80 Gerard and D'Alessio
Table 2
Optima of M-MLV RT and M-MLV H- RT a
MgC12,
mM
% Activity KCI, % Activity pH,
H ÷ H- mM H ÷ H- 22°C
% Activity
H ÷ H-
1 68 49 0 75 93 7.2
2 88 93 50 95 100 7.5
3 100 100 75 100 98 7.9
4 88 ND b 100 86 83 8.3
5 83 77 125 72 66 8.5
7.5 62 ND b 150 54 44
10 57 58 200 23 6
15 ND 33
20 20 15
90 ND b
95 ND b
97 ND b
100 ND b
98 ND b
aOptima were determined from yields (incorporation after 60 min). Optima based on
initial rates of incorporation are identical.
bND is not determined.
- 7.3. Sodium Pyrophosphate
Sodium pyrophosphate at a concentration of 4 mM stimulates the
synthesis of full-length cDNA from long mRNAs by AMV RT (40,41),
although there is disagreement on the mechanism by which sodium
pyrophosphate produces this effect (42,43). Sodium pyrophosphate
also inhibits hairpin primed double-stranded cDNA synthesis by AMV.
Sodium pyrophosphate inhibits the polymerase activity of M-MLV
RTs and should not be added to reactions catalyzed by these enzymes.
- 7.4. Polyanions
Spermidine-HC1 at 0.5 mM has been reported to stimulate the activ-
ity ofAMV RT (8) and Rauscher murine leukemia vires RT (44). How-
ever, spermine and spermidine inhibit both M-MLV RTs (8; Gerard, G.,
unpublished).
- 7.5. Bovine Serum Albumin and RNase Inhibitors
In many cDNA synthesis protocols, it is recommended that bovine
serum albumin be added to reaction mixtures to stabilize RT. Bovine
serum albumin has no effect on the activity of the M-MLV RTs. This
may be because of the high level of RT protein added to a typical
cDNA synthesis reaction.
