Antiprimer quenching-based real-time PCR and its application to the analysis of clinical cancer samples

被引:26
作者
Li, J
Wang, FF
Mamon, H
Kulke, MH
Harris, L
Maher, E
Wang, LL
Makrigiorgos, GM
机构
[1] Harvard Univ, Dana Farber Canc Inst, Dept Radiat Oncol, Sch Med, Boston, MA 02115 USA
[2] Harvard Univ, Dana Farber Canc Inst, Dept Med Oncol, Sch Med, Boston, MA 02115 USA
关键词
D O I
10.1373/clinchem.2005.063321
中图分类号
R446 [实验室诊断]; R-33 [实验医学、医学实验];
学科分类号
1001 ;
摘要
Background: Nucleic acid amplification plays an increasingly important role in genetic analysis of clinical samples, medical diagnostics, and drug discovery. We present a novel quantitative PCR technology that combines the advantages of existing methods and allows versatile and flexible nucleic acid target quantification in clinical samples of widely different origin and quality. Methods: We modified one of the 2 PCR primers by use of an oligonucleotide "tail" fluorescently labeled at the 5' end. An oligonucleoticle complementary to this tail, carrying a 3' quenching molecule (antiprimer), was included in the reaction along with 2 primers. After primer extension, the reaction temperature was lowered such that the antiprimer hybridizes and quenches the fluorescence of the free primer but not the fluorescence of the double-stranded PCR product. The latter provides real-time fluorescent product quantification. This antiprimer-based quantitative real-time PCR method (aQRT-PCR) was used to amplify and quantify minute amounts of input DNA for genes important to cancer. Results: Simplex and multiplex aQRT-PCR demonstrated linear correlation (r(2) > 0.995) down to a DNA input equivalent to 20 cells. Multiplex aQRT-PCR reliably identified the HER-2 gene in microdissected breast cancer samples; in formalin-fixed, paraffin-embedded specimens; and in plasma circulating DNA from cancer patients. Adaptation to multiplex single-nucleotide polymorphism detection via allele-specific aQRT-PCR allowed correct identification of apolipoprotein B polymorphisms in 51 of 51 human specimens. Conclusion: The simplicity, versatility, reliability, and low cost of aQRT-PCR make it suitable for genetic analysis of clinical specimens. (c) 2006 American Association for Clinical Chemistry.
引用
收藏
页码:624 / 633
页数:10
相关论文
共 30 条
[1]   Excitonic heterodimer formation in an HIV-1 oligonucleotide labeled with a donor-acceptor pair used for fluorescence resonance energy transfer [J].
Bernacchi, S ;
Piémont, E ;
Potier, N ;
van Dorsselaer, A ;
Mély, Y .
BIOPHYSICAL JOURNAL, 2003, 84 (01) :643-654
[2]  
Bernacchi S, 2001, Nucleic Acids Res, V29, pE62, DOI 10.1093/nar/29.13.e62
[3]  
Germer S, 1999, GENOME RES, V9, P72
[4]   High-throughput SNP allele-frequency determination in pooled DNA samples by kinetic PCR [J].
Germer, S ;
Holland, MJ ;
Higuchi, R .
GENOME RESEARCH, 2000, 10 (02) :258-266
[5]   Comparison of methods of measuring HER-2 in metastatic breast cancer patients treated with high-dose chemotherapy [J].
Harris, LN ;
Liotcheva, V ;
Broadwater, G ;
Ramirez, MJ ;
Maimonis, P ;
Anderson, S ;
Everett, T ;
Harpole, D ;
Moore, MB ;
Berry, DA ;
Rizzeri, D ;
Vredenburgh, JJ ;
Bentley, RC .
JOURNAL OF CLINICAL ONCOLOGY, 2001, 19 (06) :1698-1706
[6]   Real time quantitative PCR [J].
Heid, CA ;
Stevens, J ;
Livak, KJ ;
Williams, PM .
GENOME RESEARCH, 1996, 6 (10) :986-994
[7]   DETECTION OF SPECIFIC POLYMERASE CHAIN-REACTION PRODUCT BY UTILIZING THE 5'-]3' EXONUCLEASE ACTIVITY OF THERMUS-AQUATICUS DNA-POLYMERASE [J].
HOLLAND, PM ;
ABRAMSON, RD ;
WATSON, R ;
GELFAND, DH .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1991, 88 (16) :7276-7280
[8]   Real-time PCR analysis of DNA and RNA extracted from formalin-fixed and paraffin-embedded biopsies [J].
Lehmann, U ;
Kreipe, H .
METHODS, 2001, 25 (04) :409-418
[9]   Whole genome amplification of plasma-circulating DNA enables expanded screening for allelic imbalance in plasma [J].
Li, J ;
Harris, L ;
Mamon, H ;
Kulke, MH ;
Zhu, WH ;
Zhu, P ;
Makrigiorgos, GM .
JOURNAL OF MOLECULAR DIAGNOSTICS, 2006, 8 (01) :22-30
[10]   A new class of homogeneous nucleic acid probes based on specific displacement hybridization [J].
Li, QQ ;
Luan, GY ;
Guo, QP ;
Liang, JX .
NUCLEIC ACIDS RESEARCH, 2002, 30 (02) :5