Advantages of a new Taq DNA polymerase in multiplex PCR and time-release PCR

被引:49
作者
Kebelmann-Betzing, C
Seeger, K
Dragon, S
Schmitt, G
Möricke, A
Schild, TA
Henze, G
Beyermann, B
机构
[1] Humboldt Univ, Virchow Med Ctr Charite, Dept Pediat Oncol Hematol, D-13353 Berlin, Germany
[2] PE Appl Biosyst GmbH, Weiterstadt, Germany
关键词
D O I
10.2144/98241pf01
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Extensive diagnostic and scientific investigations are often restricted by limited availability of material. Therefore, methods like multiplex PCR strategies are needed to conserve as much sample as possible. Unfortunately, the establishment of such procedures poses several difficulties. Here we describe the advantages of a new enzyme, AmpliTaq Gold(TM) DNA Polymerase, in multiplex and time-release PCR. The application of this thermostable recombinant Taq DNA polymerase allows the specific amplification of DNA/cDNA targets with very high sensitivity. With our protocol, the specific amplification of 13 different cDNAs of cytokines and cytokine receptors can be realized in three multiplex PCRs (IL-2R alpha, IL-2/15R beta, gamma(c)-chain, IL-4 and IL-4R alpha; IL-10, IL-15 and IL-15R alpha; and IL-2, IFN gamma, IL-7, IL-7R alpha and IL-9R alpha). The novel application of AmpliTaq Gold DNA Polymerase in a time-release PCR protocol allows specific amplification of target DNA/cDNA when only limited amounts of material are available or only low-copy-number DNA/cDNA is suspected. No IL-9 cDNA can be detected in peripheral blood mononuclear cells (PBMC) in the absence of any stimulation, thus it was difficult to amplify this target with routine PCR protocols. Here we demonstrate the reliable and reproducible amplification of IL-9 cDNA in the Hodgkin's lymphoma cell line KM-H2, in PBMC and in stimulated PBMC. Results with AmpliTaq Gold DNA Polymerase were more sensitive and specific compared with AmpliTaq(R) DNA Polymerase, with and without manual hot-start procedure.
引用
收藏
页码:154 / 158
页数:5
相关论文
共 17 条
[1]   CYTOKINE RECEPTOR GENES - STRUCTURE, CHROMOSOMAL LOCATION, AND INVOLVEMENT IN HUMAN-DISEASE [J].
BAIRD, PN ;
DANDREA, RJ ;
GOODALL, GJ .
LEUKEMIA & LYMPHOMA, 1995, 18 (5-6) :373-383
[2]   Simplified hot start PCR [J].
Birch, DE ;
Kolmodin, L ;
Laird, WJ ;
McKinney, N ;
Wong, J ;
Young, KKY ;
Zangenberg, GA ;
Zoccoli, MA .
NATURE, 1996, 381 (6581) :445-446
[3]   PREVENTION OF PRE-PCR MIS-PRIMING AND PRIMER DIMERIZATION IMPROVES LOW-COPY-NUMBER AMPLIFICATIONS [J].
CHOU, Q ;
RUSSELL, M ;
BIRCH, DE ;
RAYMOND, J ;
BLOCH, W .
NUCLEIC ACIDS RESEARCH, 1992, 20 (07) :1717-1723
[4]   CONSTITUTIVE IN-VIVO CYTOKINE AND HEMATOPOIETIC GROWTH-FACTOR GENE-EXPRESSION IN THE BONE-MARROW AND PERIPHERAL-BLOOD OF HEALTHY-INDIVIDUALS [J].
CLUITMANS, FHM ;
ESENDAM, BHJ ;
LANDEGENT, JE ;
WILLEMZE, R ;
FALKENBURG, JHF .
BLOOD, 1995, 85 (08) :2038-2044
[5]   MAXIMIZING SENSITIVITY AND SPECIFICITY OF PCR BY PREAMPLIFICATION HEATING [J].
DAQUILA, RT ;
BECHTEL, LJ ;
VIDELER, JA ;
ERON, JJ ;
GORCZYCA, P ;
KAPLAN, JC .
NUCLEIC ACIDS RESEARCH, 1991, 19 (13) :3749-3749
[6]  
HOUSSIAU FA, 1995, J IMMUNOL, V154, P2624
[7]   FUNCTION OF THE INTERLEUKIN-2 (IL-2) RECEPTOR GAMMA-CHAIN IN BIOLOGIC RESPONSES OF X-LINKED SEVERE COMBINED IMMUNODEFICIENT B-CELLS TO IL-2, IL-4, IL-13, AND IL-15 [J].
MATTHEWS, DJ ;
CLARK, PA ;
HERBERT, J ;
MORGAN, G ;
ARMITAGE, RJ ;
KINNON, C ;
MINTY, A ;
GRABSTEIN, KH ;
CAPUT, D ;
FERRARA, P ;
CALLARD, R .
BLOOD, 1995, 85 (01) :38-42
[8]  
MERZ H, 1991, BLOOD, V78, P1311
[9]  
Michiel D F, 1992, Semin Cancer Biol, V3, P3
[10]  
MIYAJIMA A, 1992, ANNU REV IMMUNOL, V10, P295, DOI 10.1146/annurev.immunol.10.1.295