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 Mg 2+ - cofactor,  fidelity

Providing Great Care… Building Warrior Medics! The PCR Song

Providing Great Care… Building Warrior Medics! Manually intensive…

TaqMan Real-time PCR in a Nutshell 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. TA GC Complimentary Base Pairs 3` 5` Sense (forward) 3`5` Antisense (reverse) TaqPol Probe (FAM) 5`3` (TAMRA) 5`3` PrimersNucleotides (dNTPs) 5` 3` Reaction Mix 94ºC (201ºF) 3`5` 3` 5` Sense (forward) Antisense (reverse)

TaqMan Real-time PCR in a Nutshell 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 ºC ( ºF) 3` 5` 3`5` 3` TaqPol Probe (FAM) 5`3` (TAMRA) 5`3` PrimersNucleotides (dNTPs) 5` 3` Reaction Mix 3` 5` 70-60ºC ( ºF) 5` 3` 5`3` 5` 3`

TaqMan Real-time PCR in a Nutshell -e (photon) Photo detector 3`5` 3` 5` Excitation ( λ =475nm) Cleaved Emitter/Donor 3` 5` 3` 5` Receptor/Quencher 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 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 cycles corresponds to the presence of the target nucleic acids. Extending / Detecting

Typical PCR Thermocycling Algorithm Temp ºC Seconds 1 Cycle

Providing Great Care… Building Warrior Medics! 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

Electrophoresis

Providing Great Care… Building Warrior Medics! 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

Restriction Maps

Providing Great Care… Building Warrior Medics! 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! 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!