍型對的分佈。To validate the association of these 11 SNPs (SNP1 to SNP11) with the P 1 /P 2 phenotype, a large-scale association trial of different ethnic groups was conducted. This trial included 227 Taiwanese (including the analysis in the above pilot study). the eight individual), 32 Indians, 46 Caucasians and 33 African (black), the determination of this subject 338 P 1 / P 2 phenotype and genotype SNP11 SNP1 to all races of P 1 and 11 SNP-based genotype of the individual P 2 are shown in table 3. Reconstruct the haplotype of 11 SNPs in the four populations, and assign the most likely haplotype pair to each body using the PHASE program (version 2.1). Figure 1 shows the P 1 of each ethnic group. and P 2 individuals most likely distribution of haplotype pairs.
使用八個台灣人所形成的試驗性分析確認的兩個單倍型被發現是存在於更廣泛的研究中之兩個主要的單倍型，也與遍及四個族群之P1 和P2 的表現型有關。除了在四個族群中所發現的二個主要單倍型外，確認已於11個SNP之間經歷了重組的單倍型，且發現白種人和非洲人比台灣人及印度人更為頻繁。SNP1、SNP3、SNP4以及SNP7至SNP11與P1 /P2 表現型的相關性可以很容易地排除，確認遍及不同族群所分佈的多個案例(詳見表3及第1圖)為在這些SNP之間具有重組單倍型，且因此產生於其他所納入個體之大多數中所觀察到的基因型-表現型的不一致。Two haplotypes identified using experimental analysis by eight Taiwanese were found to be the two major haplotypes present in the broader study, as well as P 1 and P 2 throughout the four populations. Relevant phenotype. In addition to the two major haplotypes found in the four ethnic groups, it was confirmed that recombinant haplotypes had been experienced between 11 SNPs, and Caucasians and Africans were found to be more frequent than Taiwanese and Indians. The correlation between SNP1, SNP3, SNP4 and SNP7 to SNP11 and P 1 /P 2 phenotypes can be easily ruled out, confirming multiple cases distributed across different ethnic groups (see Table 3 and Figure 1 for details). There is a reciprocal haplotype between them, and thus an inconsistency in the genotype-phenotype observed in most of the other included individuals.
在338位所納入的個體中，僅有一例(P1 型非洲人，在 表3中以以”**”註記者及第1圖中以”*”註記者)顯示針對SNP rs8138197的基因型-表現型的不一致，在先前的研究分析208位瑞典人個體之P1 /P2 表現型中，也發現一例此SNP(rs8138197)的基因型與P1 /P2 表現型不一致。除了此P1 型非洲人之例子外，確認標記為M3的P1 型台灣人(第1圖中以”*”註記者)為具有涉及在SNP3及SNP4所發生之重組的對偶基因，隨後將其家人納入進行譜系分析，結果發現於M3所確認之經重組的對偶基因係存在於M3的父親及兄弟中，二者皆標示為具有P2 表現型(如第2圖所示)，自該譜系分析所得的結果表示SNP rs8138197並未涉及定義P1 /P2 表現型。In the 338 individuals included in only one case (P African type 1, to 3 to "**" in FIG. 1 and second injection correspondent to the "*" correspondent table) shows the genotype for SNP rs8138197 - Inconsistent phenotypes. In previous studies analyzing the P 1 /P 2 phenotypes of 208 Swedish individuals, one genotype of this SNP (rs8138197) was also found to be inconsistent with the P 1 /P 2 phenotype. In addition to the examples of this P type 1 African, the confirmation flag is P-type 1 Taiwanese M3 (FIG. 1 to the "*" reporter) having relates recombination which occurs the SNP3 and SNP4 allele, then His family was included in the pedigree analysis and found that the recombinant dual gene line identified in M3 was present in the father and brother of M3, both of which were labeled as having a P 2 phenotype (as shown in Figure 2). The results obtained from lineage analysis indicated that SNP rs8138197 did not involve the definition of the P 1 /P 2 phenotype.
