Molecular Techniques Providing Great Care… Building Warrior Medics!
Molecular Techniques Providing Great Care… Building Warrior Medics! Polymerase Chain Reaction In-vitro amplification PCR vs. qRT PCR vs. RT-PCR Molecular soup Nucleotides (dNTPs) Polymerase Reverse Transcriptase (RT-PCR) Primers Probes (for qRT-PCR) Unknown Buffer - avoid depurination Mg2+ - cofactor, fidelity Providing Great Care… Building Warrior Medics! The PCR Song http://bio-rad.cnpg.com/lsca/videos/ScientistsForBetterPCR/ Providing Great Care… Building Warrior Medics! Manually intensive… Providing Great Care… Building Warrior Medics! TaqMan Real-time PCR in a Nutshell Denaturing 3` 5` Sense (forward) Antisense (reverse) Reaction Mix 5` 3` TaqPol 5` 3` T A G C (FAM) 5` Primers Probe 3` 5` 3` (TAMRA) Nucleotides (dNTPs) Complimentary Base Pairs Any genome (dsDNA) is denatured to 2 ss at 94º (breaks H+ bonds). Polymerase is activated. Probes anneal to target sequence at 70º. Primers dock or “bookend” target sequence, and polymerase docks to primer end. TaqPol begins chemically building complimentary strands once temperature reaches 60º for 20 seconds. 94ºC (201ºF) 3` 5` Antisense (reverse) Sense (forward) 5` 3` TaqMan Real-time PCR in a Nutshell Annealing Reaction Mix 70-60ºC (158-140ºF) TaqPol 3` 5` 5` 5` 5` 3` (FAM) 5` Primers 3` 3` (TAMRA) Probe 3` 5` Nucleotides (dNTPs) 3` 70-60ºC Any genome (dsDNA) is denatured to 2 ss at 94º (breaks H+ bonds). Polymerase is activated. Probes anneal to target sequence at 70º. Primers dock or “bookend” target sequence, and polymerase docks to primer end. TaqPol begins chemically building complimentary strands once temperature reaches 60º for 20 seconds. (158-140ºF) 3` 5` 5` 3` 5` 3` 3` 5` 5` 3` TaqMan Real-time PCR in a Nutshell Extending / Detecting 3` 5` 5` 5` 3` Receptor/Quencher Excitation (λ=475nm) -e (photon) Cleaved Emitter/Donor 3` 5` 5` 3` Template Strand If target sequence was present, polymerase will cleave the fluorophore FAM via 5’ exonuclease activity as it builds complimentary bases. FAM can no longer donate an electron to TAMRA due to antiproximity once excited at a specific wavelength. Light energy given off by the electron’s movement is detected and measured, and the totality of fluorescence over 40-45 cycles corresponds to the presence of the target nucleic acids. Typical PCR Thermocycling Algorithm Temp ºC 100 90 80 70 60 50 40 30 20 10 0 1 Cycle 0 30 60 0 20 Seconds Many other techniques REs, electrophoresis Southern blotting Recombination, cloning DNA microarrays DNA fingerprinting via RFLP DNA sequencing Many more! Providing Great Care… Building Warrior Medics! Electrophoresis - + Providing Great Care… Building Warrior Medics! Electrophoresis Agarose vs PAGE Migration Factors affecting Factor chosen EtBr or “EB” UV light Danger! Ladder Providing Great Care… Building Warrior Medics! Electrophoresis Restriction Enzymes Molecular “scissors” Recognize palindromic sequences Breaks nucleic acids into smaller fragments Allows for recombination/cloning Providing Great Care… Building Warrior Medics! Restriction Maps RMs - linear sequence of sites separated by defined distances on DNA produced by REs Example usage Generate expression profile of DNA fragment (cDNA) Generate RM of both cDNA and fragment Compare both maps to discern information on introns Providing Great Care… Building Warrior Medics! Restriction Maps Blots and Hybridization Edwin Southern Press gel to filter membrane Hybridize various probes Fluorescent, radioactive Specific sequences Expose/develop filter membrane Northern, western Providing Great Care… Building Warrior Medics! Recombination and Clones Artificial plasmid, phage, BAC, YAC MCS Selectable marker Counter-selectable marker Make yourself a clone! Providing Great Care… Building Warrior Medics! Sequencing Determine exact base order/content ddNTPs Chain termination Sequencers Dye terminators Capillary electrophoresis Providing Great Care… Building Warrior Medics! Sequencing (cont.) Providing Great Care… Building Warrior Medics! RNAi Gene therapy - limited success RNAi (short hairpins) miRNAs - natural form used in gene expression siRNAs - manmade forms used in ??? Interfere with gene expression Bind with RNA - cause degradation; block translation What else do they block???!!! Providing Great Care… Building Warrior Medics! Microarrays Like micro-Southern Blot AKA “Gene Chip” Glass or silicon 1000s of probe dots Dot = gene Determine gene status for thousands Generates lots of data - what can we do? Providing Great Care… Building Warrior Medics! Bioinformatics DNA length in one cell Study generates a lot of information Computers help sort through data Conceptualization Prediction Providing Great Care… Building Warrior Medics! http://www.ncbi.nlm.nih.gov http://www.pdb.org http://kinemage.biochem.duke.edu/index.php Bioinformatics Mage Demonstration Providing Great Care… Building Warrior Medics! Molecular Jargon SNP (“snip”) Oligos (“ol-ee-goes”) Gel Het HEMONC (“heem-onk”) Allele (“uh-leel”) Loci or Locus (“low-sigh” or “low-cuss”) Hyb (“hibe” short for “hybrid” or “hybridize”) Providing Great Care… Building Warrior Medics! Molecular Jargon (cont.) Translocation e.g., t(9;22)(q34;q11) Reagent room Pre- room Post- room Amplicon Conservation RFLP (“ree-flip”) Lots more…I’ll stop now! Providing Great Care… Building Warrior Medics!
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