定量PCR实验技术Q-PCR.docVIP

定量PCR实验技术 Q-PCR Quantitative PCR Joseph SambrookPeter Maccallum Cancer Institute and The University of Melbourne, AustraliaDavid W. RussellUniversity of Texas Southwestern Medical Center, DallasExcerpted From Molecular Cloning: A Laboratory Manual Third Edition ABSTRACT Quantitative PCR involves co-amplification of two templates: a constant amount of a preparation containing the desired target sequence and varying amounts of a reference template. After amplification, the concentration of the target sequence in the preparation of nucleic acid under test is established by interpolation into a standard curve. Quantitation of nucleic acids by PCR is best performed by real-time PCR. However, the following robust protocol, which uses radioactivity to quantify PCRproducts, remains useful when a real-time instrument is unavailable. The method can be easily adapted to other methods of quantification such as fluorometry. MATERIALS Reagents and Solutions Chloroform dNTP solution (pH 8.0), containing all four deoxynucleotide triphosphates, each at a concentration of 20 mM MgCl2 (1 M) Placental RNase inhibitor (20 units/μl) Enzymes and Buffers Appropriate restriction enzymes and 10x buffers Bacteriophage T4 DNA ligase and 10x buffer Reverse transcriptase, required only if RNA is used as a template Thermostable DNA polymerase and 10x amplification buffer as supplied by the manufacturer or homemade 500 mM KCl 100 mM Tris-Cl (pH 8.3, room temperature) 15 mM MgCl2 Nucleic Acids/Oligonucleotides DNAmarkers for gel electrophoresis Externally added reference (either DNA or RNA) of known concentrationUse a DNA reference to measure the concentration of DNA sequences and, if possible, an RNA reference for RNA targets. A method to construct reference RNA is described in Protocol 15.2. Sense and antisense primers, each 20 M in H2OThere is nothing unusual about the primers used in quantitative PCR. The standard rules for primer design apply. Target nucleic acidThe target c