Direct cloning of full-length cell differentially expressed genes by multiple rounds of subtractive hybridization based on long-distance PCR and magnetic beads.

Abstract

Subtractive cloning is an ideal technique for identifying genes differentially expressed in two nuclear acids population (). The polymerase chain reaction (PCR)-based subtraction is the method of choice when the starting samples are heterogeneous or difficult to obtain, which often occurs in the tissues to be compared. PCR amplification is the easiest method for generating adequate amount of nuclear acids for multiple-round hybridization. However, the bias in the relative representation of mRNA molecules in the starting materials and the accumulation of shorter fragments become the major deficiencies for this method and should be overcome. The bias caused by PCR amplification is because of the tendency of preferentially amplifying short fragments and certain templates with unique sequences in the sample. The thermophilic polymerase that is optimized to amplify multiple genes would be helpful and the adoption of gel filtration in preparation of templates for amplification could hinder the tendency of short fragment accumulation. In addition, increasing the amount of starting samples would represent much more molecules in tissues.