Advanced PCR Lecture

8/8/2019 Advanced PCR Lecture

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A quick review of standard PCR

An enzyme for every need

The third pillar: RTases & RT-PCR

The next generation: Real-time PCR

The sky is (not) the limit

What we do for a living


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PCR is a cornerstone of modern molecular biology

The relatively simple concept of DNA amplification: cycles of denaturation, primer annealing, and extension, isbeing utilized in a myriad of applications:

Diagnostics (nucleic acid testing, pathogen detection)

Advanced research (gene expression)

Genomics (sequencing, genotyping, whole genome amplification)

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PCR remains a complex reaction involving ionic interactions, kineticconstants, and enzymatic activities.

In addition to primer design, many applications still require theoptimization of buffer conditions, type of enzyme, and cycling parameters.

Buffer considerations:

Mg2+ concentration

KCl

Ammonium sulfate

pH

Template DNA, primers

Reaction buffer

Enzyme


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Polymerase enzymes are responsible for DNA replication in the reaction

Standard PCR : Taq polymerase

Taqis a Type A polymerase isolated from the eubacterium

Thermus aquaticus capable DNA polymerization in the 5p3

direction.o Thermostable

o Generally robust, requires minimal optimization

o High yield of DNA targets


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High Fidelity PCR : Pfu polymerase

Pfu is a Type B polymerase isolated from archaeal

bacteria Pyrococcus furiosus, capable DNA polymerization

in the 5p3 direction as well as a 3p5 exonucleaseactivity (proofreading).

o Highly thermostable

o 3p5 nuclease activity removes mismatches and

greatly improves the fidelity of the reaction (2.2 x 10-6)

o Used when the amplification product will be cloned

o Requires optimization, less robust

o Typically lower yields

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Polymerase enzymes are responsible for DNA replication in the reaction


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The thermostable DNA polymerases used in standard PCR require a DNA template and istherefore limited to the analysis ofDNA samples.

The analysis of differential expression of genes and the cloning of cDNAs from rare messagesrequires RNA template.

In order to apply PCR to the study of RNA, the RNA sample must first be reverse transcribedto cDNA using a reverse transcriptase enzyme (RNA-dependent polymerase):

- Avian myeloblastosis virus (AMV)

- Moloney murine leukemia virus (M-MLV)

These enzymes are not thermostable, have low replicating fidelity, and possessRNase activity.

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Conversion of mRNA to cDNA by Reverse Transcription

or: gene-specific primeror: random hexamers


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RT-PCR CONSIDERATIONS:

The quality and purity of the starting RNA template is crucial to the success of RT-PCR.

Total RNA or poly(A)+ RNA can be used as the starting template - both must be intact and free

of contaminating genomic DNA.

Specific capture of poly(A)+ RNA will enrich a targeted message so that less of the reverse

transcription reaction is needed for the subsequent amplification.

The efficiency of the first-strand synthesis reaction, which can be related to the quality of the

RNA template, will also significantly impact the results of the subsequent amplification.

RNA cDNA DNA for analysis

Reverse TranscriptionRTase

PCRDNA polymerase

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QUANTITATION


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End Point

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Plateauphase

Linearphase

Exponentialphase

Area of detection forreal-time PCR


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Theoretically there is a quantitative relationship between the amount of starting sample and the

amount of PCR product at any given sample.

Real-time PCR detects the accumulation of amplicon during the reaction. The data is then measured

at the exponential phase of the PCR reaction rather than end-point plateau. The exponential phaseis the optimal point for analyzing data.

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Cycle threshold isrelated to the initialtarget copy number

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Applications:

Viral quantitation

Quantitation of gene expression

Microarray verification

Drug therapy efficacy

Pathogen detection

Genotyping

Advantages of real-time vs. end-point PCR:

Collects data in the exponential growth phase (vs end-point plateau)

Increase in fluorescent signal is proportional to number of amplicons generated

Increased dynamic range of detection

Does not require post-PCR processing Increased sensitivity (detection down to 2-fold change)

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Detection Assays: SYBR Green Dye

SYBR Green I binds to double-

stranded DNA. The resulting DNA-

dye-complex absorbs blue light

(max = 498 nm) and emits green light

(max = 522 nm)As DNA is amplified SYBR

fluorescence increases proportionally

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Non-specific dye used to detect the

presence or absence of an amplicon

Non-target sequence-specific detectionssystems are susceptible to false-

positives


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Dual fluorophore-labeled oligonucleotideprobe: e.g. TaqMan

TaqMan improves:

Specificity

Product quantification

Multiplex PCR

Detection Assays: Sequence-specific probes

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PCR has become a central tool for DNA analysis across all disciplines ofbiology and biochemistry

Novel enzymes and instrumentation are creating new applications forPCR

Other advanced PCR methods for research and diagnostic applications:

Hot start PCR (specificity)

Cycling sequencing (DNA sequencing)

Site-directed mutagenesis PCR

Colony PCR

Multiplex-PCR

Error-prone PCR (mutagenesis)

StEP PCR (recombination)

Emulsion PCR (cell-free cloning)


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We are developing a suite of standard enzymes for PCR:

Taq DNA polymerase

Type B polymerases (Hi-Fi): Pfu, KOD, chimera

Hot-start Taq and Type B polymerases

Long-range PCR blends

Really focuses on the engineering of novel polymerases for advancedapplications:

2nd generation Taq

SYBRTaq

Ultra-high fidelity Type B

Advanced PCR Lecture

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