COMPLEMENTARY ANCHOR PCR OF REARRANGED VARIABLE T-CELL RECEPTOR BETA-CHAIN CDNA REGIONS

Sequencible amplificates comprising the variable cDNA sequences of the rearranged T-cell receptor (TCR) beta-chain were obtained from the T- leukemia cell line Jurkat using a single-sided PCR approach based on f ive synthetic oligonucleotides derived from the flanking constant sequ ence. Double-stranded cDNA was cleaved by a restriction enzyme creatin g cohesive ends, to which an anchor oligonucleotide was ligated. Since this anchor was complementary to the antisense strand of the known co nstant region, exclusively the desired ligation product folded into a stem-loop-structure that was enzymatically extended to yield a PCR tem plate, now flanked at both ends by primer binding sites appropriate fo r nested PCR.