Molecular Biology Core

The MBCL has Applied Biosystems ViiA7 and 7500 Real-Time PCR System. Both allow real time detection of PCR products. The PCR process thus enables the accurate quantitation of DNA and RNA. The instruments can be used for various applications such as gene expression analysis and allelic discrimination (Single-Nucleic Polymorphism detection).

REAL-TIME PCR BASICS
Real-Time PCR is the most sensitive and reliable method for the detection and quantitation of nucleic acids levels. Reactions are characterized by the point in time during cycling when amplification of a PCR product is first detected rather than the amount of PCR product accumulated after a fixed number of cycles. The higher the starting copy number of the nucleic acid target, the sooner a significant increase in fluorescence is observed.

ABI equipment can run both Absolute and Relative Quantification. Absolute Quantification will quantitate unknowns based on a known quantity. It involves the creation of a standard curve from a target of known quantity (i.e. copy number) Relative Quantification can quantitate a fold difference between samples. It involves the comparison of one sample to another sample (calibrator) of significance. Relative Quantitation is useful for quantitating mRNA levels.

Relative quantitation can be performed by using standard curve method or the comparative method so called ΔΔCt method.

ALLELIC DISCRIMINATION OVERVIEW
Single nucleotide polymorphisms (SNPs) are the most common form of mutation in the genome. Allelic Discrimination Assay/SNP Genotyping detects different forms (mutation, insertion, or deletion) of the same gene. In this regard, the 5' nuclease assay (Holland et al.1991) is especially attractive because it combines PCR and detection into one step.

Probes with different reporters are used to discriminate between alleles. The aqMan® MGB probes we recommend have a minor groove binder at the 3´ end of the probe. This minor groove binder increases the Tm of probes, allowing the use of shorter probes. Consequently, the TaqMan® MGB probes exhibit greater differences in Tm values between matched and mismatched probes, which provides more accurate allele discrimination.

PRIMERS, PROBE AND DYES
Real-time detection systems for PCR were improved by probe-based (TaqMan probes), rather than intercalator-based PCR product detection (CYBR Green I). The principal drawback to intercalator-based detection of PCR product accumulation is that both specific and nonspecific products generate signal.

The TaqMan® MGB probes contain the following features: a fluorescent reporter at the 5’ end, a nonfluorescent quencher at the 3' end. Because the quencher does not fluoresce, the real-time instruments can measure the reporter dye contributions more precisely. A minor groove binder (MGB) at the 3’ end. The minor groove binder increases the melting temperature (Tm) of probes, allowing the use of shorter probes. It also makes for a more sensitive real-time assay.

The primers and probe mix could be purchased from Applied Biosystems’ TaqMan® Gene Expression Assays and Custom TaqMan® Gene Expression Assays programs.

No probe is required for SYBR Green I dye detection. It is still a good idea to design the primers using the Primer Express software. The Primer Express softwares are loaded on a Macintosh and a PC in the lab.

DU640 Spectrophotometer from Beckman Coulter is reliable equipment for semi-automated nucleic acid and protein measurement.