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| DNA sequencing
is the experimental process of determining the nucleotide sequence
of a DNA region. The automated DNA Sequencing is based on Sanger's
enzymatic method with a thermostable DNA polymerase, which allows
cycle sequencing reactions. The resulting fluorescent products
are electrophoresed and detected in a capillary instrument controlled
by a computer and the Data Collection Software. |
| EQUIPMENT |
| The sequencing reactions are
performed on an Applied Biosystems' GeneAmp PCR System
9700 thermocycler. |
| Once the fluorescent
fragments have been generated they are run on an Applied
Biosytems' 3100-Avant Genetic Analyzer. |
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| SERVICE
DESCRIPTION |
| The main
goal of the service is to maintain a DNA sequencing facility
for scientific research. We use Applied Biosystems' BigDye Terminator
v3.1 kit for the sequencing reactions of both plasmids and PCR
products. The customer sends the DNA to be sequenced and the
specific primers, and in 3 working days will receive the results
by e-mail. |
| Template
quality and quantity are the most important factors affecting
sequence length and quality. For sequencing PCR products we
require 15 µl of purified PCR product at 20-50 ng/µl, whereas
we require 15 µl of plasmid DNA at a minimum concentration of
100 ng/µl. In both cases, we require 10 µl of primers at a concentration
of 5 µM. |
| Single
run sequencing offers an excellent run quality with an average
run length of 650-700 nucleotides. |
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| Once the
customer receives the results, they can be visualized by EditView
(Macintosh) or Chromas (Windows). |
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| APPLICATIONS |
| DNA sequencing has many applications,
and among them we can highlight: |
| • Biomedical
research: Molecular characterization of genes. |
| • Human genetics: Molecular
diagnosis of hereditary disease. |
| • Clinical microbiology:
Identification of bacterial species by differential genomic
region sequencing. |
| • Molecular oncology: Mutation
detection in oncogenes and tumor suppressor genes (p53,
APC, etc.). |
| • Basic research delivery
by e-mail |
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