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Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria

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Investigating T_m Method Specificity Using Oligonucleotide Sequence Variants.

Alexandra H Heussner¹, Sarina Schuler², Gerd Berger²

¹Analytical Science Laboratory, Development Service, Vetter Pharma-Fertigung GmbH & Co. KG, 88212 Ravensburg, Germany alexandra.heussner@vetter-pharma.com.

PDA Journal of Pharmaceutical Science and Technology

|September 19, 2022

Summary

Ultraviolet-spectrometric determination of melting temperature (Tm) is a suitable sequence-specific identity test for oligonucleotides. This method accurately identifies subtle sequence variations in active pharmaceutical ingredients (APIs) for quality control.

Keywords:

Hyperchromicity Identity Melting temperature Oligonucleotide Quality control Specificity Tm

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Area of Science:

Pharmaceutical Analysis
Oligonucleotide Chemistry
Quality Control

Background:

Identity (ID) testing is crucial for active pharmaceutical ingredients (APIs) and starting materials.
Oligonucleotides, an emerging API class, require robust ID testing strategies.
Current ID testing often combines mass determination with sequence-specific methods.

Purpose of the Study:

To evaluate ultraviolet (UV)-spectrometric melting temperature (Tm) determination as a sequence-specific ID test for oligonucleotides.
To assess the method's specificity for detecting sequence variations in routine pharmaceutical quality control (QC).

Main Methods:

Design and synthesis of model oligonucleotide sequences and their variants.
Analysis of oligonucleotide duplexes using UV-spectrometric Tm determination.
Evaluation of method specificity against single base changes, deletions, and insertions.

Main Results:

Precise and specific data were obtained for oligonucleotide sequences and variants.
Even minor sequence alterations (single base changes, insertions, deletions) caused significant shifts in measured Tm.
The UV-spectrometric Tm method demonstrated high specificity towards subtle sequence modifications.

Conclusions:

UV-spectrometric Tm determination is a highly specific and applicable analytical method for oligonucleotide ID testing.
This method can be reliably integrated into pharmaceutical QC workflows for oligonucleotide APIs.