Methods

Theoretical Notes

 Experimental Design

Data Analysis

Primer Design

Assays and Sequence Detection Chemistry:


The 7900HT Sequence Detection System supports three assay types: quantification, allelic discrimination, and plus/minus. These assays can be divided into two categories (quantitative real-time PCR assays and endpoint assays) based on at what time point during the assay data is being collected.


In the quantitative real-time PCR assay, the accumulation of the PCR products is monitored and data is collected throughout the PCR process. In real-time PCR, reactions are characterized by the point in time during cycling when amplification of a target is first detected rather than the amount of target accumulated after a fixed number of cycles. There are three types of quantitative assays: DNA/cDNA quantification; one-step RT-PCR for RNA quantification; two-step RT-PCR for RNA quantification.


An endpoint assay (also called a plate read assay) measures the amount of accumulated PCR product at the end of the PCR process. An endpoint allelic discrimination assay is used to determine the genotype of the sample. With this assay type it is possible to differentiate a single nucleotide polymorphism (SNP) and classify unknown sample as homozygous or heterozygous. Another type of endpoint assay, the Plus/Minus assay, indicates the presence or absence of a specific target sequence in a sample without determination of an actual target amount.


Two types of chemistries developed by Applied Biosystems can be used to detect PCR products on SDS (sequence detection system) instruments:
· TaqMan fluorogenic 5’ nuclease chemisrtry
· SYBR green I dye chemistry


TaqMan chemistry uses a fluorogenic probe to enable the detection of a specific PCR products as it accumulates during PCR. SYBR Green chemistry, which is relatively inexpensive, will detect all double stranded DNA including nonspecific reaction products. A well optimized reactions are therefore essential for SYBR Green based assays.


Assay types that use TaqMan chemistry:
· One step RT-PCR for RNA quantification
· Two step RT-PCR for RNA quantification
· DNA quantification
· Allelic discrimination
· Plus/Minus


Assay types that use SYBR Green chemistry:
· One step RT-PCR for RNA quantification
· Two step RT-PCR for RNA quantification
· DNA quantification


The 7900HT Sequence Detection System also supports multiplex PCR based methods, in which more than one primer/probe set is being used in the same tube. Multiple PCR is most commonly used in 5’ nuclease quantification assays that involve relative quantification of gene expression.


Summary descriptions of Experimental Design and Data Analysis steps are provided. Please note that they will only refer to two-step RT-PCR for RNA quantification with the use of SYBR Green chemistry. However basic definitions such as baseline, normalization to a passive reference, normalized reporter, delta Rn apply to other quantification methods as well.

     

Methods

Theoretical Notes

 Experimental Design

Data Analysis

Primer Design

RRC Core Genomics Facility
University of Illinois at Chicago
2003