Lecture 34 PCR Product Detection

8/13/2019 Lecture 34 PCR Product Detection

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Lecture 34 PCR Product Detection

FS 362


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Components in a PCR Reaction


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Cycling Temperatures

?C

?C

?C ?C

?C

1-2 min

1-2 min

1-2 min 5-7 min

?C2-10 min

25-40 cycles

*Denaturation

*Annealing

*Extension

*Initial

denaturation *Final extension


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DNA Quantity & Quality

Presence/Absence of DNA target and PCR

product Agarose Gel Electrophoresis

Quantity of DNA target & PCR Nanodrop,

Bioanalyzer

Quality of PCR product Bioanalzyer,

Nanodrop


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PCR Product Evaluation

Before a PCR product is used in further applications(e.g., DNA sequencing) we need to make sure:

There are bands; not every PCR is initially successful

and optimization is usually required

The bands are the correct size; it is possible forprimers to anneal to an untargeted location on thegenome

There is only one band per reaction; if primers fit onother parts of the genome multiple non-specific bandsmay be present


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Detection & Analysis of PCR Products

Agarose gelelectrophoresis

PCR products

visualized whenstained EthidiumBromide (EtBr)

EtBr intercalateswith DNA bases &fluoresces uponexposure to UV light


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Agarose gels

Cast by melting agarose in buffer until solution is clear Pre-cast gels are commercially available

Gel casting tray contains combs to create wells

Upon cooling, agarose solidifes Density of agarose matrix determined by concentration of agar insolution

Negatively-charged DNA migrates through the gel matrixwhen electric field is applied.


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DNA Migration in Agarose Gels

Molecular size of DNA Larger molecules migrate more slowly than

smaller molecules Larger molecules experience more friction

Larger molecules wiggle through pores inagar matrix slower

Agarose concentration Lower concentrations allow better separation of

large fragments

Higher concentrations allow better separation ofsmaller fragments


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Voltage At low voltage, rate of DNA fragment migration is

proportional to voltage applied

As voltage increases, range of fragment separation

decreases Electrophoresis buffer

Buffers stabilize pH and provide ions for conductivity

Composition and ionic strength of electrophoresis buffer

used to make agarose gel affects mobility of DNA


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Molecular size of DNA

Larger molecules migrate more slowly than

smaller molecules Larger molecules experience more friction

Larger molecules wiggle through pores in agar

matrix slower


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Voltage applied At low voltage, rate of DNA fragments migration is

proportional to voltage applied

Range of separation of fragments decreases as voltageincreases

Electrophoresis buffer Buffers stabilize pH and provide ions for conductivity

Composition and ionic strength of electrophoresisbuffer used to make agarose gel affects mobility ofDNA



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Staining DNA in Agarose Gels

Ethidium bromide

Contain planar group that intercalates between

stacked bases of DNA

Ethidium bromide dye bound to DNA shows

increased fluorescence than dye in free

solution

DNA can be detected in the presence of free

ethidium bromide in gel


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Agarose Gel Electrophoresis of PCR

Products

pGEM N301 N302 N303 N304 N305 N306 N307 N308 N309 +con -con pGEM


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DNA & Spectrophotometry

DNA absorbs light at 260nm

Protein absorbs light at 280nm

Salts, phenol, EtOH absorb light at 230nm

Greater the absorbance, the greater theconcentration

260/280 ratio indicator DNA purity

260/230 ratio indicator of residual salts


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Nanodrop Spectrophotometry


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Nanodrop Results


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Quality

Spectrophotometry cant determine if DNA

target or PCR product is dsDNA (intact DNA)

Alternative methods are required to

determine DNA integrity Agilent

Bioanalyzer

Gel on a chip

l l


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Agilent Bioanalyzer Overview


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Agilent Bioanalyzer

Listeria monocytogenes total RNA

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Lecture 34 PCR Product Detection