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
RNA:DNA hybrid. 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)12–18as template•primer
Unit Reaction C3ndition:50 mM Tris-HCl (pH 8.3), 40 mM KCl, 6 mM MgCl , 1 mM 2
DTT, 0.5 mM [ H]dTTP, 0.1 mM poly(A), 0.1 mM oligo(dT) 12–18 0.1 mg/ml BSA, and
enzyme in 50 µl for 10 min. at 37°C.
Contents and Storage:SuperScript RNase H Reverse Transcriptase is supplied with a
vial of 5X first-strand buffer [250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl ], 2
vial of 100 mM DTT. Store at -20°C.
Reference(s):1. Gerard, G. (1989) Focus® 11: 66.2. Howland, P. et al. (1991) Mol. Brain
Res. 11 : 345.3. Gerard, G.F. et al. (1993) in Methods in Molecular Biology, Vol 16:
Enzymes of Molecular Biology (Burrell, M. Ed.), Humana Press, Totowa, N.J., p. 73.4.
Kotewicz, M. et al. (1985) Gene 35 : 249.5. Kotewicz, M.L. et al. (1988) Nucleic Acids
Res. 16 : 265.6. Houts, G. et al. (1979) J. Virol. : 519.
This is only about 30 yrs old. It is very common now. This is only about 30 yrs old. It is very common now.
Takinga DNA templateand copy into RNA
Gives idea of the size of RNA transcript
So we can measure the size of the dam
Two types: Double stranded and single stranded - we wantto measuresingle stranded nucleic acids
Nuclear run-on transcription
What processes involved in the level of transcript?
RNA is being degraded. It is very sensitive to degradation.
2' position - the OH group is much more susceptibleto attacks
We can always get rid of DNAses - put into hot water bath (around 65) but this temp will not kill RNAses.
RNAses are robust to degradation
We have to worry aboutsynthesis and degradation - how to separatethe two processes?
32P-GTP - An RNA precursor, must be labeled in alpha position 32P-GTP - An RNA precursor, must be labeled in alpha position
Labeled, then wash it with unlabeled to preventincorporation - a Pulse chase experiment
The cat reporter gene to measure transcription
Cat - enzyme that deals with chloramphenacol
Like electrophoresisbut only looking at thin layer of solvent
There are different acetylatedforms - can be based on degrees of acetylation
Other reporter genes:
GFP - green fluoresence protein
Accumulation of acetylated CAM