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Isolation by asymmetric polymerase chain reaction and partial sequencing of the common bean chloroplast trnL (UAA) gene and pseudogene
Author(s) -
Mubumbila Mfik A. V.,
Carelse Orseline,
Kempf Jules
Publication year - 1993
Publication title -
phytochemical analysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.574
H-Index - 72
eISSN - 1099-1565
pISSN - 0958-0344
DOI - 10.1002/pca.2800040402
Subject(s) - pseudogene , primer (cosmetics) , microbiology and biotechnology , chloroplast dna , gene , biology , polymerase chain reaction , exon , genetics , intron , chemistry , chloroplast , genome , organic chemistry
A polymerase chain reaction (PCR) strategy has been developed which utilizes the common bean ( Phaseolus vulgaris ) chloroplast DNA as a template and primers from (a) the 5′ exon of the broad bean chloroplast trnL (UAA) gene (EMBL ac no. X51471) and (b) the 3′ exon of the common bean chloroplast trnL (UAA) pseudogene (EMBL ac no. X58537). A 600 bp PCR Product was reamplified using the 5′ exon primer and an internal primer from the 5′ end of the intron of the common bean chloroplast trnL (UAA) pseudogene. A 130 bp PCR Product was separated into 130 bp and 106 bp DNA fragments on an 8% non‐denaturing polyacrylamide gel. Both fragments were purified by phenol extraction, reamplified by asymmetric PCR reactions and directly sequenced by the Sanger method. When compared to the broad bean trnL (UAA) gene, the 130 bp PCR product showed sequence homologies of 100% with its 5′ exon and 79% over the first 58 nucleotides of the 5′ end of the intron. The 106 bp PCR product contained deletions in its 5′ exon region. These results indicated that the PCR technique had isolated the trnL (UAA) gene and its pseudogene.