當上述結果合併考慮時，使用遍及不同族群的擴大調查，顯示與P1 /P2 表現型明確相關的SNP限縮為兩個，亦即SNP5(rs2143918)及SNP6(rs5751348)。沿著A4GALT基因之+1.3-kb至+3.7-kb區域所分佈的SNP之基因型(其涵蓋SNP1至SNP7位點)，將所納入之338位個體進行全面性調查，並無發現其他SNP顯示與P1 /P2 表現型之相關性。SNP rs2143918及SNP rs5751348之基因型，在具相當遺傳距離之四個種族之338位個體中，與P1 /P2 表現型之多型性有一致性的相關性。When the above results were considered together, an expanded survey using different populations showed that SNPs with a clear correlation with the P 1 /P 2 phenotype were reduced to two, namely SNP5 (rs2143918) and SNP6 (rs5751348). The genotype of the SNP distributed along the +1.3-kb to +3.7-kb region of the A4GALT gene (which covers the SNP1 to SNP7 sites), the 338 individuals included were comprehensively investigated, and no other SNPs were found. Correlation with the P 1 /P 2 phenotype. The genotypes of SNP rs2143918 and SNP rs5751348 were consistently consistent with the polymorphism of the P 1 /P 2 phenotype in 338 individuals of four races with considerable genetic distance.
經由在A4GALT基因中大範圍詳細及逐步的SNP篩選試驗，以及隨後使用四個種族族群的連結分析(association study)，本發明證實在A4GALT基因中SNP rs2143918及rs5751348與P1 /P2 血型有明確的關聯性，由此可知，在SNP rs2143918具有T/T及T/G基因型者與P1 表現型有關，而具有G/G基因型者與P2 表現型有關；而在SNP rs5751348具有G/G及G/T基因型者與P1 表現型有關，而具有T/T基因型者與P2 表現型有關。The SNP rs2143918 and rs5751348 and P 1 /P 2 blood types were confirmed in the A4GALT gene via extensive and detailed SNP screening assays in the A4GALT gene, and subsequent association studies using four ethnic groups. Correlation, it can be seen that SNP rs2143918 has T/T and T/G genotypes associated with P 1 phenotype, while G/G genotypes are associated with P 2 phenotype; whereas SNP rs5751348 has G / G and G / T genotype and phenotype related to P 1, and having a T / T genotype and phenotype related to P 2.
上述實施例僅例示性說明本發明之原理及其功效，而非用於限制本發明。任何熟習此項專業之人士均可在不違背本發明之精神及範疇下，對上述實施例進行修飾與改變。因此，舉凡所屬技術領域中具有此項專業知識者，在未脫離本發明所揭示之精神與技術原理下所完成之一切等效修飾或改變，仍應由後述之申請專利範圍所涵蓋。The above-described embodiments are merely illustrative of the principles of the invention and its effects, and are not intended to limit the invention. Modifications and variations of the above-described embodiments can be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, all equivalent modifications or changes made by those skilled in the art without departing from the spirit and scope of the inventions disclosed herein are still covered by the appended claims.
<110> 國立臺灣大學<110> National Taiwan University
<120> 檢測P1 /P2 血型的方法及其檢測套組<120> Method for detecting P 1 /P 2 blood type and detection kit thereof
<160> 4<160> 4
<210> 1<210> 1
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 用於擴增A4GALT基因之正向引子<223> Forward primer for amplifying the A4GALT gene
<400> 1 <400> 1
<210> 2<210> 2
<211> 18<211> 18
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 用於擴增A4GALT基因之反向引子<223> Reverse primer for amplifying the A4GALT gene
<400> 2 <400> 2
<210> 3<210> 3
<211> 18<211> 18
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 用於擴增A4GALT基因之正向引子<223> Forward primer for amplifying the A4GALT gene
<400> 3 <400> 3
<210> 4<210> 4
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 用於擴增A4GALT基因之反向引子<223> Reverse primer for amplifying the A4GALT gene
<400> 4 <400> 4
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| CN110942806A (en) * | 2018-09-25 | 2020-03-31 | 深圳华大法医科技有限公司 | Blood type genotyping method and device and storage medium |
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| US11884961B2 (en) | 2017-06-02 | 2024-01-30 | Georgia State University Research Foundation, Inc. | DNA-glycan conjugates and methods of use |
| CN110942806A (en) * | 2018-09-25 | 2020-03-31 | 深圳华大法医科技有限公司 | Blood type genotyping method and device and storage medium |
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