DENATURANTS OR COSOLVENTS IMPROVE THE SPECIFICITY OF PCR AMPLIFICATION OF A G-RICH DNA USING GENETICALLY-ENGINEERED DNA-POLYMERASES(C)

We describe conditions that improve the specificity of amplification o f a G+C-rich (57% G+C) DNA by PCR. Under standard conditions a 368-bp segment of the approx. 2.1-kb repeat unit of a satellite DNA that acco unts for approx. 3% of the genome of the Bermuda land crab, Gecarcinus lateralis, was not amplified specifically. To establish optimal condi tions for amplification of the segment of the G+C-rich satellite, we u sed two genetically engineered enzymes, AmpliTaq DNA polymerase and Am pliTaq DNA polymerase, Stoffel fragment (SF), and a number of denatura nts or co-solvents. In the absence of denaturants or co-solvents, ampl ified products of both enzymes contained nonspecific bands upon gel el ectrophoresis. Addition of certain denaturants or co-solvents to PCR m ixtures resulted in the production of the single specific band of the expected size. Reagents that improved specificity of the amplified pro duct were formamide, glycerol, DMSO, Tween-20 and NP-40; on the other hand, urea, ethanol and 1-methyl-2-pyrrolidone (NMP) inhibited amplifi cation. Of the two enzymes, SF was more specific and efficient. The pr oducts of AmpliTaq DNA, polymerase included one or more extra bands, e ven in the presence of denaturants or co-solvents, except for glycerol or DMSO.