Real Time PCR Group Members: Alanna, Susan, Jane, Sam & Rachel
General Overview of Standard PCR 1. Temperature raised to 95 C where double stranded DNA becomes single strands 2. Temperature lowered to ~60 C allowing primers to bind to the target sequence Thus polymerase has somewhere to bind!
General Overview of Standard PCR 1. Temperature is raised to 72 C (the optimal temperature for polymerase to operate at) 2. DNA polymerase extends primers to form complementary strand. 3. You now have twice as many copies 4. Temperature cycle is repeated through 40 cycles to create exponential copies of the target sequence
Reagents for Standard PCR Primers Mg2+ Nuclease-free water dntps (deoxynucleotide triphosphates) Thermophilic DNA Polymerase Additional: Reverse Transcriptase (for RNA) Appropriate Buffers
Critical Elements required for Real Time PCR Taqman based real-time PCR Primers, Taq DNA polymerase, Mg2+, Nuclease free H2O, dntps, Reverse transcriptase (if starting material is RNA), Taqman probes, appropriate buffers and DNA template. SYBR Green based real-time PCR Primers, thermophilic DNA polymerase, Mg2+, SYBR green I dye, Nuclease free H2O, dntps, Reverse transcriptase (if starting material is RNA), appropriate buffers and DNA template. Other materials include: Real-time PCR machine Computer + software for data collection + analysis
Taqman Probe and SYBR Green I Dye http://www.biosyn.com/tew/taqman-vs-sybr-green-chemistries.aspx
Applications of Real Time PCR 1. Gene expression analysis or mrna analysis 2. Detection of GMOs in food 3. Cancer or disease detection 4. Genetic variation analysis
Standard PCR vs. Real Time PCR Standard PCR Tells if a sequence is present or absent Result are semiquantitative at best Real time PCR Better detection sensitivity Elimination of post PCR processing (reduces contamination) Enables quantitative analysis of genes (tells the copy number) Faster/ more efficient results
BIOL 463 A Technique in Five Minutes TECHNIQUES PRESENTATIONS: WRITE- UP TEMPLATE 1. Names and contributions of group members: Alanna: Research, Write- up, Powerpoint Susan: Research, Write- up, Powerpoint Rachel: Research, Write- up, Powerpoint Jane: Research, Write- up, Powerpoint Sam: Research, Write- up, Powerpoint 2. Technique chosen: RT- PCR: Real time PCR or Quantitative PCR (qpcr) 3. What does this technique do? Amplification and detection of DNA simultaneously in Real Time. 4. What applications is this technique employed for? Gene expression analysis or mrna analysis Hein et al. (2001) employed real time PCR to analyze murine gamma interferon- gamma mrna (a cytokine mrna) expression in splenocytes Detection of GMOs in food Zeitler et al. (2002) identified real time PCR as a method for quantifying transgenic contaminants with herbicide resistance in conventional rape seed. Cancer or disease detection Multiplex real- time reverse transcriptase PCR is an applicable method for the detection, identification, and quantification of HBV, HCV and HIV- 1 Bernard and Wittwer (2002) used real- time PCR for detection of multiple breast cancer molecular markers Genetic variation analysis
BIOL 463 A Technique in Five Minutes Real- time PCR is able to detect mutations in the sequence, including single nucleotide polymorphisms (SNPs) through its melting point analysis. 5. What questions relating to gene regulation and/or development can be addressed using this technique? Provide two examples (peer- reviewed papers) that use this technique. Cytokine gene expression and regulation during infection: Example: Th1 and Th2 cytokine gene expression in primary infection and vaccination against Fasciola gigantica in buffaloes by real- time PCR. Cytochrome expression and regulation in different life stages (unfertilized eggs, embryos/larvae and adult tissue) and its role on neurodevelopment and plasticity: Example: Real- time PCR analysis of cytochrome P450 aromatase expression in zebrafish: Gene specific tissue distribution, sex differences, developmental programming, and estrogen regulation. 6. What critical reagents are required to use this technique? Taqman based real- time PCR Primers, Taq polymerase, Mg2+, Nuclease free H2O, dntps, Reverse transcriptase (if starting material is RNA), Taqman probes, appropriate buffers and DNA template. SYBR Green based real- time PCR Primers, thermophilic DNA polymerase, Mg2+, SYBR green I dye, Nuclease free H2O, dntps, Reverse transcriptase (if starting material is RNA), appropriate buffers and DNA template. Other materials include: Real- time PCR machine that is able to complete thermal cycling steps for PCR and collect fluorescence data simultaneously, a computer, and suitable computer software for data collection and analysis.
BIOL 463 A Technique in Five Minutes 7. What critical information is required to be able to employ this technique? Sequence data on the target sequence to design proper probes and primers. Melting temperatures for PCR products for melting curve analysis to determine amplification specificity. 8. References: Bernard PS, Wittwer CT. Real- time PCR technology for cancer diagnostics. Clin Chem 2002; 48:1178 1185. Bustin SA, Mueller R. Real- time reverse transcription PCR (qrt- PCR) and its potential use in clinical diagnosis. Clinical Science 2005; 109:365-379. Hein J, Schellenberg U, Bein G, Hackstein H. Quantification of murine IFN- γ mrna and protein expression: impact of real- time kinetic RT- PCR using SYBR Green I dye. Scand J Immunol 2001; 54:285 291 Kumar N, Raina OK, Nagar G, Prakash V, Jacob SS. Th1 and Th2 cytokine gene expression in primary infection and vaccination against Fasciola gigantica in buffaloes by real- time PCR. Parasitol Res 2013; 112:3561-3568. Sawyer SJ, Gerstner KA, Callard GV. Real- time PCR analysis of cytochrome P450 aromatase expression in zebrafish: Gene specific tissue distribution, sex differences, developmental programming, and estrogen regulation. General and comparative Endocrinology 2006; 147:108-117. Valasek MA, Repa, JJ. The power of real- time PCR. Adv Physiol 2005; 29: 151-159. Zeitler R, Pietsch K, Waiblinger H. Validation of real- time PCR methods for the quantification of transgenic contaminations in rape seed. Eur Food Res Technol 2002; 214:346-351.