Coupled amplification and sequencing of genomic DNA

Addition of dideoxyribonucleotides during the exponential phase of the PCR should result in the synthesis of two complementary sequence ladders. The authors have explored this hypothesis to develop coupled amplification and sequencing of genomic DNA. Coupled amplification and sequencing is a biphasic method for sequencing both strands of template as they are amplified. Stage I selects and amplifies a single target form the genomic DNA sample. Stage II accomplishes the sequencing as well as additional amplification of the target using aliquots from the stage I reaction mixed with end-labeled primer and dideoxynucleotiodes. They have successfully applied coupled amplification and sequencing to a 300-base-pair fragment 4 kilobases upstream from HOX2B directly from human whole genomic DNA