Services: Real-Time-PCR


Mechanisms: TaqMan Chemistry | SYBR Green I Dye Chemistry | Quantification

Primer Design | Reagents | Data Analysis

Instrument Usage Policy

This notice is to detail the policy implemented by the Functional Genomics Facility for use of the Real-Time PCR:

1. Cancellation: If you have made a reservation and wish to cancel it, please do so 12 hours in advance. You may cancel via the online scheduler, via email (sshou@bsd.uchicago.edu), or via phone (X41166). Please note that this instrument is heavily utilized and your cooperation in this matter is greatly appreciated.

2. Investigators may only book 4 hours per day. If more time is required please contact sshou@bsd.uchicago.edu  (X41166) for approval.

3. All no-shows and late cancellations will be billed for the time reserved.

 

TaqMan Chemistry

Real-time PCR analysis detects specific nucleic acid amplification products as they accumulate in real-time. Real-time PCR uses a fluorescently labeled oligonucleotide probe. During polymerization, a reporter fluorescence dye and a quencher dye are attached to a TaqMan® probe. Negligible fluorescence from the reporter dye's emission is observed once both dyes are attached to the probe. Once PCR amplification begins, DNA polymerase cleaves the probe, and the reporter dye is released from the probe. The reporter dye, which is separated from the quencher dye during every amplification cycle, generates a sequence-specific fluorescent signal. The signal increases in real time as PCR cycles continue; the fluorescence intensity increases proportionally.

The advantage of fluorogenic probes over DNA binding dyes is that specific hybridization between probe and target is required to generate fluorescent signal. Thus, with fluorogenic probes, non-specific amplification due to mis-priming or primer-dimer artifact does not generate signal. The disadvantage of fluorogenic probes is that different probes must be synthesized to detect different sequences.

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SYBR® Green I Dye Chemistry

  1. When SYBR Green I dye is added to a sample, it immediately binds to all double-stranded DNA present in the sample.
  2. During the PCR, AmpliTaq Gold® DNA Polymerase amplifies the target sequence, which creates the PCR products, or "amplicons."
  3. The SYBR Green I dye then binds to each new copy of double-stranded DNA.
  4. As the PCR progresses, more amplicons are created. Since the SYBR Green I dye binds to all double-stranded DNA, the result is an increase in fluorescence intensity proportionate to the amount of PCR product produced.

Advantages of the SYBR Green I dye

  • It can be used to monitor the amplification of any double-stranded DNA sequence.
  • No probe is required, which reduces assay setup and running costs.

Disadvantages of the SYBR Green I dye

  • The primary disadvantage of the SYBR Green I dye chemistry is that it may generate false positive signals; i.e., because the SYBR Green I dye binds to any double-stranded DNA, it can also bind to nonspecific double-stranded DNA sequences.

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Quantification

Quantification of the amount of target in unknown samples is accomplished by measuring CT and using the standard curve to determine starting copy number. The entire process of calculating CTs, preparing a standard curve, and determining starting copy number for unknowns is performed by the software of the systems that come with the equipment.

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Primer Design

Primer3

Primer3 is a web-based software designed by the Whitehead Institute of MIT. In general, the default settings that come with the software work. The only thing you need to do is to copy and paste your sequence into the sequence box and click the "pick primers" button. However, customers are encouraged to go over the settings. click here to go to the software web site.

PrimerExpress

PrimerExpress is a commercial software from Applied Biosystems, Inc. It is loaded on a Macintosh computer located in the Facility. In order to use the program, investigators will need to bring their target sequence, in a text file format, on a 100MB zip disk. Functional Genomics Facility personnel are available to provide advice on the use of the software. Click here for the manual.

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Reagents

Here is a list of reagent item/equipment and optional vendors:

  1. Primer: Operon; ABI; Eurogentec
  2. Probe: Operon; Synthegen; ABI; Eurogentec*
  3. Reverse Transcription: Invitrogen SuperScript III; Eurogentec*
  4. Quantitative real time PCR:
    1. For FAM and other molecular probes: ABI TaqMan® Universal PCR Master Mix; Eurogentec*
    2. For using SYBR Green: ABI SYBR® Green PCR Core Reagents ; Qiagen QuantiTect SYBR Green PCR kit (cat# 204143 or 204145); Eurogentec*
  5. To use ABI 7700 or 7300 machines, you need to have
    1. ABI PRISM™ Optical 96-Well Reaction Plates.
    2. ABI PRISM™ Optical Adhesive Cover Starter Pack: PN: 4313663; price: $50.
    3. ABI Optical Adhesive Cover: PN: 4311971.
  6. To use Cepheid SmartCycler machine, you need to have
    1. Smart Cycler reaction tubes: Cepheid: part number: 900-0022 for reaction volumn of 25 μl
    2. (Optional) Eurogentec kit*

* Eurogentec products exclusively distributed through VWR International. For University of Chicago discounted rate visit www.vwr.com or call 18009325000

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Data Analysis

The ABI PRISM 7700 Sequence Detection System (version 1.6 software) is not designed to construct two standard curves on the same plate. To analyze this experiment, results are exported to an Excel spreadsheet by choosing the Export option in the File menu. The exported file contains columns with the sample well number, sample description, standard deviation of the baseline, ?Rn, and CT. The FAM information is reported first with the JOE information in the rows under the FAM data.The important parameter for quantitation is the CT.

Cepheid SmartCycler experiment data can also be analyzed in the same following way.

Set up three columns as shown below listing the input amount for the standard curve samples, the log of this input amount, and the CT value.

Perform the following steps in Excel to construct a standard curve from your data.

 

Calculating the Input Amount

Perform the following steps to calculate the input amount for unknown samples

Note: this is an excerpt from ABI user manual

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