Presentation on theme: "Leeward Community College"— Presentation transcript:
1Leeward Community College Analysis of gene expression using RT-PCRDr. Tamas BorsicsABE Workshop 2006Leeward Community College9:30 am Friday, June 16, 2006
2RT – Reverse Transcription What does the term “RT-PCR” stand for?Involves two processes:RT – Reverse TranscriptionDuring this step we synthesize single stranded DNA from RNA templatePCR – Polymerase chain reactionUsing gene-specific primers we amplify a certain part of our gene of interest to get enough amount for further analysis
3DNA and RNA – What a difference! DNA: Double stranded – very stable, durableUsed in cells to store information (chromosomes)Material of inheritanceUsed in forensic scienceUsed by archeologists (~ years)
4DNA and RNA – What a difference! RNA: single stranded – unstable, strong secondary structuresUsed in cells to temporary express informationCell’s RNA profile changes from minute-to-minuteDegrades very easily – even during the isolation process
5The Central dogma of Molecular Biology The DNA replicates its information in a process that involves many enzymesUNIDIRECTIONALThe DNA codes for the production of messenger RNA (mRNA)In eucaryotic cells, the mRNA is processed (essentially by splicing) and migrates from the nucleus to the cytoplasmMessenger RNA carries coded information to ribosomes. The ribosomes "read" this information and use it for protein synthesis
6What makes the difference? Human:Roundworm:Fruit fly:Arabidopsis:Yeast: 6 300E. coli: 3 200# of genesDNA content is the same in each cell typeRNA profile is different in each cell typeEach cell uses (transcribes) only a small fraction (10-15%) of all the genes in the genome into RNA
7How transcription works? Transcriptase enzyme binds to the double stranded DNA, separates strands and the DNA 'eye' opens.Transcription begun and mRNA elongation is underwayElongation continues till the end of the transcription unit
8HOW RNA tumor viruses cause cancer??? The discovery of reverse transcriptaseVirusesDouble stranded DNA virusesSingle stranded DNA virusesRNA viruses (Retroviruses)Do not contain DNAfirst recognized to cause certain cancers in animalsTumor cells have genetic-like stabilityRNA is not suitable for stable transfectionHOW RNA tumor viruses cause cancer???
9The discovery of reverse transcriptase Already known: certain bacterial viruses could integrate their DNA into their hosts' chromosomes and persist as stable genetic elementsHoward Temin proposed that RNA tumor viruses can cause permanent alterations to cells by integrating into host chromosomes.RNAs first has to be converted into DNAs, which could then become integrated.If all the above is true there MUST be a mechanism to convert RNA into DNA !
10Is the Central dogma a “holy cow”? is there a way back????
111970: 1975: The discovery of reverse transcriptase – the breaktrough Two separate research teams, one led by Temin and the other by David Baltimore, simultaneously discovered the elusive RNA-copying DNA polymerase in purified virions – after many years of painstaking laboratory work.1975:Temin and Baltimore shared the Nobel Prize in physiology or medicine for their discovery of reverse transcriptase.
12What does reverse transcriptase do? copycccReverse transcriptase uses a single-stranded RNA template to create a double-stranded DNA.
13Reverse transcription How can it be used in research?transcriptionAAAAAmRNAmRNAtranscriptionAAAAAAAAAAmRNAtranscriptionReverse transcriptioncDNA analysisExcellent tool to study genes that are actually expressed in a certain type of cell/tissue/organ.Monitor gene expression changes due to environmental effects
14Only mRNA has a poly-Adenin tail at the 3’ end Let’s start!total RNARNA isolation~ 1%mRNArRNAtRNAMost of the RNA is unimportant for us (tRNA, rRNA)mRNA population consists of about different kindStrong secondary structure – enzyme cannot workOnly mRNA has a poly-Adenin tail at the 3’ endAAAAA
16What shall we do with our cDNA now? Problems:Vast variety of cDNA molecules (at least different kind)Only one copy DNA per mRNA strand existAbundant (housekeeping) genes give 90% of the mRNA populationThey mask our gene of interestSolution: we need a reaction that isable to amplify our low-abundance gene ANDspecific enough not to amplify other cDNAs+++ Plus we need an enzyme that is able to copy DNA
17DNA copying in a test tube The DNA replicates its information in a process called “replication”The enzyme who does the job:DNA-DNA polymeraseCan we use it on a cDNA template?
18How DNA-DNA polymerase works 5`3`5`3`5`OligomerWe made one copy strand!5`Pol3`5`3`1985: Kary B. Mullis- what if we denature and attach an oligo facing BACKWARDS…5`oligomer5`3`We made a second one!!5`oligomerPol5`3`… and repeat it over and over again?Pol
20Having good times - and inventing PCR 1985:Kary B. Mullis: “what if we attach an oligo facing backwards?”– an idea conceived while cruising the Pacific Coast Highway from San Francisco to Mendocino on a motorcycle.1993: Nobel Prize in Chemistry
21Gene-specific primers How to amplify our gene of interest from the cDNA “soup”?TTTTTAAAAAcDNATTTTTAAAAAcDNAGene-specific primersTTTTTcDNAAAAAAPCRGene #2750 bp500 bpGene #1Gel visualization
23Applications of RT-PCR Cloning genes’ expressed forms (not genomic version)Monitor a gene’s expression level in any tissueMonitor expression changes following treatmentsSophisticated RT-PCR: The real time PCRsequencing a whole mRNA profileEST (Expressed Sequence Tags) – databaseMicroarray (DNA chip)Diagnose and easily differentiate between different cancer typesEarly detection of hidden illnessesetc…
25Resources, referencesvcell.ndsu.nodak.edu/~christjo/vcell/animationSite/transcription/movie.htm….should you need more info just google around. RT-PCR is everywhere on the Web…
26PCR – A quick overviewDenaturation - separate parent strands in preparation new strand synthesisAnnealing - “stick” primers to the parent strands to prime DNA synthesisExtension - addition of nucleotides, one at a time, to the growing end of the DNA strand (3’ end) using the parent strand as the template