These tools will no longer be maintained as of December 31, 2024. Archived website can be found here. PubMed4Hh GitHub repository can be found here. Contact NLM Customer Service if you have questions.


Pubmed for Handhelds

PUBMED FOR HANDHELDS

Search MEDLINE/PubMed

  • Title: A One-Pot CRISPR/Cas9-Typing PCR for DNA Detection and Genotyping.
    Author: Gao J, Wu L, Yang D, Gong W, Wang J.
    Journal: J Mol Diagn; 2021 Jan; 23(1):46-60. PubMed ID: 33127524.
    Abstract:
    Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease Cas9 (Cas9) has high specificity to its target DNA as a gene editing tool. This characteristic makes it useful for DNA detection. Combining the advantages of CRISPR/Cas9 and PCR, this study establishes a novel CRISPR/Cas9-based DNA detection method, named CRISPR/Cas9-typing PCR version 4.0 (ctPCR4.0). This method can detect target DNA in one pot with high specificity and sensitivity. In a homogenous reaction, the target DNA is first cleaved by a pair of Cas9- single-guide RNA complexes and thus releases two single strands with free 3' ends, allowing a pair of oligonucleotides to anneal with the strands. The annealed oligonucleotides provide templates for DNA polymerization from the free 3' ends. A universal primer annealing site is thus produced at the end of two single strands. The target DNA is then amplified by PCR using a universal primer. This method was first verified by accurately detecting the cloned L1 fragments of 10 genotypes of high-risk human papilloma viruses (HPVs). This method was then validated by detecting the L1 fragments of two highest-risk HPVs, HPV 16 and HPV 18, in the genomic DNA of two HPV-positive cervical carcinoma cells, HeLa and SiHa. Finally, this method was further validated by accurately detecting 10 high-risk HPVs in 30 clinical samples.
    [Abstract] [Full Text] [Related] [New Search]