Sequencing telomeric DNA template with short tandem repeats using dye terminator cycle sequencing

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Standard DNA sequencing methods face challenges with templates containing high-frequency dinucleotide and tetranucleotide repeats, as well as telomeric DNA with tandem repeats.
• Common issues include base compression artifacts, secondary structure formation on nascent strands, and DNA polymerase slippage leading to premature chain termination in homopolymer and short tandem repeat regions.

Purpose of the Study:

• To investigate methods for improving DNA sequencing accuracy in challenging repeat-rich regions, specifically telomeric DNA.
• To evaluate the effectiveness of modified annealing times, temperatures, and denaturing conditions in overcoming sequencing artifacts.

Main Methods:

• Comparison of ABI PRISM BigDye, dGTP BigDye, and DYEnamic ET terminator chemistries.
• Modification of routine protocols by incorporating denaturants: 1 M betaine (1-carboxy-N,N,N-trimethylmethanaminium inner salt) or 5% dimethyl sulfoxide (DMSO).
• Increased annealing and denaturation times to promote primer extension and linear growth of sequencing products.

Main Results:

• Successfully sequenced DNA templates containing tandem repeats that were previously unsequenceable under standard conditions.
• Demonstrated that increased annealing/denaturation times and the use of denaturants effectively reduce secondary structure formation and polymerase slippage.
• Identified optimal conditions for sequencing telomeric DNA rich in CA- and AACCCC-type repeats, as well as dinucleotide (GT, CT) and tetranucleotide repeats.

Conclusions:

• Modified DNA sequencing protocols, including optimized thermal conditions and the addition of denaturants, can overcome limitations in sequencing repeat-rich DNA.
• These optimized methods significantly improve the ability to sequence challenging templates, such as telomeric DNA, which are critical for genetic research.
• The findings provide practical solutions for core sequencing laboratories encountering difficulties with specific DNA sequences.