Features of a novel PCR cloning method for assessing the relative levels of 20210G and 20210A mRNAs in liver from a prothrombin 20210G/A heterozygote.

Total mRNA is prepared from the liver of a 20210G/A heterozygote using a method that does not discriminate among mRNAs on the basis of poly(A) tail length. (A) Prothrombin mRNAs, containing either a G or an A at position 20210 (boxed), are reverse transcribed and PCR amplified using a forward primer within the penultimate exon (forward arrow) and an oligo(dT) primer containing a 5′-terminal EcoRI restriction site. (B) An aliquot of the initial PCR reaction is reamplified using the oligo(dT) primer and a nested, forward primer containing a 5′-terminal BamHI restriction site (indicated). (C) The resulting complementary DNAs are digested with BamHI andEcoRI (B and E, respectively) and directionally inserted into the cognate sites of pSP72. Competent DH5α Escherichia coli are transformed, and plasmid DNAs prepared from individual ampicillin-resistant colonies are sequenced to determine the G:A ratio at position 20210, reflecting the original ratio of the cellular 20210G and 20210A mRNAs.