Biology Reference
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which can be either a plasmid or λ . Commercially available reverse transcriptases
can synthesize copies of mRNA sequences that are > 3kb. However, the tran-
scripts often terminate prematurely, making clones containing the 5 end of the
mRNA rare.
Figure 6.9 outlines the synthesis of double-stranded cDNA from mRNA.
Messenger RNA can be prepared for cDNA cloning by affinity chromatogra-
phy on oligo(dT) cellulose (or by commercially available kits). The reaction is
preceded by a brief heat denaturation of the mRNA to eliminate its second-
ary structure, because reverse transcriptase is inhibited if the mRNA exhibits
a secondary structure. The polyadenylated mRNA, the primer, and the reverse
transcriptase are combined. The primer in this case is a short sequence of (dT)
residues. The product of the first strand synthesis is a hybrid of mRNA and the
synthesized cDNA. The first strand is used as a template-primer complex to make
the second strand of DNA. The enzyme RNase H is used to introduce gaps in
the mRNA strand. At the same time, DNA polymerase I uses the primer-template
complexes formed by RNase H to synthesize a double-stranded DNA.
Once the double-stranded DNA is synthesized, it is inserted into a vector. To
insert it into a vector, the synthesized molecule needs to have ends that can be
ligated into the vector. One option is to make the cDNA blunt-ended by end fill-
ing with the Klenow fragment of DNA polymerase I and then ligating it into a
vector that has been cut with a restriction enzyme that produces a blunt end.
Another option involves the addition of cohesive ends to the cDNA so that it
will ligate into a vector more easily. There are three methods to add cohesive
ends to the ds DNA: 1) tailing with terminal transferase, 2) adding linkers, and
3) adding adaptors. The details of carrying out these procedures can be found in
many cloning protocols.
There are 10,000 different mRNA molecules in an average insect cell. At
least 200,000 cDNA clones should be generated to be sure that a representative
cDNA library is constructed. If the desired clone is a single-copy gene, then it
will be rare, so powerful screening methods are required to isolate the clone of
interest.
Isolating RNA is more difficult than isolating DNA. Preparation of mRNA
requires the absolute elimination of ribonucleases (RNases) from glassware,
pipets, tips, and solutions. Anything that might contaminate the reactions with
RNase must be eliminated, including hair, dust, and sneezes. Even fingerprints
contain enough RNase to degrade your RNA. Furthermore, RNase is a very hardy
enzyme and difficult to eliminate. Phenol extraction followed by ethanol precip-
itation was a common technique for isolating RNA, but various kits designed for
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