| 摘要 |
<p>The present invention provides for methods for rapid subtraction hybridization of two pools of double-stranded cDNA to isolate a differentially expressed gene which comprises: isolating RNA from a first and a second population of cells; producing a first and second double-stranded cDNA pool; admixing the first and second pools of cDNA separately with a first restriction enzyme; admixing the digested first pool and second pool of cDNA with an appropriate amount of a double-stranded oligonucleotide; wherein the oligonucleotide sequence comprises (i) an internal recognition site for a second restriction enzyme and (ii) a ligatable cohesive end at one end which is capable of hybridizing with a ligatable cohesive end resulting from digestion of the first pool of cDNA with the first restriction enzyme; performing two separate polymerase chain reactions (PCR) using an aliquot of the ligation mixture (tester cDNA or driver cDNA) admixed with a PCR primer; digesting the PCR products of the tester cDNA with the second restriction enzyme under appropriate digestion conditions; admixing an aliquot of the digestion product (tester cDNA) with an excess of driver cDNA PCR products under hybridization conditions, so as to isolate a differentially expressed gene from among the two pools of double-stranded DNA.</p> |
