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
First step – PCR amplification:
Use our online Primer Designer™ free Tool to search for the right PCR/Sanger sequencing primer pair from a database of ~650,000 predesigned primer pairs for resequencing the human exome and human mitochondrial genome.
Second step – Clean-up of PCR reaction:
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:
  1. A  5 minutes protocol
  2. One-tube, one-step PCR cleanup
  3. 100% recovery of PCR products
  4. Novel enzyme technology
  5. Eliminate spin columns or beads 
Want to learn how to clean up PCR Reactions with ExoSAP-IT Express Reagent? 
Here is a quick peek 

Interested to hear more about the next steps Sanger sequencing?
Stay Tuned! 

Feel free to contact us via rtpcr@rhenium.co.il, and we would be happy to help you with your questions

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