Lecture 7 Applied Molecular Biology Realtime PCR (QPCR) quantitative PCR - Measures how much of a specific DNA sequence is in a sample (Measurement is via a ct (cq) value. Can be used to measure native DNA or mRNA (after conversion to cDNA with reverse transcriptase) this measures expression of a gene. Uses fluorescent probes, whose fluorescence is measured within the PCR machine How it works: · Many different probes . Most common are o SYBR Green (cheapest) " Fluoresces only when binding to ds-DNA, binds to all ds-DNA and is non-specific. " As product in PCR increases as cycles go on, more SYBR binds and fluoresces more. So more fluorescence = more product " However as nonspecific can bind to any contaminates just as well. " Melting point analysis to check specificity o TaqMan " 2 primers prime target DNA sequence " TaqMan probe binds in the middle of DNA sequence. Has a fluor at 5' end and quencher a 3' end · If fluor physicallay close to quencher we don't see any fluorescence as quencher absorbs. So when TaqMan probe in one piece no fluorescence " As 5' primer extended it has 5' to3' exonuclease and cuts up probe " Fluor away from quencher and releases signal TaqMan probe 5' 3' in is 5' 3' . 1 Fluor 1 5' Quencher A The less template DNA you have the more cycles are required to reach the ct value Fluorescence What is a Ct (cycle threshold value) . The number of cycles at which fluorescence is above background. This should be just above background but often it is drawn much higher - this will have significant effect on Ct value obtained Cycle . Care must be taken when setting background levels otherise ct can become meaningless . In simple terms the lower the ct the more template DNA present in original cycle · Allways look at trace. All samples should plateau at same place if not problem with experiment
Applications · Clinical/Research: SNP genotyping, genotyping, copy number etc · Microbiology: rapid determination identification of microbes (e.g. food contamination) · Plus many others 1st gen sequencing · Sangar sequencing worked by enzymatic incorporation of chain-terminating dideoxynucleotides, followed electrophoretic separation of the products · Used by human sequencing project · Expensive 500ft per megabase = 1millionbp Roche 454 sequencing (pyrosequencing) · Like sangar sequencing there is a specific sequence primer annealed to the template DNA and then extended enzymatically with DNA polymerase · Template DNA attached to little beads . One nucleotide is washed over the template DNA at a time . If the template calls for that nucleotide to be incorporated, one molecule of pyrophosphate (PPi) will be released per incorporation · The pyrophosphate is then turned into light via ATP synthesis and luciferase . Plate imaged by camera that can quantitate the amount of light released by each base addition in each addition · Cost ft20 be mb Illumina Sequencing - · Dteails similar to sangar sequencing - a