The Brigham and Women's Hospital, Inc.

Ryoji Kojima

Steven R Gullans

Jeffrey Randall

A method for conducting sequential nucleic acid hybridization steps is described, whereby the ability of earlier-used primers or probes to participate in subsequent hybridization steps can be minimized, even though the differences between primer lengths are relatively small. It also relates to a rapid and quantitative method for the sequential synthesis of polynucleotide sequences by using a plurality of oligonucleotide primers, with the earlier utilized primers causing a minimum of interference with the subsequent primed synthesis reactions, yet without the need for intermediate purification steps. One preferred embodiment described is a method for differential display reverse-transcription polymerase chain reaction (DDRT-PCR), wherein complementary DNAs (cDNAs) are first synthesized using oligo-dT-primed reverse transcription (RT), and selected subsets of said cDNAs are then amplified using a second primer in a polymerase chain reaction (PCR), with a minimum degree of background being caused in the PCR step by residual amounts of the oligo-dT primer.

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Boston, MA, United States of America

Ryoji Kojima