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DNA SEQUENCING (1).pptx
- 1. PCR The Polymerase ChainReaction (PCR) provides an extremely sensitive means of amplifying relatively large quantities of DNA First described in 1985, Nobel Prize for Kary Mullis in 1993 The technique was made possible by the discovery of Taq polymerase, the DNA polymerase that is used by the bacterium Thermus aquaticus that was discovered in hot springs The primary materials, or reagents, used in PCR are: - DNA nucleotides, the building blocks for the new DNA - Template DNA, the DNA sequence that you want to amplify - Primers, single-stranded DNAs between 20 and 50 nucleotides long (oligonucleotides) that are complementary to a short region on either side of the template DNA - DNA polymerase, a heat stable enzyme that drives, or catalyzes, the synthesis of new DNA PCR DNA sequencing Microarrays Mass-spec
- 2. The PolymeraseChain Reaction (PCR) provides an extremely sensitive means of amplifying relatively large quantities of DNA First described in 1985, Nobel Prize for Kary Mullis in 1993 The technique was made possible by the discovery of Taq polymerase, the DNA polymerase that is used by the bacterium Thermus aquaticus that was discovered in hot springs The primary materials, or reagents, used in PCR are: - DNA nucleotides, the building blocks for the new DNA - Template DNA, the DNA sequence that you want to amplify - Primers, single-stranded DNAs between 20 and 50 nucleotides long (oligonucleotides) that are complementary to a short region on either side of the template DNA - DNA polymerase, a heat stable enzyme that drives, or catalyzes, the synthesis of new DNA
- 3. PCR The cycling reactions: There are three major steps in a PCR, which are repeated for 20 to 40 cycles. This is done on an automated Thermo Cycler, which can heat and cool the reaction tubes in a very short time. Denaturation at around 94°C : During the denaturation, the double strand melts open to single stranded DNA, all enzymatic reactions stop (for example the extension from a previous cycle). Annealing at around 54°C : Hydrogen bonds are constantly formed and broken between the single stranded primer and the single stranded template. If the primers exactly fit the template, the hydrogen bonds are so strong that the primer stays attached Extension at around 72°C : The bases (complementary to the template) are coupled to the primer on the 3' side (the polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added complementary to the template) PCR DNA sequencing Microarrays Mass-spec
- 4. PCR The different stepsof PCR PCR DNA sequencing Microarrays Mass-spec
- 5. PCR Exponential increase ofthe number of copies during PCR PCR DNA sequencing Microarrays Mass-spec
- 6. Every cycleresults in a doubling of the number of strands DNA present After the first few cycles, most of the product DNA strands made are the same length as the distance between the primers The result is a dramatic amplification of a the DNA that exists between the primers. The amount of amplification is 2 raised to the n power; n represents the number of cycles that are performed. After 20 cycles, this would give approximately 1 million fold amplification. After 40 cycles the amplification would be 1 x 1012
- 7. Verification of PCRproduct PCR DNA sequencing Microarrays Mass-spec
- 8. The mostimportant consideration in PCR is contamination Even the smallest contamination with DNA could affect amplification For example, if a technician in a crime lab set up a test reaction (with blood from the crime scene) after setting up a positive control reaction (with blood from the suspect) cross contamination between the samples could result in an erroneous incrimination, even if the technician changed pipette tips between samples. A few blood cells could volitilize in the pipette, stick to the plastic of the pipette, and then get ejected into the test sample Modern labs take account of this fact and devote tremendous effort to avoiding cross-contamination
- 9. Optimizing PCR protocols WhilePCR is a very powerful technique, often enough it is not possible to achieve optimum results without optimizing the protocol Critical PCR parameters: - Concentration of DNA template, nucleotides, divalent cations (especially Mg2+) and polymerase - Error rate of the polymerase (Taq, Vent exo, Pfu) - Primer design PCR can be very tricky PCR DNA sequencing Microarrays Mass-spec
- 10. Primer design General noteson primer design in PCR Perhaps the most critical parameter for successful PCR is the design of primers Primer selection Critical variables are: - primer length - melting temperature (Tm) - specificity - complementary primer sequences - G/C content - 3’-end sequence Primer length - specificity and the temperature of annealing are at least partly dependent on primer length - oligonucleotides between 20 and 30 (50) bases are highly sequence specific - primer length is proportional to annealing efficiency: in general, the longer the primer, the more inefficient the annealing - the primers should not be too short as specificity decreases PCR DNA sequencing Microarrays Mass-spec
- 11. DNA Sequencing The differentsteps in cycle sequencing PCR DNA sequencing Microarrays Mass-spec
- 12. DNA Sequencing Cycle sequencing chaintermination Taken from: http://www.mbio.ncsu.edu/JWB/MB409/ lecture/lecture04/lecture04.html PCR DNA sequencing Microarrays Mass-spec
- 13. DNA Sequencing Sequencing systems PCR DNAsequencing Microarrays Mass-spec LICOR DNA 4300 ABI 3100
- 14. DNA Sequencing Snapshots ofthe detection of the fragments on the sequencer four-dye system single-dye system PCR DNA sequencing Microarrays Mass-spec
- 15. DNA Sequencing Chromatogram file PCR DNAsequencing Microarrays Mass-spec
- 16. DNA Sequencing The linearamplification of the gene in sequencing PCR DNA sequencing Microarrays Mass-spec
- 17. Massively parallel measurementsof gene expression: microarrays • Defining the “transcriptome” • The northern blot revisited • Detecting expression of many genes: arrays • A typical array experiment • What to do with all this data? Brown and Botstein (1999) “Exploring the new world of the genome with DNA microarrays” Nature Genetics 21, p. 33-37.
