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ARMS^TM Allele-specific Amplification-based Detection of Mutant p53 DNA and mRNA in Tumors of the Breast,

¹ AstraZeneca Diagnostics, Gadbrook Park, Northwich CW9 7RA, United Kingdom

² Department of Surgery, Medical School, University of Newcastle-upon-Tyne, Newcastle-upon-Tyne NE24HH, United Kingdom

³ Department of Surgery, Queen Elizabeth Hospital, Gateshead NE965X, United Kingdom

⁴ AstraZenecaPharma UK, King’s Court, Wilmslow, Cheshire SK104TG, United Kingdom

⁵ AstraZeneca Research, Safety of Medicines, Alderley Park, Macclesfield, Cheshire SK104TG, United Kingdom

^a address correspondence to this author at: Renovo Ltd, Manchester Incubator Building, 48 Grafton St., Manchester M13 9XX, United Kingdom; fax 44-161-606-7333

^b address correspondence to this author at: AstraZeneca Diagnostics, Gadbrook Park, Northwich CW9 7RA, United Kingdom

Alterations in the p53 gene have been reported in approximately one-half of human tumors (1). Much debate surrounds the clinical significance of p53 mutations as measured directly or inferred by immunohistochemical staining (2). Many studies have addressed p53 mutational status of tumors with regard to prognosis (3)(4)(5)(6) and optimizing treatment (7)(8) in cancer.

Current recommendations categorize primary breast cancer patients on the basis of tumor size, estrogen receptor status, and histological grade (9). Improvement in the predictive power would aid patient management, particularly in cases where chemotherapy could be predicted to have no benefit. Prognosis and treatment outcomes have correlated with specific p53 mutations, particularly those affecting DNA binding (10)(11)(12). Identification of the particular p53 mutations present in a tumor may therefore have more value than monitoring for any p53 mutation or protein overexpression. Furthermore, to be clinically useful, mutations must be detectable in the presence of significant quantities of "wild-type" (wt) sequences from healthy tissues.

We extracted RNA and DNA in parallel from breast tumors and used real-time ARMS^TM allele-specific amplification (13)(14)(15)(16) technology to obtain quantitative data on mutant p53 sequences in both nucleic acid pools.

Oligonucleotides were prepared by Oswel (Southampton, United Kingdom) or AstraZeneca Diagnostics (Abingdon, United Kingdom). Molecular biology-grade reagents were obtained from Sigma unless indicated. ARMS buffer consisted of 1.2 mmol/L MgCl₂, 10 mmol/L Tris-HCl, 50 mmol/L KCl, pH 8.3. The reaction buffer was identical to ARMS buffer except that it contained 3.5 mmol/L MgCl₂. The cassette dilution buffer consisted of 10 mmol/L Tris-HCl, 50 mmol/L KCl, 1 g/L bovine serum albumin, pH 8.3.

Blood from 50 healthy volunteers, who had . . . [Full Text of this Article]

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