公开(公告)号：US5851805A

公开(公告)日：1998-12-22

申请号：US784751

申请日：1997-01-16

申请人： Dong Wei ,  Veronica M. Maher ,  Joseph Justin McCormick

发明人： Dong Wei ,  Veronica M. Maher ,  Joseph Justin McCormick

IPC分类号： C12N15/10 ,  C12Q1/68 ,  C07H19/00 ,  C07H21/00 ,  C07H21/04 ,  C12P19/34

CPC分类号： C12Q1/686 ,  C12N15/1096 ,  C12Q1/6809 ,  C12Q1/6855 ,  Y10S435/81

摘要： An improved method for amplifying cDNA fragments corresponding to the mRNA species expressed in cells is described. The method involves priming the mRNA with a first labeled synthetic oligodeoxynucleotide and then reverse transcribing the mRNA to produce a mRNA:cDNA hybrid; and then producing a double-stranded DNA from the hybrid. This DNA is digested with a restriction endonuclease and then a synthetic DNA linker, carrying a second label, is ligated at the cut to the DNA strand containing the 5' phosphate group. The DNA is then isolated with a first reagent which binds the second label. The DNA fragments of interest are then isolated using a second reagent which binds the first label. The isolated DNA fragments are amplified using polymerase chain reaction, and then separated on a sequencing gel. The method is particularly useful for comparing the mRNA species expressed in the cells of various samples.

摘要翻译： 描述了用于扩增与细胞中表达的mRNA种类相对应的cDNA片段的改进方法。 该方法包括用第一标记的合成寡脱氧核苷酸引发mRNA，然后逆转录mRNA产生mRNA：cDNA杂交; 然后从杂种产生双链DNA。 该DNA用限制性内切核酸酶消化，然后携带第二标记的合成DNA接头在切割处连接到含有5'磷酸基团的DNA链上。 然后用与第二标记物结合的第一试剂分离DNA。 然后使用结合第一标记的第二试剂分离感兴趣的DNA片段。 使用聚合酶链反应扩增分离的DNA片段，然后在测序凝胶上分离。 该方法对于比较各种样品的细胞中表达的mRNA种类特别有用。