Real-Time PCR is used for studying known genes. Consequently, the sequence of a specific target gene of interest must be known (so you can design the PCR primers).
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- First step: PCR amplification of sequencing template.
- Second step: Clean-up of PCR reaction.
Please find here the next steps:
Third step: Cycle Sequencing:
The sequencing reaction is performed on the thermal cycler, using the PCR products as a template. A master mix containing labeled ddNTPs, dNTPs, a single primer, a high-performance DNA sequencing polymerase, and the DNA template will be used. Note that unlike in PCR, which uses two primers, only a single primer is used to generate single-stranded fragments during each cycle of sequencing.
Whether for PCR or cycle sequencing, it is important to design primers that will bind their target sequence during thermal cycling.
Fourth step: Cycle sequencing clean-up:
A sequencing clean-up step is then performed prior to capillary electrophoresis. This is different from the PCR reaction clean-up; this step is needed to remove unincorporated ddNTPs from the reaction, as well as salts and other contaminants. If the fluorescent ddNTPs are not removed, their fluorescent signals interfere with the signals from the desired fragments.
The capillary electrophoresis (CE) is performed on a Genetic Analyzer instrument that utilizes small glass capillaries filled with polymer.
The electrophoresis within these capillaries separates the fluorescent labeled chain-terminated fragments by length. CE can separate these molecules with single-nucleotide resolution
Once the run is finished, the instrument will generate an ab1 file. Sequencing Analysis software will convert the fluorescent peaks of each nucleotide into a sequence.
In summary, Sanger sequencing can be a powerful tool for determining the sequence of a small number of genes, the basics of which are primer design, DNA template preparation, PCR and clean-up, cycle sequencing and clean-up, CE, and data analysis.
As RT-PCR, the Sanger Sequencing technology will provide you with very useful genomic information.
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