Biomedical Engineering Reference
In-Depth Information
characteristics of these various producer cells and their advantages and disadvantages, along with
factors taken into account when choosing one for biopharmaceutical production, are points con-
sidered in Chapter 5. In the remainder of this chapter we will review the basic molecular biology
techniques that underpin the isolation, identifi cation, cloning and expression of a target protein-
encoding gene sequence. We will fi rst overview the classical approach to cloning, which entails
the generation of genomic libraries as described immediately below. We will then consider an
alternative approach that has now come to the fore, and which is based upon the polymerase chain
reaction (PCR) technique.
3.4 Classical gene cloning and identifi cation
The basic approach to cloning a segment of DNA entails:
1.
Initial enzyme-based fragmentation of intact genomic DNA (usually chromosomes isolated as
described earlier in this chapter) so that it is broken down into manageable fragment sizes for
further manipulation. Ideally all/most fragments will contain one gene.
2.
Integration of the various fragments generated into cloning vectors, which are themselves small
DNA molecules capable of self-replication. Typically, these are plasmids or viral DNAs and the
composite or engineered DNA molecules generated are called rDNA.
3.
Introduction of the vectors housing the DNA fragments into host cells.
4.
Growing these cells on agar plates.
5.
Screening/identifi cation of the host cell colonies containing the rDNA molecules (i.e. screening
the 'library' of clones generated) in order to identify the specifi c colony containing the target
DNA fragment, i.e. the target gene (Figure 3.12).
We will now look at each of these stages separately. The initial fragmentation of genomic DNA
is undertaken using enzymes known as restriction endonucleases (REs). Some 800 different
REs have been identifi ed thus far. These enzymes recognize, bind and cut DNA sequences
which exhibit a defi ned base sequence (Table 3.2). These sequences normally exhibit a twofold
symmetry around a specifi c point and are usually 4, 6 or 8 bp in length. Such areas are often
termed palindromes. In general, the larger the recognition sequence the fewer such sequences
present in a given DNA molecule and, hence, the smaller the number of DNA fragments that
will be generated. Depending upon the specifi c RE utilized, DNA cleavage may yield blunt ends
(e.g. BsaAI and EcoRV in Table 3.2) or staggered ends - the latter are often referred to as sticky
ends.
An essential feature of the cloning vector used is that it must be capable of self-replication
in the cell into which it is introduced, which is usually
E. coli
. Two of the most commonly
used types of vector in conjunction with
E. coli
are plasmids and bacteriophage λ. Plasmids
are circular extra-chromosomal DNA molecules, generally between 5000 and 350 0000 bp
in length, that are found naturally in a wide range of bacteria. They generally house several
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