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Figure 1. Sequencing with PCR primer and standard primer tail.

During capillary electrophoresis, the products of the cycle sequencing reaction are injected electrokinetically into capillaries filled with polymer. High voltage is applied so that the negatively charged DNA fragments move through the polymer in the capillaries toward the positive electrode.

A high voltage is applied so that the negatively charged DNA fragments move through the polymer in the capillaries toward the positive electrode (Figure 1). Capillary electrophoresis can resolve DNA molecules that differ in molecular weight by only one nucleotide.

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Figure 2. Sequencing with PCR primer and standard primer tail.

Shortly before reaching the positive electrode, the fluorescently labeled DNA fragments, separated by size, move through the path of a laser beam. The laser beam causes the dyes on the fragments to fluoresce. An optical detection device on Applied Biosystems DNA analyzers detects the fluorescence (Figure 2).

The Data Collection Software converts the fluorescence signal to digital data, then records the data in a *.ab1 file. Because each dye emits light at a different wavelength when excited by the laser, all four colors, and therefore, all four bases, can be detected and distinguished in one capillary injection.

Capillary Electrophoresis instrument selection guide

  3730xl
3730xL
3730
3730
3500xl
3500xL
3500
3500
3130xl
3130xL
3130
3130
310
310
  Instrument
Number of Capillaries 96 48 24 8 16 4 1
Compatible Applications:
(S) Supported; (A) AB Demonstrated; (C) Customer Demonstrated; (N) Not Supported
BAC End Sequencing S S S S S S S
Checking Clone Constructs S S S S S S S
de novo Sequencing S S S S S S S
Heterozygote Detection & Resequenicng (SNP-based) S S S S S S A
HLA Typing N N S S S S C
Methylation S S S S S S C
mtDNA Sequencing S S S S S S S
Resequencing
- Comparative Genomics S S S S S S S
- Indels S S S S S S S
SAGE™ Method C C C C C C A
SNP Analysis S S S S S S S
General Sizing of PCR Products S S S S S S S
Fragment Length Polymorphism
- AFLP S S S S S S S
- RFLP A A A A A A A
Large Fragment Analysis
- BAC Fingerprinting A A A A A A N
- VNTR C C C C C C C
Microsatellite
- Genotyping S S S S S S S
- Analysis for Forensics/HID N N S S S S S
- Instability/RER A A A A A A A
Chimerism Studies C C C C C C C
Conformation Analysis
- SSCP N N N N A A S
Relative Fluorescent Quantitation
- Loss of Heterozygosity (LOH) S S S S S S S
SNP Genotyping
- SNaPshot® System A A S S S S S
- SNPlex™ System S S S S S N N
DNA Protein Binding Assays & DNA Fingerprinting C C C C C C C