Manipulation of a Large DNA Molecule using the Phase Transition

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Conventional DNA sequencing methods are limited to short fragments (up to 1000 base pairs).
• Sequencing long DNA requires fragmentation, leading to loss of order information.
• Preparing fragments from the terminus of long DNA can preserve order.

Purpose of the Study:

• To develop a method for preparing DNA fragments from the terminus of long DNA molecules.
• To enable DNA sequencing with preserved order information.
• To improve manipulation and cutting of large genomic DNA.

Main Methods:

• Genomic DNA manipulation using reversible globular transformation for stability and handling.
• Sequential spinning of coiled DNA from globular form.
• Fixation of stretched DNA onto a glass surface in a desired pattern.
• Development of a method for cutting DNA from the terminus of stretched molecules.
• Electrochemical supply of magnesium ions to activate restriction enzymes for precise DNA cutting.

Main Results:

• Successful manipulation and stabilization of large genomic DNA via globular transformation.
• Demonstrated sequential spinning and fixation of DNA onto a glass surface.
• Developed and validated a method for cutting DNA fragments from stretched molecules.
• Achieved targeted DNA cleavage using electrochemically controlled restriction enzyme activation.

Conclusions:

• The developed method allows for the preparation of DNA fragments from the terminus of long DNA molecules, preserving their order.
• This technique overcomes limitations of conventional DNA sequencing by enabling manipulation and precise cutting of large DNA.
• The approach holds potential for advancing genomic studies requiring long-read sequencing with positional information.