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Molecular Techniques

Molecular Techniques. 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 Mg 2+ - cofactor,  fidelity.

joshua-mercer
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Molecular Techniques

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  1. Molecular Techniques

  2. 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

  3. The PCR Song http://bio-rad.cnpg.com/lsca/videos/ScientistsForBetterPCR/

  4. Manually intensive…

  5. TaqMan Real-time PCR in a Nutshell Complimentary Base Pairs 94ºC (201ºF) 3` 3` 5` 5` Reaction Mix Sense (forward) Sense (forward) G C T A (FAM) 5` 3` (TAMRA) TaqPol 5` 5` 3` 3` Probe Antisense (reverse) Antisense (reverse) Primers Nucleotides (dNTPs) 5` 3` 5` 3` Denaturing 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.

  6. TaqMan Real-time PCR in a Nutshell Reaction Mix 70-60ºC (158-140ºF) (FAM) 5` 3` (TAMRA) TaqPol 3` Probe 5` 5` 5` 3` 3` Primers Nucleotides (dNTPs) 5` 3` 70-60ºC (158-140ºF) 5` 3` 5` 3` 5` 5` 3` 3` 3` 5` 5` 3` Annealing 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.

  7. TaqMan Real-time PCR in a Nutshell 3` 5` Receptor/Quencher 5` 3` 5` 3` 5` 5` 3` Extending / Detecting 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 anti-proximity 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. Photo detector Excitation (λ=475nm) -e (photon) Cleaved Emitter/Donor Template Strand

  8. Typical PCR Thermocycling Algorithm Polymerization Temp ºC Denaturation 1 Cycle Read Hotstart Seconds

  9. Many other techniques • REs, electrophoresis • Southern blotting • Recombination, cloning • DNA microarrays • DNA fingerprinting via RFLP • DNA sequencing • Many more!

  10. Electrophoresis - +

  11. Electrophoresis • Agarose vs PAGE • Migration • Factors affecting • Factor chosen • EtBr or “EB” • UV light • Danger! • Ladder

  12. Electrophoresis

  13. Restriction Enzymes • Molecular “scissors” • Recognize palindromic sequences • Breaks nucleic acids into smaller fragments • Allows for recombination/cloning

  14. 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

  15. Restriction Maps

  16. Blots and Hybridization • Edwin Southern • Press gel to filter membrane • Hybridize various probes • Fluorescent, radioactive • Specific sequences • Expose/develop filter membrane • Northern, western

  17. Recombination and Clones • Artificial plasmid, phage, BAC, YAC • MCS • Selectable marker • Counter-selectable marker • Make yourself a clone!

  18. Sequencing • Determine exact base order/content • ddNTPs • Chain termination • Sequencers • Dye terminators • Capillary electrophoresis

  19. Sequencing (cont.)

  20. 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???!!!

  21. 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?

  22. Bioinformatics • DNA length in one cell • Study generates a lot of information • Computers help sort through data • Conceptualization • Prediction

  23. http://www.ncbi.nlm.nih.gov

  24. http://www.pdb.org

  25. http://kinemage.biochem.duke.edu/index.php

  26. Bioinformatics Mage Demonstration

  27. 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”)

  28. 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!

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