Biochemistry 2280A Lecture Notes - Ribonuclease H, Ecori, Sticky And Blunt Ends
Document Summary
Bacteria cannot handle introns, thus if genomic dna is to be used, the introns would have to be eliminated. Introns are a problem in expressing genes in e. coli cdna cdna is a replicate dna copy of mrna c = complementary cdna does not contain introns. The cdna can be cloned or used in pcr. First step is to purify rna through multiple steps: major contaminant is dna so it takes multiple steps to remove it, end up with pure rna starting point to make cdna. Second step is to perform a hybridization or annealing step: hybridize or anneal with a poly(t) primer, primer is about 10 ts in length. Third step is to synthesize dna copy with reverse transcriptase: reverse transcriptase native enzyme that converts rna into dna for integration into the host genome. Fourth step is to degrade rna with rnase h, which degrades rna in an rna-rna duplex: or can be digested with sodium hydroxide.