Because of their unique properties, several molecular biology methods utilize dideoxynucleotides. For example, the dideoxynucleotide chain-terminating method uses deoxyribonucleotides lacking a hydroxyl group (OH) at the 3′-positions of the ribose sugar. Oligonucleotides modified with chain terminator or end blocker dideoxynucleotides at the 3’-end block ligation or prevent polymerase extension from the 3’-terminus. For oligonucleotides modified with a deoxyribonucleotide, a phosphodiester bond cannot form with a 5′-hydrogen resulting in a chain elongation stop.
The lack of a 3'-OH group on the ribose sugar makes dideoxynucleotides a valuable tool for the following applications:
1. DNA sequencing: The Sanger sequencing method, a widely used technique to determine the sequence of DNA molecules, uses dideoxynucleotides.
2. Site-directed mutagenesis: Dideoxynucleotides help to create mutations in specific regions of DNA. This technique incorporates a dideoxynucleotide into the growing DNA strand during replication, which terminates the chain and introduces a mutation.
3. In vitro transcription: Dideoxynucleotides can terminate RNA synthesis during in vitro transcription producing RNA molecules that have a defined 5'- and 3'-end.
4. Primer extension: In primer extension assays, dideoxynucleotides allow the determination of the position of specific nucleotides in a DNA or RNA molecule. In this technique, a primer is annealed to the target molecule, and DNA polymerase extends the primer in the presence of dideoxynucleotides. Incorporating a dideoxynucleotide at a specific position terminates the extension reaction, indicating the position of the nucleotide of interest.
The Sanger DNA sequencing method uses dideoxynucleotides as chain-elongating inhibitors or chain terminators of DNA polymerase. The abbreviation of dideoxynucleotides is ddNTPs (ddGTP, ddATP, ddTTP, or ddCTP). Because the ribose's 2'- and 3’-position do not contain hydroxyl groups, dideoxynucleotides are also known as 2', 3’-dideoxynucleotides.
Dideoxynucleotides can be labeled with a radioactive or nonradioactive tag to visualize fragments containing ddNTPs.
The earlier Taq polymerases used were deficient in two respects:
(i) During sequencing, the enzymes incorporate each of the four dideoxynucleoside 5′ triphosphates (ddNTPs) at widely different rates (ddGTP, for example, was incorporated ten times faster than the other three ddNTPs), and
(ii) The enzymes exhibited uneven band-intensity or peak-height patterns in radio-labeled or dye-labeled DNA sequence profiles; therefore, Li et al., in 1999, created Taq polymerase variants with improved biotechnological specificities converting these polymerases to functional tools:
[1] With a better extension of guanine (G) bases, and
[2] a more consistent band-intensity pattern allowing for more accurate sequencing results.
As a result, during genome sequencing, using Taq DNA polymerases mutated at position 660 helped limit errors and reduce the requirement for redundancy, thereby decreasing cost and labor.
Reference
Li Y, Mitaxov V, Waksman G. Structure-based design of Taq DNA polymerases with improved properties of dideoxynucleotide incorporation. Proc Natl Acad Sci U S A. 1999 Aug 17;96(17):9491-6. [PMC]
Sanger, F. (8 December 1980). "Determination of Nucleotide Sequences in DNA (Nobel lecture)".
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