Biomedical Engineering Reference
In-Depth Information
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5'cap
mRNA (primary transcript)
3'
Removal of introns
(splicing out)
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Polyadenylation
AAA(A)
n
AAA
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Figure 3.9
Overview of the transcription of eukaryote genes and subsequent mRNA editing. Prior to the
completion of the synthesis of the primary transcript, a 5' cap (7-methylguanosine) is enzymatically added
at the 5' end of the growing RNA chain. This helps to prevent mRNA degradation by nuclease enzymes. The
non-coding introns (see main text) are enzymatically removed from the primary transcript (a process called
splicing), yielding a mature mRNA sequence coding for the intended polypeptide. Finally, the mRNA 3' end is
also modifi ed by the addition of a poly A tail, comprising 80-250 adenylate residues, which again likely helps
protect the mRNA from degradation
approach adopted entails initial liberation of the nucleic acid by disruption of any cell wall present
(or viral capsid) and of the cellular plasma membrane, followed by selective precipitation and often
chromatography. In the context of plants and some microorganisms, initial disruption of the cell
wall may require application of physical or other vigorous disruptive infl uences (see Chapter 6).
This can potentially complicate DNA purifi cation, particularly as it can cause physical shear-
ing (fragmentation) of the extremely long DNA chromosome. The gentlest method of cell lysis
usually involves incubation with cell-wall-degrading enzymes, and the addition of detergent will
solubilize the plasma membrane. Following cellular disruption, initial purifi cation steps normally
entail solvent-based extraction/precipitation. For example, shaking in the presence of phenol (or
a mixture of phenol and chloroform), followed by standing or centrifugation (to achieve phase
separation) results in extraction of the (now denatured) proteins into the phenol phase and/or
accumulation at the interphase, with nucleic acids remaining in the upper, aqueous phase. Fur-
ther purifi cation may be achieved by selective precipitation of the nucleic acids using ethanol or
isopropanol as precipitant. If DNA is required, then the RNA present may now be removed by
the addition of the enzyme ribonuclease, which selectively degrades RNA. On the other hand, if
(eukaryotic) mRNA is required, then affi nity-based purifi cation may be undertaken using an oligo
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