GENE CLONING AND DETERMINATION OF DNA BASE SEQUENCE

摘要

PURPOSE: To provide a gene cloning method dispensing with preliminary Southern hybridization treatment and provide a DNA base sequence determination method enabling the simultaneous determination of sequences. ;CONSTITUTION: A 1st primer having a prescribed base pair length in the cloning direction in a DNA region having known sequence and a 2nd primer are synthesized, the genom DNA to be cloned is digested with a restriction enzyme to obtain a DNA fragment having a sequence recognized with the enzyme, the DNA fragment is bonded to a block having a sequence pairing with the above sequence, containing a primer A and a primer B in the sequence and having a known sequence as a whole, the 1st PCR is performed by using the 1st primer in the target gene and the primer A of the block, the 2nd PCR is carried out on a part of the above PCR product by the 2nd primer and the primer B of the block, these fragments are amplified and purified and the base sequence of the DNA is determined. In the DNA base sequence determination method described above, the genom to be cloned is partly digested with a restriction enzyme recognizing 4 bases and the digestion frequency is adjusted by a size fractionation.;COPYRIGHT: (C)1996,JPO