Submit Article |Resources | Link to us | Sitemap | Search | Language
Back to Home
Home >> Recombinant DNA Technology >> Automated DNA Sequencing
Back to Home

Automated DNA Sequencing - Automated DNA sequencing is based on the Sanger Coulson method, with two notable differences from the standard procedure. The first difference concerns the labelling of the products of polymerase chain reaction: automated procedures use fluorescent labels in the place of radioactive labelling used in the standard procedure.

The fluorescent labels are usually attached to the four dideoxynucleotides used for chain termination. In the four track system of automated DNA sequencing, each of the four dideoxynucleotides is used in a separate reaction, and the products are run in four adjacent lanes of the gel.

If a different fluorochrome is attached to each of the four dideoxynucleotides, all of them could be used in the same reaction in place of preparing a separate reaction for each dideoxynucleotide. This is called the single track system since the reaction products are run in a single gel lane or capillary.

Generally, the DNA to be sequenced is subjected to thermal cycle sequencing to generate the chain terminated polynucleotides required for sequencing. The reaction products are subjected to polyacrylamide gel electrophoresis under denaturing conditions or loaded into a capillary filled with a sequencing gel.

The bands produced in the polyacrylamide gel/capillary are identified with the help of a fluorescence detector, which identifies the fluorescent signal emitted by each band. The fluorochromes are excited by a laser beam and the resulting fluorescence signal is sensed by a photovoltaic cell.

The resulting data are fed into a computer, which, in turn, converts these signals into the base sequence of the DNA molecule. The sequence information could be printed out or stored in a data storage device for future use; this is the second major deviation from the standard Sanger Coulson procedure.

In the four track system, the sequence can be recognized from the raw data, but it has to be interpreted using an appropriate computer programme in the single track system; this becomes necessary because the shifts in mobility due to the different fluorochromes have to be compensated for.

Automated DNA sequencers can read upto 96 DNA sequences in a 2 hours period, which is extremely fast as compared to manual DNA sequencing.
Automated DNA sequencing has the following advantages over manual DNA sequencing:
(1) radioactivity is not used,
(2) gel processing after electrophoresis and autoradiography are not needed,
(3) the tedius manual reading of gels is not required as data are processed in a computer,
(4) the sequence data is directly fed into and stored in a computer,
(5) the separation of the same reaction products can be repeated to recheck the results in cases of doubt since they can be stored for a long period of time, and
(6) it is extremely fast.
 

 
Submit Article |Resources | Link to us | Sitemap | Search | Language
English|Français|Español|Deutsch|Italiano|Portugues|Japanese|Korean|Sim-Chinese| Language