DETECTION AND ANALYSIS OF NUCLEIC ACIDS - A Guide to Methods in the Biomedical Sciences - page 37

while the abundance of the protected fragment reveals the abundance
of the RNA transcript.
S1 analysis
S1 analysis is a method used to map exon-intron boundaries and
determine the ends of RNA. S1 analysis usually relies on the use
of a single stranded DNA probe that has been labeled on its end by
a kinase. The probe is added to RNA, and the non-hybridizing portion
of the probe is digested by S1 nuclease, fractionated by PAGE and
analyzed by means similar to RNase protection.
Primer extension
This method is designed to quantitate the levels of a given RNA in a
cell as well as to map the end of the RNA. Primer extension is used
to confirm RPA and S1 mapping of the ends of RNA. Primer exten-
sion uses reverse transcriptase (RT; see below) and a primer (a short
single stranded DNA) complementary to a sequence in the RNA. After
hybridization, the reverse transcriptase (together with dNTP precursors)
is used to synthesize (“extend”) a cDNA copy of the RNA. Because RT
is a polymerase, it synthesizes the cDNA copy in the direction,
which is directed toward the 5end of the RNA. Consequently, when the
RNA template terminates, extension will end. The length of the cDNA
synthesized identifies the length from the primer to the end of the
RNA, and the amount of product synthesized is a direct reflection of the
abundance of the RNA.
Other methods to quantitate RNA use modifications of the polymerase
chain reaction, and are described in Chapter 3.
Reverse transcriptase (RNA-dependent
DNA polymerase)
Reverse transcriptase (RT) is an enzyme isolated from retroviruses
(Human Immunodeficiency Virus, or HIV, is one example of a retrovirus).
Retroviruses contain a RNA genome, but their life cycle is dependent on
the production of a DNA intermediary that integrates into the DNA of the
host genome. RT is responsible for transcribing the RNA genome into
the first strand of this DNA. A double stranded copy is then synthesized
and the proviral DNA is integrated into the host cell genome.
The isolation and characterization of reverse transcriptase was a ma-
jor breakthrough in our understanding of the life cycle of retroviruses. Its