TY  - JOUR
AU  - Xue, Xiaodong
AU  - Li, Qinxia
AU  - Wu, Zhen
AU  - Fan, Na
PY  - 2025
DA  - 2025/03/31
TI  - Key Techniques and Efficiency Analysis of Amplification of Flanking Unknown Sequences by Inverse PCR
JO  - Agricultural Science and Food Processing
T2  - Agricultural Science and Food Processing
JF  - Agricultural Science and Food Processing
VL  - 2
IS  - 1
SP  - 47
EP  - 55
DO  - 10.62762/ASFP.2024.865235
UR  - https://www.icck.org/article/abs/ASFP.2024.865235
KW  - brachypodium distachyon
KW  - inverse PCR
KW  - unknown sequence
KW  - flanking sequence
AB  - Inverse PCR (IPCR) is a reliable, straightforward, and effective technique for acquiring unknown sequences. In this study, we used the model monocot Brachypodium distachyon (ecotype Bd21) to standardize the conditions and materials necessary for the successful execution of IPCR. The analysis of the amplified sequences resulted in the following conclusions. First, the distance between the nearest primer and the boundary of the known-unknown sequence is crucial for determining whether the target sequence can be expanded in the second round of IPCR. Specifically, this distance should exceed 100 bp, ideally around 200 bp. Second, because the random cleavage of a 6 bp endonuclease occurs at a greater distance than that of a 4 bp endonuclease, the use of a 6 bp endonuclease in IPCR results in larger but often inconsistent bands, while maintaining good specificity. Therefore, if the goal is to amplify longer sequences or achieve high accuracy, it is advisable to select endonucleases with 6 bp restriction sites. Third, IPCR is a viable technique that can be effectively utilized to obtain unknown DNA sequences. The experimental conditions established in this study serve as a theoretical basis for the amplification of unknown genome sequences of Gramineae crops and other species.
SN  - 3066-1579
PB  - Institute of Central Computation and Knowledge
LA  - English
ER  - 

@article{Xue2025Key,
  author = {Xiaodong Xue and Qinxia Li and Zhen Wu and Na Fan},
  title = {Key Techniques and Efficiency Analysis of Amplification of Flanking Unknown Sequences by Inverse PCR},
  journal = {Agricultural Science and Food Processing},
  year = {2025},
  volume = {2},
  number = {1},
  pages = {47-55},
  doi = {10.62762/ASFP.2024.865235},
  url = {https://www.icck.org/article/abs/ASFP.2024.865235},
  abstract = {Inverse PCR (IPCR) is a reliable, straightforward, and effective technique for acquiring unknown sequences. In this study, we used the model monocot Brachypodium distachyon (ecotype Bd21) to standardize the conditions and materials necessary for the successful execution of IPCR. The analysis of the amplified sequences resulted in the following conclusions. First, the distance between the nearest primer and the boundary of the known-unknown sequence is crucial for determining whether the target sequence can be expanded in the second round of IPCR. Specifically, this distance should exceed 100 bp, ideally around 200 bp. Second, because the random cleavage of a 6 bp endonuclease occurs at a greater distance than that of a 4 bp endonuclease, the use of a 6 bp endonuclease in IPCR results in larger but often inconsistent bands, while maintaining good specificity. Therefore, if the goal is to amplify longer sequences or achieve high accuracy, it is advisable to select endonucleases with 6 bp restriction sites. Third, IPCR is a viable technique that can be effectively utilized to obtain unknown DNA sequences. The experimental conditions established in this study serve as a theoretical basis for the amplification of unknown genome sequences of Gramineae crops and other species.},
  keywords = {brachypodium distachyon, inverse PCR, unknown sequence, flanking sequence},
  issn = {3066-1579},
  publisher = {Institute of Central Computation and Knowledge}
}