Biology Reference
In-Depth Information
Target sequence
a
Template
b
c
200 bp amplicon
500 bp amplicon
(a)
1
2
3
1000 bp
800 bp
600 bp
500 bp
400 bp
300 bp
500 bp
200 bp
200 bp
100 bp
(b)
Figure 5.4
(a) The target sequence on the template DNA molecule has been amplified using two
different primer pairs: using primers a
+
c generates a 500 bp product and the a
+
bprimerpair
leads to a 200 bp product. (b) Following amplification of template DNA in two separate reactions
the products were separated on a 2% agarose gel and stained with ethidium bromide. The amplified
products can be seen in lane 2 (200 bp) and lane 3 (500 bp); lane 1 contains a 100 bp ladder
will co-purify and in some cases inhibit the
Ta q
polymerase. Potent inhibitors of
the
Ta q
polymerase include haem compounds from blood [33 - 38]; bile salts and
complex polysaccharides from faeces and plant material [38 - 41]; humic substances
from soil, which is commonly co-extracted from bone [36, 42 - 44]; urea from urine
[38, 45 - 47]; melanin from hair and skin [48]; blue dye in clothing such as denim,
called indigo [49]; collagen from tissue and bones [50]; and high concentrations of
ions, in particular calcium and magnesium [46]. EDTA is used in high concentrations
for the isolation of DNA from bone and will also inhibit PCR unless removed as it
binds ions such as magnesium ions that are essential for PCR [36].
Extraction methods have been developed to remove commonly encountered PCR
inhibitors and, for example the silica-based methods that are commonly used in
forensic analysis are effective at removing most inhibitors, whereas the methods that
produce a cruder extract such as the Chelex
resin are more prone to inhibition.
When it is not possible to remove all the potential inhibitors from a DNA extract, the
addition of the protein bovine serum albumin (BSA) to the PCR can in many cases
prevent or reduce the inhibition of the
Ta q
polymerase. The BSA acts as a binding
