BIO372H5 Lecture Notes - Lecture 3: Ribonuclease H, Ferrari 250, Sanger Sequencing
Document Summary
Sanger sequencing is an example of primer extension. Description:superscript reverse transcriptase (rt) is a dna polymerase that synthesizes a complementary dna strand from single-stranded rna, dna, or an. This enzyme is produced from a cloned m-mlv rt gene from which the rnase h sequence has been deleted, reducing the rnase h activity. This structural modification prevents degradation of the rna molecule during first-strand cdna synthesis (figure 1). Purified from e. coli expressing the pol gene of m-mlv (4,5), mutagenized to reduce the rnase h activity (6). Unit description:one unit incorporates 1 nmol of deoxyribonucleotide into acid- precipitable material in 10 min. at 37 c using poly(a) oligo(dt)12 18 as template primer (7). Unit reaction condition:50 mm tris-hcl (ph 8. 3), 40 mm kcl, 6 mm mgcl2, 1 mm. Dtt, 0. 5 mm [3h]dttp, 0. 1 mm poly(a), 0. 1 mm oligo(dt)12 18, 0. 1 mg/ml bsa, and enzyme in 50 l for 10 min. at 37 c.
