Cycle Sequencing
Definition
A Sanger sequencing format using a thermostable DNA polymerase and repeated denaturation-annealing-extension cycles (typically 25-35 cycles). Produces exponentially more termination products from the same template than a single-pass reaction, reducing the template requirement and enabling sequencing from PCR products and small forensic samples.
- Method
- Sanger sequencing variant
- Enzyme
- Thermostable DNA polymerase
- Cycle count
- Typically 25 to 35 cycles
- Advantage
- Lower template input requirement
Common questions
Why does cycle sequencing suit forensic casework better than single-pass Sanger sequencing?+
Forensic samples are often low-quantity or degraded, and repeated denaturation-annealing-extension cycles generate far more termination products from the same limited template than one round would, making a readable trace possible from otherwise insufficient DNA.
Does cycle sequencing amplify the target DNA the way PCR does?+
It uses linear, not exponential, amplification of sequencing products because only one primer is used per reaction, so the yield increase per cycle is additive rather than doubling, which distinguishes it from a standard PCR amplification step.
Related terms
- 2',3'-Dideoxynucleotide Triphosphate (ddNTP)
- A nucleotide analogue lacking the 3'-hydroxyl group present in normal dNTPs. When incorporated by DNA polymerase into a growing chain, it prevents...
- Big Dye Terminator (BDT) V3.1
- Applied Biosystems' (now Thermo Fisher Scientific's) commercial cycle sequencing kit using four spectrally distinct dichlororhodamine dyes coupled to the four ddNTPs. The...
- EMPOP (ENBISI Mitochondrial Population Database)
- The ENFSI-curated reference database of forensic-grade mtDNA haplotypes used to estimate the frequency of a given control-region profile in a specified population....
- Phred Quality Score
- A logarithmic estimate of base-calling error probability (Phred Q = -10 log10 P). Q20 = 1% error rate; Q30 = 0.1% error...
- rCRS (Revised Cambridge Reference Sequence)
- The standard reference sequence for the human mitochondrial genome (NC_012920.1, 16,569 bp), used as the coordinate system for reporting all forensic mtDNA...
- Sequencing-by-Synthesis (SBS)
- The Illumina detection paradigm: reversibly fluorescent 3'-blocked nucleotides are incorporated one cycle at a time into clonally amplified clusters on a flow...