Question

Purpose:
In this laboratory you will carry out a quantitative real-time PCR (QPCR), using SYBR Green, on the samples that you purified last laboratory session. QPCR serves several purposes in a forensic laboratory and is included in the sample processing workflow for all/most DNA samples. QPCR ultimately measures (either in absolute numbers or relatively) the amount of DNA in a sample and the presence of any inhibitors (either partial or complete). This allows the forensic scientist to determine if more purification is needed, to remove inhibitors, and how much sample to add to the STR PCR to individualise the sample.

SYBR Green is a fluorescent dye that binds double stranded DNA and sits in the minor grove of DNA. This property is the reason that SYBR Green will not bind single stranded (or denatured) DNA. QPCR works by incorporating SYBR Green into a PCR and detecting the fluorescence after each cycle of amplification. As the number of amplicons (sections of DNA copied during PCR) increase, there will be more double stranded DNA, more SYBR Green will bind, and the fluorescence will increase. The signal of the fluorescence is predictable based on the amount, in either ng or copy number, of DNA present, although a DNA standard series needs to be incorporated to every run to ensure accuracy in this measurement. By using a standard series of known DNA concentrations, unknown samples can then be compared and a starting concentration value for those unknowns can be determined.

Need help with 'introduction and aims' part.

What is the purpose of the experiment? What technique are you using? Why is it appropriate? (1 paragraph)

Answer 1

Quantitative real-time PCR (qPCR) is a powerful method for interrogation of gene expression. Accurate quantification is essential for correct interpretation of qPCR data. Due to its sensitivity, qPCR has become the standard method for measuring levels of gene expression. Quantification of PCR may be relative or absolute. The experiment is done mainly for monitoring amplification and quantification of targeted DNA. It is done mainly using two metods:

1. non-specific fluorescent dyes.
2. sequence specific DNA probes.

Relative quantification using intercalating dyes is the most common method used. The dye used is SYBR Green. It binds to only double stranded and show its fluorescence. but in case of DNA probes it binds only to specific sequence and these DNA probes will be labelled with fluorescent reporter.

In forensics it is mainly used for

• quantification used for normalisationof DNA profiling PCR.
• control of amplifiability.
• indication of DNA degradation.

This can be used to indicate the purity of the sample being amplified. If the sample collected is very low, it can be amplified and the amout of sample which we need can be detected easily.  Comparison between the sample from crime scene and sample of the suspect is possible. It is simple and cheap to perform, but relies on the use of reference gene, against which the sample gene[gene of interest(GOI)] concentrations are normalised.

quantification is performed by constructing a standard curve for each gene of interest and plotting the quantification cycle values against log[quantity] of a dilution series of known GOI.

The standard curve provides efficiency of the amplification primers and amout of GOI in the unknown sample. As this method is very cheap and easily interpretable, it is widely used.