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Providing Great Care… Building Warrior Medics! Molecular Techniques

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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. - PowerPoint PPT Presentation

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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 Songhttp://bio-rad.cnpg.com/lsca/videos/ScientistsForBetterPCR/

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.

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

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

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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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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

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

http://www.pdb.org

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

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Bioinformatics

Mage Demonstration

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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”)

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!