Efficient complementary DNA amplification and expression using polymerase chain reaction technology.

摘要: Publisher Summary This chapter describes the detailed methodology for bacterial expression and isolation of heterologous proteins in Escherichia coli ( E. ). To carry out described techniques, a tissue or cell culture that expresses gene interest is required. In addition, limited amount sequence information must be available, minimal requirement being short segment protein allows one to deduce nucleotide sequence, which mixed population defined sequences owing codon degeneracy. Initially, 3´ end cDNA amplified by polymerase chain reaction (PCR) PCR product sequenced. results specific can used generate new oligonucleotides 5' amplification. The coding region then directly using complex mixture mRNA as template. presents fast efficient laboratory protocol amplification cDNA, followed expression, quantitation, proteins, starting with produces interest.