Quantum Seq

Sanger Sequencing Workflow

PCR amplification of targeted region (to be sequenced)

• Amplification primers are typically outside the sequencing primer binding sites
• ExoSAP-IT commonly used for amplicon clean-up

Cycle sequencing of amplicon

• 2 reactions per amplicon (forward and reverse strands)
• Amplicon is mixed with target-specific sequencing primers and cycle sequencing reaction mix from QuantumDye kit for amplification of fluorescent-labeled dideoxy-terminated sequence products
• Cycle sequencing reaction mix is diluted with ddH2O and 5x Sequencing Buffer
• ABI manual recommends 8 µl of cycle sequencing mix/reaction
• Most customers use much less to save on reagent costs – from 1.0 to 0.5 µl (1:8 – 1:16 dilution)
• This means a 24-reaction kit can be used to sequence from 96 – 192 amplicons (2 reactions per sample)
• Customers often purchase 5x Sequencing Buffer separately depending upon how much they dilute the cycle sequencing mix

Sequencing product clean-up

• Remove unincorporated primers, dNTPs and fluorescent-labeled dideoxy terminators
• Magnetic beads/alcohol precipitation (EtOH or isopropanol)
• Size exclusion resin – small molecules are trapped while large one flow through
• Mixed bed ion exchange resins – small molecular binds to resin

Capillary electrophoresis

• Separation and basecalling of sequence products
• 3730 and 3730xl are the most common instruments in high throughput labs with 96 capillaries
• Other instruments include:
• 3130 and 3130xl – 16 capillaries
• SeqStudio – 4 capillaries