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- 19. 1) Study alltranscripts at same time 2) Transcript abundance usually correlates with level of gene expression--much gene control is at level of transcription 3) Changes in transcription patterns often occur as a response to changing environment--this can be detected with a microarray
- 20. Detection of mRNAtranscripts • Northern Blot -- immobilize mRNA on membrane, detect specific sequence by hybridization with one labeled probe--requires a separate blotting for each probe • DNA microarray -- immobilize many probes (thousands) in an ordered array, hybridize (base pair) with labelled mRNA or cDNA
- 21. Generating an arrayof probes Identify open reading frames (orfs) 1) PCR each orf (several for each orf), attach (spot) each PCR product to a solid support in a specific order (pioneered by Pat Brown’s lab, Stanford) 2) Chemically synthesize orf-specific oligonucleotide probes directly on microchip (Affymetrix)
- 22. http://derisilab.ucsf.edu/microarray/ (Derisi Lab atUCSF) The chip defines the genes you are measuring The hybridization represents the measurement The RNA comes from the cells and conditions you are interested in
- 24. A print headfor generating arrays of probes Print head travels from DNA probe source (microtiter plate) to solid support (treated glass slide) Small amount of DNA probe is put on a specific spot at a specific location Each spot (DNA probe sequence) has a specific “address” QuickTime™ and a TIFF (Uncompressed) decom press or are needed to s ee this picture. QuickTime™ anda TIFF ( Uncompressed) decompressor are neededtosee this pictur e. Print head Printing needles
- 27. A yeast arrayexperiment vegetative sporulating Isolate mRNA Prepare fluorescently labeled cDNA with two different- colored fluors hybridize read-out
- 28. Example microarray data Green:mRNA more abundant in vegetative cells Red: mRNA more abundant in sporulating cells Yellow: equivalent mRNA abundance in vegetative and sporulating cells
- 29. Mass spectrometry foridentifying proteins in a mixture From J.R. Yates 1998 “Mass spectrometry and the age of the proteome” J Mass Spec. 33, p 1-19 Liquid chromatography and tandem mass spectrometry Software for processing data
- 30. Shotgun sequencing Shotgunsequencing dispenses with the need for mapping and so is much faster. It involves chopping the DNA into fragments of size c. 2000 base pairs (bps) and 10000 bps, sequencing the first and last 500 bps of each fragment. It then uses computer algorithms to assemble the entire sequence from the sequenced fragments.
- 31. Speed and accuracyof sequencing Shotgun sequencing is much faster- it took a matter of months to obtain a draft sequence of the fruit-fly, Drosophila Melanogaster (135Mbps), when the state-funded conventional sequencing effort had taken several years to achieve a similar level of completion. BUT assembly of pieces, in eg. the human (3x109 bps), requires very powerful computers AND repetitive DNA, which is common in eukaryotic genomes, causes great difficulties in the assembly process – may get it wrong.
- 32. Conclusions Sequencing DNAinvolves: Amplifying it by PCR or cloning Chopping it up into manageable bits Replicating it with fluorescently-tagged dideoxynucleotides Running the different length fragments on a gel and reading this Assembling the pieces (sequences of manageable bits). Shotgun sequencing is faster than mapping-based assembly methods, but can have accuracy problems.
- 33. Sequence datais stored in online databases Extracting useful information and patterns from such data is part of bioinformatics and often employs intelligent systems techniques.