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Sequencing of DNA Using Modified Nucleotides

Technology #1827

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Jingyue Ju PhD
Managed By
Peter Golikov

“Lead Inventor: Jingyue Ju, Ph.D.

Inexpensive and accurate DNA sequencing to identify nucleotides in a single strand of DNA:
Despite many recent advances, inexpensive and accurate DNA sequencing remains a critical goal in both industry and academia. Consequently, a number of approaches for identifying the nucleotides in a single strand of DNA have been developed. Pyrosequencing, an extension of the sequencing by synthesis approach, allows for the light-based, quantitative analysis of DNA sequences via the luciferase-based detection of a pyrophosphate generated by a polymerase reaction. However, this method can be inaccurate when multiple identical nucleotides are sequentially repeated in a DNA strand. Furthermore, the firefly luciferase detection system can produce false signals in the presence of excess deoxyadenosine triphosphate. Due to these significant drawbacks, the development of an error-proof pyrosequencing method is highly desirable.

Variant of the DNA pyrosequencing technique ttakes advantage of modified nucleotides:
The invention describes a dramatically improved variant of the pyrosequencing technique, which takes advantage of 3' modified nucleotides. The DNA template is hybridized to a primer and incubated with a cocktail of enzymes, which includes mutant DNA polymerase and firefly luciferase. Subsequently, four deoxynucleotide triphosphates featuring a blocking group at the 3' hydroxyl are sequentially added to this mixture. Notably, the 3' modified deoxyadenosine triphosphate is not a substrate for luciferase and does not impede its activity. Upon the addition/incorporation of a complementary blocked nucleotide, the released pyrophosphate is converted to a light signal, and the sequencing reaction is terminated. This temporary stop avoids the simultaneous readout of multiple bases in a homopolymeric region. The blocking group is then removed chemically or with light, and the cycle is repeated, allowing for the accurate pyrosequencing of the entire DNA strand.

• Detection of single-nucleotide polymorphisms, insertion/deletions, short tandem repeats, and pooled allele frequencies
• Human leukocyte antigen typing for guided disease treatment
• Gene expression/regulation profiling including identification of gene copy number, allelic imbalances in RNA, or methylation status
• Improved drug development for pharmacogenomics and chemogenomics
• Screening for bacterial resistance

• Relative to typical pyrosequencing, errors/background signals are minimized allowing for the identification of any DNA sequence and a vastly improved read length
• Compatibility with microarrays affords low cost and high throughput DNA sequencing

Patent Status: Patent Pending (WO/2007/002204) ~ see link below.

Licensing Status: Available for Licensing and Sponsored Research Support

Publications: Wu J, Zhang S, Meng Q, Cao H, Li Z, Li X, Shi S, Kim DH, Bi L, Turro NJ, Ju J. 3'-O-modified Nucleotides as Reversible Terminators for Pyrosequencing (2007) PNAS 104: 16462-16467