John M. S. Bartlett.pdf - Bio-Nica.info

Sequencing 339
sequencing approach and use fluorescent rather than radioactive labels. This has the
advantages of greater safety, generation of machine-readable data, and greater reagent
stability. The downside of this is the relative expense of the equipment required.
Fluorescent dyes can be incorporated as labeled primers, dNTPs. or ddNTPs. The use
of four different dyes, one for each of the ddNTPs (dye terminator sequencing), has
allowed one of the biggest improvements in throughput of these systems. Now, rather
than the base-specific reactions being kept separate and run down individual lanes
of a gel, they can be performed in the same reaction tube and analyzed in one lane,
with fragments being separated by size and distinguished by the wavelength of the
fluorescent emission. Automated sequencers based on capillary electrophoresis have
been developed, which dispense with the gel-making step. Many specialized centers
now use robotic systems to extract DNA template, perform PCR and sequencing reactions,
and load 96-capillary sequencers. Sequence data generated can then be directly
imported into databases and processed with little or no hands-on intervention.
These developments still continue. Research is under way to develop the technology
of mass spectrometry for DNA sequencing, and sequencing by hybridization is the
subject of a great deal of development work.
6. Direct Sequencing of PCR Products
Unlike methods where the PCR product is cloned and a single clone is sequenced,
direct sequencing of PCR products is usually unaffected by the relatively high error
rate of Taq DNA polymerase because (unless there are only a few starting copies of
template, and a misincorporation occurs in an early round of PCR) the vast majority of
the amplified product will consist of the correct sequence.
Direct sequencing of PCR products has significant advantages over the cloning
strategy. It is a simple procedure that can be easily standardized and only a single
sequence needs to be determined for each sample. Indeed, the procedures are so well
standardized that there has been an exponential growth in the number of laboratories
offering core-sequencing services for PCR products. Although the majority of such
core laboratories provide an excellent service, there are a number of factors that will
affect their ability to produce good quality data.
7. Requirements for Good Quality Sequence from PCR Products
• Optimize the PCR reaction to yield only a single product. If the same oligos are used
to sequence as primed the PCR, they will also sequence from any nonspecific products,
interfering with data quality. Primer-dimers may not interfere with the identification of
a PCR product on agarose gel electrophoresis, but they serve as efficient template for
sequencing, resulting in characteristic noise in sequence data close to the primer.
• PCR should be performed with as little primer as possible. If labeled dideoxyterminator
sequencing is used (probably the most common approach), residual PCR primer can serve
as sequencing primer. Thus, even when only one PCR product is present, it is sequenced
from each strand simultaneously, again interfering with data quality. There are a number of
techniques available for the removal of unincorporated primers, from enzymatic digestion
to column purification, but these are generally unnecessary if the primer concentration
is limited in the PCR.
• If PCR products are isolated from a gel prior to sequencing, great care should be taken
to minimize the amount of salt carried over from the isolation procedure. High-salt