公开(公告)号：US5747246A

公开(公告)日：1998-05-05

申请号：US263413

申请日：1994-06-21

申请人： Christophe Pannetier ,  Madeleine Cochet ,  Sylvie Darche ,  Philippe Kourilsky

发明人： Christophe Pannetier ,  Madeleine Cochet ,  Sylvie Darche ,  Philippe Kourilsky

IPC分类号： C12Q1/68

CPC分类号： C12Q1/6851

摘要： The present invention relates to a process for determining the quantity of a DNA fragment of interest in a sample wherein: 1) a standard DNA fragment can be amplified with the same oligonucleotide primers is added to the sample to be analyzed containing the DNA fragment of interest, the standard DNA fragment and the fragment of interest differing in sequence and/or in size by not more than 10%, 2) the DNA fragment of interest and the standard fragment are coamplified with the same primers, preferably to saturation of the amplification of the DNA fragment of interest, 3) one or more labeled oligonucleotide primer(s), specific for the DNA fragment of interest and the standard fragment and different from the primers of step 2), is/are added to the reaction medium obtained in step 2), so that, after denaturation of the DNA, said primer(s) hybridize(s) with said fragments at a suitable site in order that an elongation with the DNA polymerase generates labeled DNA fragments of different sizes and/or sequences or with different labels according to whether they originate from the DNA fragment of interest or the standard fragment, respectively, and then 4) the initial quantity of DNA fragment of interest is determined as being the product of the initial quantity of standard DNA fragment and the ratio of the quantity of amplified DNA fragment of interest to the quantity of amplified standard DNA fragment.

摘要翻译： 本发明涉及一种测定样品中感兴趣的DNA片段的量的方法，其中：1）可以用相同的寡核苷酸引物扩增标准DNA片段，将其加入待分析的样品中，其含有感兴趣的DNA片段 ，标准DNA片段和感兴趣的片段在序列和/或大小上不同于10％，2）感兴趣的DNA片段和标准片段用相同的引物共扩增，优选扩增的饱和度 感兴趣的DNA片段，3）在步骤2中获得的反应介质中加入一种或多种针对目的DNA片段特异的标记寡核苷酸引物和与步骤2）的引物不同的标准片段） 2），使得在DNA变性后，所述引物在合适的位点与所述片段杂交，以便DNA聚合酶的延伸产生不同大小的标记的DNA片段 s和/或序列或不同标记，根据它们是否源自目的DNA片段或标准片段，然后4）目的DNA片段的初始量被确定为初始量的 标准DNA片段和目标扩增DNA片段的量与扩增的标准DNA片段的量的比例。

公开(公告)号：US5635354A

公开(公告)日：1997-06-03

申请号：US435529

申请日：1995-05-05

申请人： Philippe Kourilsky ,  Christophe Pannetier ,  Madeleine Cochet

发明人： Philippe Kourilsky ,  Christophe Pannetier ,  Madeleine Cochet

IPC分类号： C07K14/725 ,  C12Q1/68 ,  C12P19/34

CPC分类号： C07K14/7051 ,  C12Q1/6876 ,  C07K2317/24 ,  C07K2319/00

摘要： In a method for describing repertoires of antibodies (Ab) and T cell recers (TcR) of the immune system of an individual, reverse transcription is carried out on the mRNA contained in a biological sample, separate amplifications are then carried out on the transcription product (or directly on the DNA extracted from the sample) by a PCR type method for each primer pair V, C, where V corresponds to a variable segment of the repertoire of interest and C hybridizes on the constant segment of the repertoire of interest. For each J segment of the labelled repertoire, an elongation step is carried out on each of the amplification products using a specific oligonucleotide of this segment J as primer and the amplification product as matrix. For each elongation product corresponding to a triplet (V, C)J thus obtained, the size of the different elongation products is revealed. The description of the repertoires corresponds to a VCJ triplet and to the element size for each element of the repertoire.

摘要翻译： 在描述个体免疫系统的抗体（Ab）和T细胞受体（TcR）的所有组成成分的方法中，对生物样品中所含的mRNA进行逆转录，然后在转录产物上进行单独的扩增 （或直接在从样品提取的DNA上）通过PCR型方法对每个引物对V，C，其中V对应于感兴趣的所有组成成分的可变区，C杂交在所关心的所有组成部分的恒定区段。 对于标记图谱的每个J片段，使用该片段J的特异性寡核苷酸作为引物并且扩增产物作为基质，对每个扩增产物进行延伸步骤。 对于对应于由此获得的三重峰（V，C）J的每个伸长率产物，显示不同伸长率产物的尺寸。 所有曲目的描述对应于VCJ三元组，并且对应于曲目的每个元素的元素大小。