研究目的
To propose a method for accurately replicating and detecting the targeted location of a DNA strand from individual cells at the single-cell level through laser-induced heating and dielectrophoresis.
研究成果
The developed microfluidic platform successfully replicated local DNA sequences in situ with high efficiency and detected small quantities of products with high sensitivity. The technique could be applied to rapidly perform gene sequencing for new plant varieties in agricultural biology.
研究不足
The method may have limitations in the amount of stretched and immobilised DNA on the electrode, particularly for higher thermal cycles, leading to higher error bars in fluorescence intensity calculations. Gel electrophoresis could not detect replicated products due to insufficient quantity.
1：Experimental Design and Method Selection:
The study integrated laser-induced heating and dielectrophoresis to develop a microfluidic platform for in situ replication and detection of target DNA sequences. Aluminium electrodes were manufactured to stretch and immobilise DNA strands. Laser-induced heating was used for thermal cycles to replicate local DNA sequences.
2：Sample Selection and Data Sources:
λDNA was used as an experimental sample, dyed with nucleic acid fluorescent dye. Biotinylated primers were designed for DNA replication.
3：List of Experimental Equipment and Materials:
Included an infrared laser, fluorescence microscope, charge-coupled device camera, and various chemicals like avidin, bovine serum albumin, and quantum dots.
4：Experimental Procedures and Operational Workflow:
Involved coating coverslips with avidin, stretching and immobilising DNA with dielectrophoresis, replicating DNA with laser-induced heating, and detecting products with Qdots.
5：Data Analysis Methods:
Fluorescence intensity of Qdots was calculated to detect replicated DNA products.
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