44
KAI-OLAF NETZER
Subprotocol 7
RNA-Dependent DNA Polymerases
Reverse transcriptase
Enzymes which preferably copy RNA templates into DNA are called reverse
transcriptases (RT). These RNA-dependent DNA polymerases were discovered in retroviruses independantly by Temin and Baltimore in 1970. The
reverse transcriptases currently used in molecular biology are purified
from avian myeloblastosis virus (AMV) or Moloney murine leukemia virus
(MML V). The latter is available as a recombinant enzyme. Both enzymes
have 5'-+3' polymerase activity and RNaseH activity. RNaseH activity is a
bidirectional exoribonuclease activity on RNA:DNA hybrids. However,
both enzymes lack 3'-+ 5' proofreading activity which explains their considerable error rate of w- 4 in vitro. Reverse transcriptase is used to transcribe
mRNA into single-stranded or double stranded cDNA (complementary
DNA). Oligo (dT), random primers, or specific oligo nucleotides can serve
to prime the reaction. While firststrand cDNA synthesis is performed, the
RNA template is removed bythe RNaseH activityofRT. Alternatively, when
using genetically engineered reverse transcriptase lacking RN aseH activity,
the RNA template is digested after reverse transcription in a second reaction
with RNaseH. Second oligonucleotides or self priming initiate the second
strand synthesis which is necessarywhen a cDNA library is tobe generated.
Second strand synthesis is not necessary if subsequent PCR is planned.
Protocol for the generation of first strand cDNA by using MMLV reverse
transcriptase
The quality of the RNA template is the limiting factor in cDNA synthesis.
Therefore, great care should be taken to avoid RNA degradation during
preparation. Our experience is that for first-strand synthesis with subsequent cDNA amplification, total cellular extracts of RNA are sufficient
(e. g. using RNAzol™). Successful reactions have been performed with
RNA from as little as 10 4 cells. With nested PCR, even fewer cell numbers
may suffice. A useful amount of total cellular RNA to start with is 5 J.Lg.
KAI-OLAF NETZER
Subprotocol 7
RNA-Dependent DNA Polymerases
Reverse transcriptase
Enzymes which preferably copy RNA templates into DNA are called reverse
transcriptases (RT). These RNA-dependent DNA polymerases were discovered in retroviruses independantly by Temin and Baltimore in 1970. The
reverse transcriptases currently used in molecular biology are purified
from avian myeloblastosis virus (AMV) or Moloney murine leukemia virus
(MML V). The latter is available as a recombinant enzyme. Both enzymes
have 5'-+3' polymerase activity and RNaseH activity. RNaseH activity is a
bidirectional exoribonuclease activity on RNA:DNA hybrids. However,
both enzymes lack 3'-+ 5' proofreading activity which explains their considerable error rate of w- 4 in vitro. Reverse transcriptase is used to transcribe
mRNA into single-stranded or double stranded cDNA (complementary
DNA). Oligo (dT), random primers, or specific oligo nucleotides can serve
to prime the reaction. While firststrand cDNA synthesis is performed, the
RNA template is removed bythe RNaseH activityofRT. Alternatively, when
using genetically engineered reverse transcriptase lacking RN aseH activity,
the RNA template is digested after reverse transcription in a second reaction
with RNaseH. Second oligonucleotides or self priming initiate the second
strand synthesis which is necessarywhen a cDNA library is tobe generated.
Second strand synthesis is not necessary if subsequent PCR is planned.
Protocol for the generation of first strand cDNA by using MMLV reverse
transcriptase
The quality of the RNA template is the limiting factor in cDNA synthesis.
Therefore, great care should be taken to avoid RNA degradation during
preparation. Our experience is that for first-strand synthesis with subsequent cDNA amplification, total cellular extracts of RNA are sufficient
(e. g. using RNAzol™). Successful reactions have been performed with
RNA from as little as 10 4 cells. With nested PCR, even fewer cell numbers
may suffice. A useful amount of total cellular RNA to start with is 5 J.Lg.