20.109(S15):Diagnostic primer analysis (Day8): Difference between revisions
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Real-time reverse transcription-polymerase chain reaction (RRT-PCR) allows researchers to monitor the results of PCR as amplification is occurring (this technique is also referred to as quantitative or real-time PCR). During RRT-PCR data are collected throughout the amplification process using a fluorescent dye. The fluorescent dye is highly specific for double-stranded DNA and when bound to DNA molecules the fluorescence intensity increases proportionate to the increase in double-stranded product. In contrast, the data for traditional PCR are simply observed as a band on a gel (remember back to M1D5). | Real-time reverse transcription-polymerase chain reaction (RRT-PCR) allows researchers to monitor the results of PCR as amplification is occurring (this technique is also referred to as quantitative or real-time PCR). During RRT-PCR data are collected throughout the amplification process using a fluorescent dye. The fluorescent dye is highly specific for double-stranded DNA and when bound to DNA molecules the fluorescence intensity increases proportionate to the increase in double-stranded product. In contrast, the data for traditional PCR are simply observed as a band on a gel (remember back to M1D5). | ||
[[Image:Screen Shot 2015-01-27 at 4.04.38 PM.png|thumb|550px|center|'''The fluorescent dye binds to double-stranded DNA during the cycles of PCR.''' At the annealing temperature the primer (blue arrow) binds to the template (black line). During an incubation at the extension temperature the new copy of DNA (orange dashed arrow) is sythesized by the polymerase enzyme. The inactive fluorescent dye molecules present in the reaction (grey stars) bind to the newly generated double-stranded DNA and become activated (green stars). An animation of this process is linked [http://www.sigmaaldrich.com/life-science/molecular-biology/pcr/quantitative-pcr/sybr-green-based-qpcr/syber-green-animation.html here].]] | [[Image:Screen Shot 2015-01-27 at 4.04.38 PM.png|thumb|550px|center|'''The fluorescent dye binds to double-stranded DNA during the cycles of PCR.''' At the annealing temperature the primer (blue arrow) binds to the template (black line). During an incubation at the extension temperature the new copy of DNA (orange dashed arrow) is sythesized by the polymerase enzyme. The inactive fluorescent dye molecules present in the reaction (grey stars) bind to the newly generated double-stranded DNA and become activated (green stars). To eliminate clutter, the basepairs between the DNA strands were omitted. An animation of this process is linked [http://www.sigmaaldrich.com/life-science/molecular-biology/pcr/quantitative-pcr/sybr-green-based-qpcr/syber-green-animation.html here].]] | ||
==Protocols== | ==Protocols== | ||
Revision as of 14:28, 27 January 2015
Introduction
Real-time reverse transcription-polymerase chain reaction (RRT-PCR) allows researchers to monitor the results of PCR as amplification is occurring (this technique is also referred to as quantitative or real-time PCR). During RRT-PCR data are collected throughout the amplification process using a fluorescent dye. The fluorescent dye is highly specific for double-stranded DNA and when bound to DNA molecules the fluorescence intensity increases proportionate to the increase in double-stranded product. In contrast, the data for traditional PCR are simply observed as a band on a gel (remember back to M1D5).
Protocols
Part 1: AIV screening analysis
Part 2: Bird microbiome analysis
- Use any extra time to continue working on your bird microbiome analysis.
Homework
Due on M1D9
- Trees
Due on M2D3
- You will report your findings for the AIV screening portion of this module as a Primer Design Memo. Review the guidelines for this assignment and feel free to get an early start!
Reagent List
- Your brains!
Next Day: Journal club II
Previous Day: Phylogenetic analysis
