DNA polymorphic analysis from extremely small amounts of DNA samples using a semi-nested PCR
使用半巢式 PCR 对极少量 DNA 样本进行 DNA 多态性分析
基本信息
- 批准号:04670352
- 负责人:
- 金额:$ 1.34万
- 依托单位:
- 依托单位国家:日本
- 项目类别:Grant-in-Aid for General Scientific Research (C)
- 财政年份:1992
- 资助国家:日本
- 起止时间:1992 至 1993
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
Since jeffreys et al. reported that DNA with hypervariable minisatellite loci could be used for identification of individuals, DNA typing systems have been developed. Large amounts(micrograms)of gemonic DNA must be analyzed in these methods, so application of these methods to forensic science samples, consisting of degraded or very small amounts of DNA, is not always possible. The polymerase chain reaction(PCR)procedure has provided a solution to this problem, enabling the amplification of a target DNA region by 100,000 times. However, one finds that the large number of PCR cycles required to amplify extremely small amounts of DNA such as a single sperm cell makes for even less efficiency of the reaction, often resulting in insufficient amounts of PCR products. The nested PCR method is known to increase the amplification sensitivity and can be applied to various DNA analyzes.In the present study, we have improved the procedure of DNA purification or PCR to minimize the inhibition of the contaminated substances. Furthermore, we devised a new PCR system with a semi-nested set of primers in the second PCR to improve the amplification sensitivity of HLA-DQAI gene.Using the semi-nested PCR, more than 2 or 3 pg of template DNA could be amplified and typed correctly. The semi-nested PCR technique was found to enhance the sensitivity and efficiency of the amplification reaction and allowed the successful typing of the HLA-DQA1 gene. This is helpful for genotyping from samples with extremely small amounts of DNA, such as forensic or ancient DNA samples.
自从jeffreys等报道具有高变小卫星基因座的DNA可用于个体鉴定以来,DNA分型系统已经被开发出来。在这些方法中必须分析大量(微克)的基因组DNA,因此将这些方法应用于由降解或非常少量的DNA组成的法医学样品并不总是可能的。聚合酶链式反应(PCR)程序已经提供了解决该问题的方案,使得能够将靶DNA区域扩增100,000倍。然而,人们发现,扩增极少量的DNA(如单个精子细胞)所需的大量PCR循环使得反应效率更低,通常导致PCR产物的量不足。巢式PCR方法可以提高扩增灵敏度,适用于各种DNA分析,本研究对DNA纯化或PCR方法进行了改进,以减少污染物质对扩增的抑制作用。为了提高HLA-DQAI基因扩增的灵敏度,我们设计了一套新的PCR体系,在第二次PCR中采用半套式引物,可扩增出2 ~ 3 pg以上的模板DNA,并能正确分型。半巢式PCR技术提高了扩增反应的灵敏度和效率,并使HLA-DQA 1基因的分型成功。这有助于对含有极少量DNA的样本(例如法医或古代DNA样本)进行基因分型。
项目成果
期刊论文数量(30)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
小島俊典ら: "唾液斑痕を試料としたHLAクラスII遺伝子のDNA型判定" 日本法医学雑誌. 47(5). 380-386 (1993)
Toshinori Kojima 等人:“使用唾液染色样本进行 HLA II 类基因的 DNA 分型”,《日本法医学杂志》47(5) 380-386 (1993)。
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- 影响因子:0
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- 通讯作者:
Okajima,H.et al.: "Amplification of HLA-DQA1 gene from bloodstains by polymerase chain reaction." Japanese Journal of Legal Medicine. 47(1). 6-12 (1993)
Okajima,H.et al.:“通过聚合酶链式反应从血迹中扩增 HLA-DQA1 基因。”
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- 影响因子:0
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Uchihi,R.et al.: "Deoxyribonucleic acid(DNA) typing of human leukocyte antigen (HLA)-DQA1 from single hairs in Japanese." Journal of Forensic Sciences. 37(3). 853-859 (1992)
Uchihi,R.等人:“日本人单根毛发中人类白细胞抗原 (HLA)-DQA1 的脱氧核糖核酸 (DNA) 分型。”
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- 影响因子:0
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玉木 敬二: "日本におけるバイオテクノロジー(17)ーDNAによる個人識別法ー" 現代医学. 40. 169-174 (1992)
Keiji Tamaki:“日本生物技术(17)-基于DNA的个人识别方法”现代医学40。169-174(1992)。
- DOI:
- 发表时间:
- 期刊:
- 影响因子:0
- 作者:
- 通讯作者:
Uchihi,R.et al.: "Deoxyribonucleic acid(DNA)typing of human leukocyte antigen(HLA)-DQA1 from single hairs in Japanese." Journal of Forensic Sciences. 37(3). 853-859 (1992)
Uchihi,R.et al.:“从日本人的单根毛发中对人类白细胞抗原 (HLA)-DQA1 进行脱氧核糖核酸 (DNA) 分型。”
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- 影响因子:0
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KATSUMATA Yoshinao其他文献
KATSUMATA Yoshinao的其他文献
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{{ truncateString('KATSUMATA Yoshinao', 18)}}的其他基金
Establishment of high-sensitive multiplex typing systems for Y-STRs and its application to a very small amount of specimens
Y-STR高灵敏多重分型系统的建立及其在极少量标本中的应用
- 批准号:
13670421 - 财政年份:2001
- 资助金额:
$ 1.34万 - 项目类别:
Grant-in-Aid for Scientific Research (C)
Forensic molecular biological applications using single nucleotide polymorphisms (SNPs) regions
使用单核苷酸多态性 (SNP) 区域的法医分子生物学应用
- 批准号:
12557041 - 财政年份:2000
- 资助金额:
$ 1.34万 - 项目类别:
Grant-in-Aid for Scientific Research (B)
Establishment of paternity test system in Japan
日本亲子鉴定制度的建立
- 批准号:
11307008 - 财政年份:1999
- 资助金额:
$ 1.34万 - 项目类别:
Grant-in-Aid for Scientific Research (A).
The selection of new microsatellite loci suitable for fortune forensic exam ination and the establishment of its wide use
适合命理检验的新微卫星位点的筛选及其广泛应用的建立
- 批准号:
10557046 - 财政年份:1998
- 资助金额:
$ 1.34万 - 项目类别:
Grant-in-Aid for Scientific Research (B)
Studies on the origin of the Japanese based on the analysis of MVR-PCR alleles in asian populations
基于亚洲人群MVR-PCR等位基因分析的日本人起源研究
- 批准号:
09470123 - 财政年份:1997
- 资助金额:
$ 1.34万 - 项目类别:
Grant-in-Aid for Scientific Research (B)
The development of a new HLA-DNA typing method using DNA direct sequencing.
使用 DNA 直接测序开发新的 HLA-DNA 分型方法。
- 批准号:
04557031 - 财政年份:1992
- 资助金额:
$ 1.34万 - 项目类别:
Grant-in-Aid for Developmental Scientific Research (B)