SNP/RTPCR


The ABI Prism® 7900HT sequence detection system is a high throughput, real time PCR instrument designed for automated, high throughput detection of fluorescent PCR products. An Automation Accessory combined with 384-well plate capability makes the 7900HT system ideally suited to meet the requirements of high throughput research and diagnostic applications.Key applications for this instrument include:
• gene expression quantification
• detection of single nucleotide polymorphism (SNPs)
• dissociation (melt) curve analysis of PCR products
Fluorescent chemistries used for PCR product detection in the 7900HT include the intercalating dye SYBR Green I or sequence specific (Taqman®) probes. SYBR Green is a double stranded DNA intercalating dye. It fluoresces only when bound to DNA and is used to ‘track’ the amount of amplicons produced during PCR, i. e. in real time. SYBR Green is also used for dissociation (melt) curve analysis of PCR products.

Taqman probes are based on the 5' nuclease assay. The 5' nuclease assay utilises the 5'-3' exonuclease activity of Taq polymerase to cleave a sequence specific probe which has been designed to hybridise to sequence that is flanked by the forward and reverse primers. The Applied Biosystems Taqman probe is comprised of a short oligodeoxynucleotide that is labelled with a reporter dye at the 5' end and a quencher dye at the 3' end of the probe. When the probe is intact, the reporter and quencher dyes remain in close proximity resulting in suppression of the reporter dye fluorescence via energy transfer (FÖster Resonance Energy Transfer). If the target sequence is present during PCR, the probe anneals to its complement sequence. As the primers hybridise to their complement sequence the Taq polymerase extends from the primers and 5'-3' nucleolytic activity cleaves the probe, resulting in separation of the reporter dye from the quencher dye and producing an increase in reporter dye emission intensity. The probe fragments are displaced from the target sequence, and polymerization of the strand continues. The 3' end of the probe is phosphorylated to prevent it acting as primer and prevents Taq polymerase extension during PCR. Note that an increase in fluorescent signal is detected ONLY if the target sequence is complementary to the probe and is amplified during the PCR process. Thus the fluorescence based sequence specific detection is very sensitive and accurate. Figure 1 shows an example of the format of the data produced for SNP analysis.
  

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The 7900HT uses an argon-ion laser for the excitation of the PCR chemistries and a CCD camera measures the fluorescence spectrum and intensity from each reaction. Continuous wavelength detection from 500-660 nM enables the use of multiple fluorescent chemistries in a single reaction. The contribution of each individual dye and the background is differentiated using a ‘virtual filter’. The virtual filter consists of a series of algorithms to separate the composite spectra from the raw spectral profile and then to determine the contribution of each dye in the raw data. The term ‘multicomponenting’ is used to describe this process of spectral profile resolution.
Minimum reaction volumes of 5 µl can be used on the 7900HT. To enable accuracy in setting up such small-scale reaction volumes SUPAMAC has purchased a Beckman Coulter Biomek FX robot. Preparation of both SNP analysis reactions and quantitative PCR analysis are carried out on the Biomek FX.
SNP detection utilizes plate read only on the 7900HT and the capacity is 84 plates in 3 hours, which equates to 10 752 genotypes per hour. For real time PCR the thermal cycling is carried out in the 7900HT and all 384 wells are monitored continuously throughout the cycling, which takes approximately 2 hrs per run. Bar coding at various points throughout the sample processing and analysis is used for efficient sample tracking and reporting of results.


SNPlex information
The SNPlex System is based on a proprietary OLA (Oligonucleotide Ligation Assay) technology combined with electrophoretic detection. The highly specific, multiplexed OLA interrogates the target SNP in a DNA sample, and then a universal PCR reaction is used to amplify the resulting ligation products. Detection is carried out using fluorescent-labeled, universal reporter probes in conjunction with high sensitivity electrophoresis technology. The data are analyzed using an enhanced version of the Applied Biosystems GeneMapper™ Analysis Software 3.5, which offers new tools for automated allele-calling and quality control.