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Related Experiment Video

Updated: May 26, 2026

Identifying Mutations by High Resolution Melting in a TILLING Population of Rice

Identifying Mutations by High Resolution Melting in a TILLING Population of Rice

Published on: September 2, 2019

Computation-assisted SiteFinding- PCR for isolating flanking sequence tags in rice.

Hongru Wang¹, Jun Fang, Chengzheng Liang

¹State Key Laboratory of Plant Genomics and National Center for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China.

|December 14, 2011

Summary

This study optimized SiteFinding-PCR for isolating flanking sequence tags (FSTs) from T-DNA insertion lines. The new method significantly improves FST isolation efficiency in plants like rice.

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Last Updated: May 26, 2026

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Published on: September 2, 2019

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Area of Science:

Molecular Biology
Plant Genetics
Bioinformatics

Background:

SiteFinding-PCR is a key technique for identifying flanking sequence tags (FSTs) in T-DNA insertion lines.
Improving the efficiency of FST isolation is crucial for genetic studies and crop improvement.
Current SiteFinding-PCR methods require optimization for broader applicability and higher success rates.

Purpose of the Study:

To enhance the efficiency of SiteFinding-PCR for isolating FSTs from T-DNA insertion lines.
To develop a computation-assisted primer design strategy for SiteFinding-PCR.
To adapt the optimized method for use in various plant species.

Main Methods:

Computational screening of the rice genome to identify optimal primer sequences.
Design of a modified random primer incorporating a specific 3' end sequence (GCATG).
Application of the optimized SiteFinding-PCR in 168 transgenic rice lines.

Main Results:

The modified SiteFinding-PCR yielded 107 specific products, including 64 FSTs from 168 lines.
A 73.0% increase in FST isolation efficiency was observed compared to the previous method (P < 0.01).
Computational data provided for adapting the method to maize, sorghum, Arabidopsis, and other grasses.

Conclusions:

The computation-assisted primer design significantly improves SiteFinding-PCR efficiency for FST isolation.
The optimized method offers a more effective approach for characterizing T-DNA insertions in rice.
The adaptability of the modified SiteFinding-PCR to multiple plant species facilitates broader genetic research.