Mutagenesis, Vol. 17, No. 5, 365-374,
September 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press
Ligation of a primer at a mutation: a method to detect low level mutations in DNA
Department of Radiation Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA, USA and 1 School of Dentistry, University of California Los Angeles, Los Angeles, CA, USA
Detection of low frequency mutations following exposure to mutagens or during the early stages of cancer development is instrumental for risk assessment and molecular diagnosis. We present a sensitive new method to detect trace levels of DNA mutations induced within a large excess of wild-type sequences. The method is based on mutation-induced generation of new restriction enzyme recognition sites. A DNA sequence is amplified from genomic DNA or cDNA using a high fidelity polymerase. The purified PCR product is digested with a restriction enzyme that recognizes the newly generated restriction site, partially dephosphorylated and ligated with an oligonucleotide at the position of the mutation. The ligated oligonucleotide is then utilized in two rounds of PCR to amplify the mutated DNA but not the wild-type allele that contains no restriction site. An A
T polymorphism in mRNA (tenascin gene, A2366T, AsnIle) and a GA polymorphism in genomic DNA (Ku gene, G74582A, ValIle), both of which generate a restriction site for the enzyme SAU3A1, demonstrate the application. Eleven patient samples pre-characterized for the G74582A polymorphism in the repair gene Ku are used to demonstrate the reliability of this approach. This technique quantitatively detects the Ku GA polymorphism at a mutant frequency of 1.6x10-6 relative to the wild-type allele. Mutations in p53 that are frequently induced by mutagens can readily be detected using the present method. As an example, using a second enzyme BbvI, a mutation frequently encountered in human cancers (G14154A mutation, p53 codon 245, ArgGln) was detected in patient samples. The process does not require radioactivity, utilizes established procedures and overcomes several factors known to produce false positives in RFLP-based assays. The present amplification via primer ligation at the mutation (APRIL-ATM) has potential applications in the detection of mutagen-generated genetic alterations, early detection of tumor marker mutations in bodily discharges and the diagnosis of minimal residual disease.
2 To whom correspondence should be addressed at: Longwood Radiation Oncology Center, Brigham–Dana Farber Children's Hospitals, Level L2, Radiation Therapy, 75 Francis Street, Boston, MA 02115, USA. Tel: +1 617 6326905; Fax: +1 617 6326900; Email: mmakrigiorgos{at}partners.org
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  J. Li, R. Berbeco, R. J. Distel, P. A. Janne, L. Wang, and G. M. Makrigiorgos s-RT-MELT for rapid mutation scanning using enzymatic selection and real time DNA-melting: new potential for multiplex genetic analysis Nucleic Acids Res., June 9, 2007; 35(12): e84 - e84. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W.-H. Liu, M. Kaur, G. Wang, P. Zhu, Y. Zhang, and G. M. Makrigiorgos Inverse PCR-Based RFLP Scanning Identifies Low-Level Mutation Signatures in Colon Cells and Tumors Cancer Res., April 1, 2004; 64(7): 2544 - 2551. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. V. Harden, D. C. Thomas, N. Benoit, K. Minhas, W. H. Westra, J. A. Califano, W. Koch, and D. Sidransky Real-Time Gap Ligase Chain Reaction: A Rapid Semiquantitative Assay for Detecting p53 Mutation at Low Levels in Surgical Margins and Lymph Nodes from Resected Lung and Head and Neck Tumors Clin. Cancer Res., April 1, 2004; 10(7): 2379 - 2385. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Kaur and G. M. Makrigiorgos Novel amplification of DNA in a hairpin structure: towards a radical elimination of PCR errors from amplified DNA Nucleic Acids Res., March 15, 2003; 31(6): e26 - e26. [Abstract] [Full Text] [PDF]
|
|