Biomedical Engineering Reference
In-Depth Information
Figure 3.10 Affi nity-based purifi cation of mRNA. The unpurifi ed mRNA-containing solution is percolated
through a column packed with cellulose beads (a), to which a short chain of deoxythymidylate (an oligo dT
chain) has been attached. Any mRNA present is retained in the column due to complementary base pairing
between its 3' poly A tail and the immobilized oligo dT, (b). Non-bound material can then be washed out
of the column, with subsequent desorption of the mRNA by passing a low-salt buffer through the column.
The mRNA collected may then be precipitated out of solution using ethanol, followed by its collection via
centrifugation. An alternative, and now more commonly used variation, entails the direct addition of oligo
(dT)-bound magnetic beads directly into the cell lysate and 'pulling out' the mRNA using a magnet. The
method is rapid, thus minimizing contact time of the mRNA with degradative ribonucleases present naturally
in the cytoplasm
(dT) column (Figure 3.10). Nucleic acids absorb UV light maximally at 260 nm (compared with
280 nm in the case of proteins); thus, absorbance at 260 nm can be used to quantify the amount of
nucleic acid present and to follow the purifi cation protocol. The ratio of absorbance at 260 nm ver-
sus 280 nm can also be used to determine how contaminated the nucleic acid preparation is with
protein. The ratio A 260 / A 280
1.8 for pure DNA and 2.0 for pure RNA preparations; lower ratios
usually indicate the presence of contaminant protein. DNA can also be detected and quantifi ed by
the addition of the chemical ethidium bromide. Ethidium bromide molecules intercalate (bind) in
between DNA bases and fl uoresce when illuminated with UV light.
3.2.3 Nucleic acid sequencing
The determination of the exact base sequence present in a stretch of nucleic acid (particularly in
DNA) underpins much of modern molecular biology. Sequencing plays a central role in rDNA
cloning experiments, as well as in determining genome data. Two approaches have been developed
to sequence DNA: the Maxam-Gilbert chemical sequencing method and the Sanger-Coulson
enzymatic sequencing method. Both involve the ultimate generation of a full set of fragments of
the DNA strand to be sequenced, as illustrated in Figure 3.11. The methodologies employed ensure
that the identity of the fi nal (3 ) base in each fragment is known. The fragments are then sepa-
rated on the basis of their size by electrophoresis and, because the identity of the end base in each
fragment is already known, the full sequence can simply be read from the ladder of fragments
generated. Full details of sequencing methodologies are outside the scope of this topic, but they
are included in all core molecular biology and biochemistry student textbooks. RNA is sequenced
by an enzyme-based method somewhat similar to the enzyme-based DNA method.
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