Biology Reference
In-Depth Information
development, quantitation of mRNA from specific tissues, rearrangements of
DNA during cell differentiation, and RNA processing.
8.5.4 Analysis of mRNA Polyadenylation
Measurement of poly(A) tail length is important when studying mRNA stability.
A simple PCR-based method has been developed (
Eguchi and Eguchi 2000
).
8.5.5 Cloning a Gene
The PCR can generate microgram quantities of a specific DNA fragment and
these can be cloned (
Scharf 1990
), although many products of the PCR are
“recalcitrant to cloning” (
Sambrook and Russell 2001
). One reason is that sev-
eral of the DNA polymerases used in the PCR have the ability to add a single,
unpaired nucleotide at the 3
′
end of the PCR product (terminal transferase
activity). The nucleotide added depends both on the adjacent base and on the
particular polymerase used; for example, when the 3
′
-terminal base of the tem-
plate DNA is cytidine,
Taq
will add an adenine to the end of the completed PCR
product (
Sambrook and Russell 2001
).
One solution to this problem is to use the 3
′
to 5
′
exonuclease activity of bacte-
riophage T4 DNA polymerase or
Pfu
DNA polymerase to “polish” the ends of the
PCR products that contain the added bases; the polished DNA fragments can then
be phosphorylated by T4 kinase and cloned into a blunt-ended dephosphorylated
vector (
Costa and Weiner 1994, Costa et al. 1994
). Unfortunately, blunt-ended
cloning is notoriously inefficient (10- to 100-fold less efficient than cloning with
DNA fragments with cohesive termini). Furthermore, blunt-ended cloning allows
no opportunity to direct the orientation of the fragment within the vector.
A second reason for potential difficulties in cloning a PCR product is that
Taq
(and perhaps other polymerases) can survive extraction with methods used to
purify the PCR products (
Bennett and Molenaar 1994, Sambrook and Russell
2001
). The residual polymerase and dNTPs may make it difficult to tailor the
ends of the amplified DNA for cloning.
Currently, the most popular and efficient method for cloning PCR products
involves a method that relies on ligation of cohesive ends of the PCR product
and a vector (
Sambrook and Russell 2001
).
Taq
DNA polymerase typically adds
an A at the 3
′
end of the product. If a plasmid vector is used with a protruding
3
′
T residue at each of its ends, cloning is more efficient because the T and A
can base-pair. It is perhaps surprising that pairing of single bases is sufficient to
increase efficiency of cloning, but it is estimated to be
≈
50-fold more efficient
than blunt-ended cloning.
