Question

4. What relationship does the Ct value have to DNA concentration?
5. How is the sample concentration determined using real time PCR methods?

Sample Name	Target Name	Cт	Quantity ng/ul	M:F Ratio	Degradation Index
Knife handle	IPC	26.89
Knife handle	Large Autosomal	30.29	0.02		14.50
Knife handle	Small Autosomal	28.54	0.29		14.50
Knife handle	Y	28.18	0.23
Knife blade	IPC	33.70
Knife blade	Large Autosomal	undetermined
Knife blade	Small Autosomal	26.02	1.55
Knife blade	Y	undetermined
Cigarette Butt	IPC	27.67
Cigarette Butt	Large Autosomal	26.06	0.44		1.02
Cigarette Butt	Small Autosomal	27.77	0.45		1.02
Cigarette Butt	Y	27.28	0.44
Bathroom sink stain	IPC	27.57
Bathroom sink stain	Large Autosomal	23.89	1.89		0.72
Bathroom sink stain	Small Autosomal	26.21	1.36		0.72
Bathroom sink stain	Y	25.80	1.24
Q Reagent Blank	IPC	27.73
Q Reagent Blank	Large Autosomal	undetermined
Q Reagent Blank	Small Autosomal	undetermined
Q Reagent Blank	Y	undetermined
Suspect Standard	IPC	28.00
Suspect Standard	Large Autosomal	24.34	1.39		1.06
Suspect Standard	Small Autosomal	26.08	1.48		1.06
Suspect Standard	Y	25.72	1.31
Victim Standard	IPC	28.05
Victim Standard	Large Autosomal	25.82	0.51		0.76
Victim Standard	Small Autosomal	27.98	0.39		0.76
Victim Standard	Y	undetermined
K Reagent Blank	IPC	28.02
K Reagent Blank	Large Autosomal	undetermined
K Reagent Blank	Small Autosomal	undetermined
K Reagent Blank	Y	undetermined

1. For each sample, list the concentration of DNA in the sample according to the small autosomal assay.

Answer 1

Answer;

4)

In a real time PCR assay a positive reaction is detected by accumulation of a fluorescent signal. The Ct(cycle threshold) is defined as the number of cycles required for the fluorescent signal to cross thethreshold (ie exceeds background level). Ct levels are inversely proportional to the amount of targetnucleic acid in the sample (ie the lower the Ct level the greater the amount of target nucleic acid in thesample).

Example;

WVDL real time assays undergo 40 cycles of amplification.
Cts < 29 are strong positive reactions indicative of abundant target nucleic acid in the sample
Cts of 30-37 are positive reactions indicative of moderate amounts of target nucleic acid
Cts of 38-40 are weak reactions indicative of minimal amounts of target nucleic acid which couldrepresent an infection state or environmental contamination.

And we can take example from given table like;

Knife handle (large autosomal) have CT value 30.29 while the concentration is 0.02.

5)

Quantitative PCR (qPCR) uses real-time fluorescence to measure the quantity of DNA present at each cycle during a PCR. A wide variety of approaches have been developed for generating a fluorescent signal, the most common of which use either hydrolysis probes (e.g., TaqMan^®), or a double-stranded DNA binding dye, (e.g., SYBR^® Green). At a point where the qPCR fluorescence signal is detectable over the background fluorescence, a quantification cycle, or Cq value, can be determined. Cq values can be used to evaluate relative target abundance between two or more samples. Alternatively, they can be used to calculate absolute target quantities in reference to an appropriate standard curve, derived from a series of known DNA dilutions.

Real-time PCR uses an increase in the intensity of a fluorescent signal generated by an intercalating dye or from the breakdown of a dye-labeled probe during amplification of a target sequence to detect nucleic acids either for their presence or absence or for their amount. The PCR cycle number where fluorescent signal becomes discernable above background noise is called the CT value. Between two samples, a decrease in a CT value (ΔCT) of one cycle represents a doubling of the amount of target.

Real-time PCR (RT-PCR) is also called quantitative PCR or qPCR. The key feature in RT-PCR is that amplification of DNA is detected in real time as PCR is in progress by the use of fluorescent reporter. The fluorescent reporter signal strength is directly proportional to the number of amplified DNA molecules.

There are two detection methods of RT-PCR, the first is based on sequence-specific probe such as TaqMan probe, molecular beacon; the second is based on generic non-sequence-specific double-stranded DNA-binding dye such as SYBR green. RT-PCR is a very sensitive and powerful DNA analysis tool. RT-PCR can be divided into four stages: linear ground phase, early exponential phase, linear exponential phase (log phase) and plateau phase. In the first phase, PCR is just starting, fluorescent signal has not risen above background. The second phase is where fluorescent signal just rise significantly above background, the cycle at which occurs is called cycle threshold (Ct). In linear exponential phase, PCR is in its optimal amplification stage with doubling PCR products in every cycle. The last phase is when substrates are exhausted and Taq DNA polymerase is in its end of life, fluorescent signal will no long increase.

??

Or in simple;

Real-time quantitative PCR.

(1) Real-time PCR is used to measure accurately the different amounts of a target gene product present in independent samples. In the case of real- time RT-PCR, the samples are cDNA previously reverse-transcribed from RNA preparations. (2) Each sample undergoes a PCR amplification together with a fluorochrome present in the reaction mixture, which fluoresces only when the specific product is recognized or synthesized (Fig. ). A fluorescence threshold is determined in the linear part of the curve, corresponding to the phase with the best efficiency of amplification. A ‘Ct’ is defined as the number of cycles necessary to reach this threshold of fluorescence. Ct = f (log 10 initial concentration of the specific target).