providing great care… building warrior medics! molecular techniques
TRANSCRIPT
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
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The PCR Songhttp://bio-rad.cnpg.com/lsca/videos/ScientistsForBetterPCR/
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.
T A
G CComplimentary
Base Pairs
3` 5`Sense(forward)
3`5`Antisense(reverse)
TaqPolProbe
(FAM) 5` 3` (TAMRA)
5` 3`Primers Nucleotides (dNTPs)
5`3`
Reaction MixReaction 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.
70-60ºC(158-140ºF)
3` 5`
3`5`
5` 3`
TaqPolProbe
(FAM) 5` 3` (TAMRA)
5` 3`Primers Nucleotides (dNTPs)
5`3`
Reaction MixReaction Mix
3`
3`5`
70-60ºC(158-140ºF)
5`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`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 40-45 cycles corresponds to the presence of the target nucleic acids.
Extending / Detecting
0102030405060708090
100
0 30 60 0 20
Typical PCR Thermocycling Algorithm
Temp ºC
Seconds
1 Cycle1 Cycle
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Many other techniques
REs, electrophoresis
Southern blotting
Recombination, cloning
DNA microarrays
DNA fingerprinting via RFLP
DNA sequencing
Many more!
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Electrophoresis
Agarose vs PAGE
Migration Factors affecting
Factor chosen
EtBr or “EB” UV light
Danger!
Ladder
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Restriction Enzymes
Molecular “scissors”
Recognize palindromic sequences
Breaks nucleic acids into smaller fragments
Allows for recombination/cloning
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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
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Blots and Hybridization
Edwin Southern Press gel to filter
membrane Hybridize various probes
Fluorescent, radioactive Specific sequences
Expose/develop filter membrane
Northern, western
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Recombination and Clones
Artificial plasmid, phage,
BAC, YAC
MCS
Selectable marker
Counter-selectable
marker
Make yourself a clone!
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Sequencing
Determine exact base
order/content
ddNTPs
Chain termination
Sequencers Dye terminators
Capillary electrophoresis
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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???!!!
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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?
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Bioinformatics
DNA length in one cell
Study generates a lot of
information
Computers help sort
through data
Conceptualization
Prediction
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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”)