Automated direct sequencing of polymerase chain reaction-amplified fragments of the human Ha-ras gene

We present modifications to polymerase chain reaction-based DNA sequence analysis which avoid the need for M13 cloning and allow one set of sequencing primers to be used for analysis of any desired DNA sequence. This procedure employs nested amplification primers including short 5′-terminal sequences suitable for the attachment of fluorescent markers or for sequencing with M13 universal and reverse sequencing primers. Our modifications provided adequate single-stranded DNA for reliable automated sequence analysis of selected Ha-ras gene regions, starting with less than 1 μg of genomic DNA.