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!