Biomedical Engineering Reference
In-Depth Information
tion of molecules pose little danger to the accurate interpretation of data. How-
ever, for sequence analysis of cloned PCR products, errors owing to
misincorporation may sometimes complicate data interpretation. Thus, it is
advisable to analyze multiple clones from a single PCR or to clone PCR prod-
ucts from several independent amplifications. Another application in which
misincorporation may result in error of interpretation is in the amplification of
low-copy-number targets (e.g., single-molecule PCR). In these situations, if a
misincorporation happens in an early PCR cycle (the extreme case being in
cycle 1), the error will be passed on to a significant proportion of the final PCR
products. Hence, in these applications, the amplification conditions should be
carefully optimized.
10. PCR Thermocyclers
One of the main attractions of PCR is its ability to be automated. A number
of thermocyclers are available from different manufacturers. These
thermocyclers differ in the design of the cooling systems, tube capacity, num-
ber of heating blocks, program memory, and thermal uniformity. Units with
multiple heating blocks are very valuable for arriving at the optimal cycling
profile for a new set of primers, as multiple conditions can be tested simulta-
neously. Tube capacity generally ranges from 32 to 384 wells and should be
chosen with the throughput of the laboratory in mind. Most thermocyclers
nowadays have heated covers, making the addition of oil to the PCR mixture
obsolete. A variant of thermocycler using the convection of PCR mixtures
between the annealing and denaturing temperature has been developed
(
15
,
16
)
.
This convection-driven PCR can significantly shorten the time for a PCR, and
the exponential amplification can reach 100,000-fold within 25 min
(
17
)
.
11. Analysis and Processing of PCR Product
The amplification factor produced by PCR simplifies the analysis and
detection of the amplification products. In general, analytical methods for
conventional DNA sources are also applicable to PCR products.
11.1. Electrophoresis
Agarose gel electrophoresis followed by ethidium bromide staining repre-
sents the most common way to analyze PCR products. A 1.5% agarose gel is
adequate for the analysis of PCR products from 150 to 1000 bp. DNA markers
of different size ranges are available commercially. The development of capil-
lary electrophoresis has significantly increased the throughput and resolution
of PCR product analysis. The improved resolution of capillary electrophoresis
has allowed the discrimination of a single nucleotide difference in size.
