66 Cook and Slatersystem (with the omission of the radiolabeled triphosphate, as described
in Section 4.2.) and hybridized with an excess of the single-stranded
DNA primer. The primer and the RNA are then incubated at 85°C for
10 min to denature the nucleic acids, and annealed by incubating at a
suitable temperature, for example, 55°C for 8-12 h. The exact tem-
perature needs to be optimized by preliminary experiments. The length
and GC content of the primer are used to determine the temperature of
annealing; the standard temperature of 55°C is suitable for a 20 base
oligonucleotide sequence with a GC content of approx 50% (46,47).
The primer:RNAhybrid is then dissolved in reverse transcriptase buffer,
approx 50 U of reverse transcriptase added to extend the primer to
produce a eDNA copy complementary to the RNA template and incu-
bated at 42°C for 90 min. If analyzing more than 10 lag of RNA in order
to prevent distortion of the sequencing gel, a RNase digestion is needed
at this stage.When the reverse transcriptase reaches the end of the
sequence (the cap site), it "falls off." The length of the resulting end-
labeled eDNA is measured by denaturing gel electrophoresis usually
on a DNA sequencing gel, hence, the sequence prior to the cap site can
also be determined.
Single-stranded DNA primers are preferred for experiments, since
they eliminate the formation of DNA:DNA hybrids and can be designed
to hybridize to specific sequences of the mRNA.
4.4. Nuclease $1 RNA Mapping
The enzyme S 1 nuclease degrades single-stranded DNA to yield 5'
phosphoryl mono or oligonucleotide (48). Primer extension cannot be
used to identify the 3' end of a transcript. Nuclease S 1 mapping is
therefore the method of choice for this purpose. The procedure was
originally described by Berk and Sharp in 1977 (49); it has been used
to map the location of 5' and 3' termini of mRNA or DNA templates,
and locate 5' and 3' splice points (50).
End-labeled RNA or DNA probes derived from various segments of
the genomic DNA are hybridized to mRNA favoring DNA:RNA hybrid
formation (51). Products of the hybridization are digested with nucle-
ase S 1 under conditions favoring single-stranded nucleic acid degra-
dation only. Then the products are analyzed by gel electrophoresis to
yield important quantitative and qualitative information about the
mRNA structure. The basic method is as follows: Probe and RNA are