Real-Time PCR or Sanger sequencing?
Two technologies widely used in genomics, both providing very useful information. However, which tool do you need for your project?
While RT-PCR or qPCR allows DNA sequence detection and quantitation, extensively used for DNA copy number detection, gene expression, SNP genotyping, Sanger sequencing, also known as sequencing by capillary electrophoresis, allows to determine the sequence of the whole gene of interest.
Thus, when choosing which technology to be used in a certain project, one should decide whether there is a need for detection and trends of expression or the full sequencing of the gene in study.
Sanger sequencing with 99.99% base accuracy is considered the gold standard for sequencing technology and is used to support a diverse range of applications:
- Determining the accuracy of CRISPER- and TALEN-mediated genome editing techniques.
- Confirming next-generation sequencing (NGS) variants.
- Enabling reliable genotyping of the genetically diverse HIV-1 virus.
- Detecting minor allele fractions down to 5%.
- low-level variant detection in material containing minimal amounts of DNA, such as formalin-fixed, paraffin-embedded (FFPE) tissues for Molecular profiling of cancers.
- Mitochondrial DNA sequencing
The technology of Sanger sequencing uses fluorescently labeled oligonucleotide primers to seek out specific DNA regions to conduct targeted sequencing of up to 1,000 bases.
There are six steps in the Sanger sequencing workflow from sample to data
- PCR amplification of sequencing template
- Clean-up of PCR reaction
- Cycle sequencing
- Cycle sequencing clean-up
- Capillary electrophoresis
- Data analysis
It is necessary to remove excess primers and unincorporated nucleotides from the PCR reaction prior to sequencing. The ExoSAP-IT Express reagent, an enzymatic cleanup method offers:
- A 5 minutes protocol
- One-tube, one-step PCR cleanup
- 100% recovery of PCR products
- Novel enzyme technology
- Eliminate spin columns or beads
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.
