Gene Cloning

Key Tools for Gene Cloning 77
DNA sequence information before you begin. As the amount of sequence
data available in public databases increases this is becoming a less significant
constraint. There are many applications of PCR that involve amplifying a
region of DNA where the sequence is known; in these cases the experiment is
often designed to look for polymorphisms (small changes in DNA sequence in
the same gene or region of DNA which are found within the population of a
particular species). Examples of this type of application are in genetic fingerprinting
and detection of genetic diseases (Chapter 13). It is also possible to
use PCR to amplify a gene from one organism by designing primers based on
the sequence of the same gene in a closely related organism.
The other main limitation to PCR is the length of the region that can be
amplified. PCR works well over short stretches of DNA up to about 2 kb.
With longer target regions it is necessary to increase the extension time to
allow DNA polymerase to complete the synthesis of the longer product.
There are PCR kits on the market, which improve the effective range of the
PCR reaction. These usually use a second thermostable DNA polymerase
with proofreading ability (Box 3.5) in conjunction with Taq polymerase and
use PCR programs which increase the extension time in the later cycles.
These systems make it possible to amplify target regions of up to 20 kb. The
use of PCR primers with melting temperatures above 58°C also increases
the efficiency of long range PCR as primers with low melting points
increase the chance of non-specific annealing leading to preferential
amplification of short PCR products.
Taq polymerase does not have proofreading capability (Box 3.5). As a
result it has a relatively high error rate: on average it incorporates the wrong
nucleotide every 9000 nucleotides. As the products of each cycle are used as
template in successive cycles this can lead to a cumulative error rate of 1 in
300 nucleotides over 30 cycles. For many applications this is not a problem.
However, the error rate inherent in PCR is especially important when PCR
products are to be cloned. In this case a single molecule will be selected by
the cloning procedure and copied many times; clearly if a mutation has
occurred by mis-incorporation of a base during the PCR process, this will be
perpetuated as the cloned DNA is copied in the host. This problem can be
avoided by using alternative high fidelity thermostable DNA polymerases
with proofreading activity. Generally speaking, even when using high
fidelity polymerases it is essential to sequence the PCR product after cloning
to ensure that no errors have been incorporated in the product.
Questions and Answers
Q3.1. If you had a culture of E. coli, some containing only pBR322 and some
only pUC18, how would you select only those with pBR322?
A3.1. From Figure 3.1, you can see that pBR322 carries genes for resistance to
both tetracycline and ampicillin, whereas pUC18 only carries the gene for