Resonance ionization spectroscopy for multiplex sequencing of tin-labeled DNA

Area of Science:

• Biochemistry
• Molecular Biology
• Analytical Chemistry

Background:

• DNA sequencing methods are crucial for genetic analysis.
• Stable isotope labeling offers a non-radioactive alternative for biomolecule detection.
• Efficient attachment of labels to oligonucleotide primers is essential for sequencing accuracy.

Purpose of the Study:

• To develop and validate a tin-based reagent for DNA sequencing.
• To assess the impact of the tin reagent on DNA electrophoresis and sequencing fidelity.
• To demonstrate the utility of stable isotope labeling in DNA sequencing using advanced detection techniques.

Main Methods:

• Synthesis of triethylstannylpropanoic acid (TESPA) and its conjugation to oligonucleotide primers.
• Electrophoresis of DNA fragments labeled with tin isotopes ([116Sn]-TESPA and [120Sn]-TESPA).
• Simultaneous detection of DNA bands using autoradiography ([35S]-dTTP) and resonance ionization spectroscopy (RIS) for [120Sn]-TESPA.

Main Results:

• The presence of [116Sn]-TESPA on oligonucleotide primers did not significantly alter DNA mobility or sequencing ladder integrity during electrophoresis.
• Simultaneous application of [120Sn]-TESPA and [35S]-dTTP in the Sanger sequencing procedure allowed for dual detection of DNA bands.
• Resonance ionization spectroscopy confirmed the coincidence of signals with autoradiography, validating the accurate localization of tin-labeled DNA fragments.

Conclusions:

• Triethylstannylpropanoic acid (TESPA) is an effective reagent for labeling oligonucleotide primers in DNA sequencing.
• Stable isotope labeling with tin, detected by RIS, provides a viable and complementary method to radioactive labeling in DNA sequencing.
• This approach confirms previous findings on stable isotopes and demonstrates their practical application in actual DNA sequencing products.