One-Step PCR Sequencing. Final Technical Progress Report for February 15, 1997 - November 30, 2001

PDF Version Also Available for Download.

Description

We investigated new chemistries and alternate approaches for direct gene sequencing and detection based on the properties of boron-substituted nucleotides as chain delimiters in lieu of conventional chain terminators. Chain terminators, such as the widely used Sanger dideoxynucleotide truncators, stop DNA synthesis during replication and hence are incompatible with further PCR amplification. Chain delimiters, on the other hand, are chemically-modified, ''stealth'' nucleotides that act like normal nucleotides in DNA synthesis and PCR amplification, but can be unmasked following chain extension and exponential amplification. Specifically, chain delimiters give rise to an alternative sequencing strategy based on selective degradation of DNA chains ... continued below

Physical Description

vp.

Creation Information

Shaw, B. R. April 16, 2004.

Context

This report is part of the collection entitled: Office of Scientific & Technical Information Technical Reports and was provided by UNT Libraries Government Documents Department to Digital Library, a digital repository hosted by the UNT Libraries. More information about this report can be viewed below.

Who

People and organizations associated with either the creation of this report or its content.

Author

Sponsor

Publisher

Provided By

UNT Libraries Government Documents Department

Serving as both a federal and a state depository library, the UNT Libraries Government Documents Department maintains millions of items in a variety of formats. The department is a member of the FDLP Content Partnerships Program and an Affiliated Archive of the National Archives.

Contact Us

What

Descriptive information to help identify this report. Follow the links below to find similar items on the Digital Library.

Description

We investigated new chemistries and alternate approaches for direct gene sequencing and detection based on the properties of boron-substituted nucleotides as chain delimiters in lieu of conventional chain terminators. Chain terminators, such as the widely used Sanger dideoxynucleotide truncators, stop DNA synthesis during replication and hence are incompatible with further PCR amplification. Chain delimiters, on the other hand, are chemically-modified, ''stealth'' nucleotides that act like normal nucleotides in DNA synthesis and PCR amplification, but can be unmasked following chain extension and exponential amplification. Specifically, chain delimiters give rise to an alternative sequencing strategy based on selective degradation of DNA chains generated by PCR amplification with modified nucleotides. The method as originally devised employed template-directed enzymatic, random incorporation of small amounts of boron-modified nucleotides (e.g., 2'-deoxynucleoside 5'-alpha-[P-borano]- triphosphates) during PCR amplification. Rather than incorporation of dideoxy chain terminators, which are less efficiently incorporated in PCR-based amplification than natural deoxynucleotides, our method is based on selective incorporation and exonuclease degradation of DNA chains generated by efficient PCR amplification of chemically-modified ''stealth'' nucleotides. The stealth nucleotides have a boranophosphate group instead of a normal phosphate, yet behave like normal nucleotides during PCR-amplification. The unique feature of our method is that the position of the stealth nucleotide, and hence DNA sequencing fragments, are revealed at the desired, appropriate moment following PCR amplification. During the current grant period, a variety of new boron-modified nucleotides were synthesized, and new chemistries and enzymatic methods and combinations thereof were explored to improve the method and study the effects of borane modified nucleotides on polymerase and unmasking mechanisms. This approach takes advantage of differences in reactivity of the normal and modified nucleotidic linkages to generate PCR sequencing fragments that terminate at the site of incorporation of the modified nucleotide. In principle, the position of the modified nucleotide in each PCR product can be revealed in two ways, either by enzymatic unmasking (as previously described) or by chemical unmasking. We identified reagent sets for enzymatic or chemical conversion of boronated PCR products into mono- and bidirectional sequencing fragments. (a) We developed a new modified cytidine boranophosphate analogue that is (i) compatible with PCR, but more resistant to exonuclease III read-through than unmodified cytidine and (ii) permits better base calling; (b) We developed chemical methods for DNA and RNA cleavage at boronated nucleotide sites; and (c) We developed methods to quantify and detect stealth boranophosphate groups in DNA and RNA. Key advantages of boranophosphates as sequence delimiters in PCR are that they (1) delineate the DNA sequence yet (2) do not obstruct exponential amplification, and they (3) permit direct PCR sequencing, cycle sequencing, or RNA sequencing. They are also compatible with most sequencing platforms.

Physical Description

vp.

Notes

OSTI as DE00825879

Source

  • Other Information: PBD: 16 Apr 2004

Language

Item Type

Identifier

Unique identifying numbers for this report in the Digital Library or other systems.

  • Report No.: DOE/ER/62376-1
  • Grant Number: FG02-97ER62376
  • DOI: 10.2172/825879 | External Link
  • Office of Scientific & Technical Information Report Number: 825879
  • Archival Resource Key: ark:/67531/metadc787364

Collections

This report is part of the following collection of related materials.

Office of Scientific & Technical Information Technical Reports

Reports, articles and other documents harvested from the Office of Scientific and Technical Information.

Office of Scientific and Technical Information (OSTI) is the Department of Energy (DOE) office that collects, preserves, and disseminates DOE-sponsored research and development (R&D) results that are the outcomes of R&D projects or other funded activities at DOE labs and facilities nationwide and grantees at universities and other institutions.

What responsibilities do I have when using this report?

When

Dates and time periods associated with this report.

Creation Date

  • April 16, 2004

Added to The UNT Digital Library

  • Dec. 3, 2015, 9:30 a.m.

Description Last Updated

  • Aug. 5, 2016, 3:17 p.m.

Usage Statistics

When was this report last used?

Yesterday: 0
Past 30 days: 0
Total Uses: 2

Interact With This Report

Here are some suggestions for what to do next.

Start Reading

PDF Version Also Available for Download.

Citations, Rights, Re-Use

Shaw, B. R. One-Step PCR Sequencing. Final Technical Progress Report for February 15, 1997 - November 30, 2001, report, April 16, 2004; United States. (digital.library.unt.edu/ark:/67531/metadc787364/: accessed December 15, 2017), University of North Texas Libraries, Digital Library, digital.library.unt.edu; crediting UNT Libraries Government Documents Department.