Helicos has developed a novel solution to the analysis of homopolymers (stretches of the same nucleotide within a DNA strand, e.g. ...CCCCC...). In the presence of ordinary labeled deoxynucleotide triphosphates, highly processive polymerases normally "run through" homopolymers, preventing the accurate counting or even detection of identical bases in series.
Helicos has developed proprietary fluorescent nucleotide analogues, called Virtual Terminator™ Nucleotides, which counteract the processivity of a DNA polymerase, and inhibit it from incorporating the next base in a homopolymer. Using both traditional capillary electrophoresis and True Single Molecule Sequencing (tSMS)™, we demonstrate their ability to accurately allow polymerase to step through a homopolymer, base-by-base. Additionally, the total chemistry time for incorporating a Virtual Terminator Nucleotide, and reversing the inhibition, takes about 20 minutes!
Demonstration of Virtual Terminator Nucleotides Using Capillary Electrophoresis
Using Virtual Terminator Nucleotides to sequence a DNA template containing
five 'G's in a row, analyzed on an ABI 3700
capillary electrophoresis instrument.
The DNA polymerase and 100 nM of the Virtual Terminator dCTP are added to the primer/template mixture and incubated for 10 minutes at 37°C and the product is analyzed by capillary electrophoresis (Panel 1). The termination is reversed, and the next Virtual Terminator dCTP incubation is carried out (Panel 2). This process is repeated three additional times (Panels 3, 4, and 5) to step through the five 'G' homopolymer, one base at a time.
Single Molecule Sequencing using Virtual Terminator Nucleotides
Using Virtual Terminator Nucleotides to sequence single molecule
DNA templates containing five 'G's in a row, analyzed by single molecule imaging.
The indicated Virtual Terminator dNTP is added and an image is acquired after every incubation (representative 10 strands shown). Single fluorescent nucleotide incorporations (white spot in center) are detected by the automated base caller (indicated by a white square in the lower left of each image). The resulting base calls are indicated below the images. After every image acquisition step, the termination is reversed, and the next Virtual Terminator dNTP incubation is carried out. On every single molecule strand, five incubations of Virtual Terminator dCTP (quads 2 – 6) are required to step through the five 'G' homopolymer, one base at a time.