Methods in Molecular Biology • 16 Enzymes of Molecular Biology

82 Gerard and D'Alessio

Table 3

Comparison of the Reaction Optima of M-MLV RT,

M-MLV H- RT, and AMV RT

Enzyme

Parameter M-MLV RT M-MLV H- RT AMV RT

pH 8.3 8.3 8.4
[KC1] 75 mM 75 mM 50-100 mM
[MgCI2] 3 mM 3 mM 10 mM
Temperature 37°C 37°C (42°C) a 42°C
[DTT] 10 mM 10 mM 1 mM
[dNTPs] 0.5 mM 0.5 mM 1 mM
Other components None None 4 mM NaPPi
0.5 mM
Spermidine-HCl
aM-MLV H- RT functions equally well at 37 and 42°C.

M-MLV RT and M-MLV H- RT can be used at incubation tempera-
tures as high as 45 and 50°C, respectively.

2. 7.10. Summary
   Table 3 summarizes the reaction optima of M-MLV RT, M-MLV H-
   RT, and AMV RT.


3. The Second-Strand Reaction
   We have chosen to describe exclusively a protocol (see Section 4.)
   for a one-tube format double-stranded cDNA synthesis procedure
   because it is fast, maximizes DNA product recovery, and yields prod-
   uct that can be modified and cloned into most vectors. The second-
   strand cDNA is synthesized by nick-translation replacement of mRNA.
   First described by Okayama and Berg (47), and later popularized by
   Gubler and Hoffman (48), second-strand synthesis is catalyzed by E. coli
   DNA polymerase I in combination with E. coli RNase H and E. coli
   DNA ligase. Inclusion of E. coli DNA ligase in the reaction has been
   shown to improve the cloning efficiency of double-stranded eDNA
   synthesized from longer (>2 kb) mRNAs (18).
   We must make several additional comments concerning the second-
   strand reaction. The reaction is incubated at 16°C to reduce the tendency
   of DNA polymerase I at higher temperatures to strand displace rather
   than nick translate. The pH of the first-strand reaction is reduced in the