Biomedical Engineering Reference
In-Depth Information
gene of interest. Genome projects now mean that such sequence information is known for many
proteins. Alternatively, likely base sequences can be deduced if a partial amino acid sequence of
the protein is known. Hybridization studies are usually initiated by physically pressing a nitrocel-
lulose paper onto the agar plates containing the recombinant colonies. A replica of the plate is
thus created on the paper, as some cells from each colony adhere to it. Subsequent treatment of
the paper with alkali lyses the cells, releasing and denaturing the DNA within. The DNA adsorbs
tightly to the paper. The paper is then exposed to a solution containing the labelled DNA probe
under conditions that allow it to anneal to the target DNA, if it is present. After washing (to re-
move unbound probe), any probe retained on the paper surface can de detected by an appropriate
visualization technique (e.g. autoradiography if the probe is radiolabelled), and the positioning of
the label on the paper surface pinpoints which colony on the agar surface houses the desired DNA
fragment. Cells from the appropriate colony can then be grown up in larger amounts by submerged
fermentation (see Chapter 5) in order to produce larger amounts of the desired (now cloned) gene.
The cells can be collected, lysed and the vector therein recovered by standard microbiological
techniques. The cloned gene can then be excised from the vector via treatment with an appropriate
RE and purifi ed by standard molecular techniques.
3.4.1 cDNAcloning
An alternative to cloning genomic DNA, as outlined in the sections above, entails beginning
the process not with chromosomal fragments but with mRNA. This is often an approach taken
when cloning eukaryotic genes in particular. As described in Figure 3.10, total eukaryotic cellular
mRNA can be purifi ed from the cell via an affi nity-based mechanism. The mRNAs recovered in
this way refl ect only the polypeptide-encoding genes that are expressed in the cells at the time
of their extraction. Incubation of the mRNA with the enzyme reverse transcriptase results in the
conversion of the single-stranded mRNA into double-stranded DNA known as cDNA (see also
Figure 3.1). These cDNA fragments can then be cloned to generate a cDNA library, and the de-
sired cDNA clone can be identifi ed by means similar to those already described in the context of
cloning genomic DNA. cDNA libraries are smaller than genomic libraries as they are derived only
from expressed genes. Non-coding regions in the genome (as well as quiescent genes and genes
coding for rRNA and tRNA) are not represented in the library. Therefore, cDNA libraries are
more manageable to work with, assuming that the gene of interest is being expressed.
3.4.2 Cloning via polymerase chain reaction
An enormous number of individual genes have been sequenced over the last two decades or more.
Of latter years in particular, genome projects have also begun to make available the sequence of
the entire complement of genes present in many species. The bulk of this sequence information has
been made publicly available by its deposition in sequence databases. As a result, scientists who
now wish to clone a particular gene will usually have prior access to partial/entire sequence infor-
mation from the relevant organism or a closely related species. This sequence information allows
them to obtain large amounts of the gene of interest by using the PCR technique (Figure 3.15). This
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