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The Center for Genomic Technologies : Real-Time-PCR technologies

The Technology:
Real Time PCR enables amplification with detection and quantification of one or more specific sequences in a DNA sample. Data is collected during the exponential growth (log) phase of PCR when the quantity of the PCR product is directly proportional to the amount of template nucleic acid.
Applied Biosystems 7900HT Real-Time PCR System is designed for detection of fluorescent PCR-related chemistries. The light from laser excites the fluorescent dyes present in each well of plate. The resulting emission is collected by CCD camera and then data analysis algorithm is applied. The system can distinguish between 5,000 and 10,000 template copies with a 99.7% confidence level.
 
The Center offers  different applications:
  1. Real-Time PCR
    1. Real-Time PCR using the SYBR Green I chemistry
    2. Real-Time PCR using the TaqMAN Probe
  2. Microfluidic card (Low Density Microarray)
  3. SNP (Single Nucleotide Polymorphism) detection using the TaqMAN Probe
Consumables and Disposables
Use only original Applied Biosystems products.
  • ABI PrismTM 96-well Optical Reaction Plates sealed with Optical Adhesive Covers
  • ABI PrismTM 384-well Optical Reaction Plates sealed with Optical Adhesive Covers
  • Micro Fluidic Cards

 Real-Time PCR

Types of Real-Time PCR experiments

 Absolute quantitation
In absolute quantitation, you quantitate unknowns based on a known quantity. First you create a standard curve; then you compare unknowns to the standard curve and extrapolate a value.
Relative quantitation
In relative quantitation, you analyze changes in gene expression in a given sample relative to another reference sample (such us untreated control sample)
Dissociation curve analysis
Detects small differences in PCR-product melting (dissociation) curves.
 
Sample submission:
There are three options :
1.       Running a 96-wells PCR plate, the assay is done by the customer
2.       RNA samples are submitted. RT-PCR amplifications and Real-Time PCR are performed at the
         Center, with primers provided by the customer.
3.       cDNA samples are submitted. Real-Time PCR is performed at the Center, with primers provided by
         the customer.


Real-Time PCR using the SYBR Green I chemistry SYBR®-Green Based Detection
Uses SYBR Green I dye, a highly specific, double-stranded DNA binding dye, to detect PCR product as it accumulates during PCR cycles. Detects all amplified double-stranded DNA, including non-specific reaction products.
  
Requirements for Primers (Primers can be designed by Primer Express Software that is available at the Center):
Primers with 30 to 80% G/C content, Melting temperature (Tm) for the primers 58 to 60°C.
The five nucleotides at the 3´ end of primer should have no more then two G or C bases.
Recommended primer concentration 50nM.  Amplicons are in the 50- to 150-basepairs range.
 
Real-Time PCR using the TaqMAN chemistry
 
The TaqMan chemistry uses a fluorogenic probe to enable the detection of a specific PCR product as it accumulates during PCR cycles.
 
Requirements for Primers and Probe (Primers and Probe can be designed by Primer Express Software that is available in our lab):
Primers and probe with 30 to 80% G/C content
Melting temperature (Tm) for the primers 58 to 60°C, Tm for the probe 8 to 10°C higher.
Do not select probes with G on the 5´ end.
The five nucleotides at the 3´ end of primer should have no more then two G or C bases.
Recommended primer concentration 900nM and probe concentration 250nM.
Amplicons are in the 50- to 150-basepairs range.
 
Assay-On-Demad and Assay-By-Design are commercially available at Agentek (Galit Schwartz).
 
Microfluidic card (Low Density Microarray)
 
The TaqMan Low Density Array is an easy-to-use micro fluidic card for quantitative real-time PCR. Arrays are delivered with TaqMan assays pre-loaded into each of the 384 reaction wells according to the selected format.
 
The micro fluidic technology utilizes eight samples loading ports, each connected to 48 reaction chambers.
 
Microfluidic card’s design and order from Agentek (Galit Schwartz).
 
Sample submission: 
cDNA samples are submitted. Real-Time PCR is performed at the Center, with card provided by the customer. Concentration of cDNA between 25-50 ng / µl. Amount of cDNA 100 ng / port (48 reaction chambers)
 
 When submitting your samples, always enclose:
Sample submission form for Cards   (type the details to prevent reading errors)
 
 
SNP (Single Nucleotide Polymorphism) detection using the TaqMAN Probe
 
TaqMan SNP genotyping Assays provide fast, accurate genotyping data and simplest workflow available anywhere, based on proven TaqMan primer and probe chemistry, These assays produce high-confidence results in a wide variety of genotyping applications, including screening, candidate region, candidate gene, and fine mapping studies. Selecting TaqMan SNP genotyping assays is easy using the SNPbrowser Software It is a free download at www.allsnps.com/snpbrowser.
 
Sample submission:
Genomic DNA samples are submitted and the assay is performed at the Center, with SNP genotypng assay provided by the customer.
When you submit your DNA samples always enclose the following:
 
1. DNA samples
The DNA concentration should be 10-20 ng/µl.
The volume of the samples depends on the number of requested SNPs but not less than 20 µl.
DNA samples should arrive in a 96-wells plate even if the plate is not full.
The order of the samples in the plate should always be as follows:
          Sample 1 is in position A1
          Sample 2 is in position B1
          .....................................
          Sample 96 is in position H12
 
2. SNP Assay
Assay-On-Demad and Assay-By-Design are commercially available at Agentek (Galit Schwartz).
 
 
Galit Schwartz
Product Line Manager
Applied Biosystems
Agentek-Israel
Tel: 972 (0)3 649 3111
Fax: 972 (0)3 648 1257
Mobile: 972 (0)54 6708106
 
 
 
 

 
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