DNA Quantitation

Discuss two reasons why DNA must be quantified prior to polymerase chain reaction

• the FBI’s Quality Assurance Standard 9.4 requires human-specific DNA quantitation to determine the appropriate amount of DNA template to include in PCR amplification of STRs

• Establishes that human DNA (not bacterial) has been isolated

1.       What is normalization and why is it important for PCR-based DNA typing?

Normalization is the process of achieving a DNA concentration that fits the optimal window for analysis

  A DNA sample is quantified and determined to have a DNA concentration of 0.2 ng/μL. To normalize the extract to a DNA concentration of 0.0333 ng/μL, you will dilute 15 μL of the extract in TE buffer. How much TE buffer should you use to dilute the extract?

75 ul

two disadvantages of UV absorbance and yield gel methods of DNA quantification

• are not sensitive

• consume a significant sample volume

• not human specific

1.       How does the slot blot quantitation assay detect DNA and estimate DNA quantity? What is the probe used in this assay?

detects DNA by immobilizing denatured DNA on a nylon membrane and then hybridizing it using a human-specific probe. The amount of DNA in a sample is then determined by the intensity of the signal produced when the hybridized complex is detected. The signal intensity is compared to a set of standards and an estimation is produced. The probe used in this assay is a 40 base pair probe bound to a region on chromosome 17 called D17Z1.

What kind of dye is commonly used in end-point PCR?

SYBR Green

How does SYBR Green attach to double-strand DNA and allow for fluorescent detection

It attaches to dsDNA by intercalating between base pairs and its fluorescence increases when bound. The detection of PCR products can then be seen by measuring the intensity of the fluorescence.  

What are the three stages of real-time PCR analysis? Explain what is occurring at each stage.

exponential amplification, linear amplification and plateau region

In the exponential stage, the amount of DNA doubles every cycle. The fluorescent signal from the DNA-binding probe increases proportionally to the amount of PCR product generated. Next, in the linear amplification stage, reagents start to become limited and enzyme efficiency decreases resulting in the reaction efficiency slowing down. The increase in PCR product is no longer doubling but increasing at a slower linear rate. Lastly, the plateau stage is when the reaction reaches a point where amplification stops

Which stage is used for quantification of DNA?

Exponential stage because PCR amplification is the most efficient and reliable at this point which allows for accurate determination of initial DNA concentration based on Ct value

Define cycle threshold (Ct) value

PCR cycle number at which fluorescent signal of the target DNA crosses a predefined threshold

What is the relationship between Ct value and DNA quantity?

Ct inversely related to the starting amount of DNA, with a higher Ct value indicating less DNA and a lower value indicating a high initial DNA conc.

Plot of log[DNA] vs Ct gives a linear graph w/negative slope

The Quantifiler Trio DNA Quantification kit contains an internal PCR control. What are two things that IPC results can tell you about real-time PCR results?

IPC can tell you if PCR inhibitors are present and it confirms that PCR reagents and thermal cycling conditions are working correctly. If IPC signal is delayed or absent, it indicates PCR inhibition

What is the primary focus?

determine the appropriate amount of template DNA to include in PCR amp of STRs to avoid off-scale data and artifacts

What is the purpose of qPCR?

determine amount of “amplifiable” DNA

What instruments do we use for qPCR?

Tecan and ABI7500

What are the three phases of qPCR?

(1) Exponential

(2) Linear

(3) Plateau

What is standard 9.4?

Thou shall quant

What are two reasons why DNA must be quantified prior to PCR?

(1) QAS 9.4: requires human-specific DNA quantitation to determine appropriate amount of DNA template to include in PCR amp of STRs

(2) Quantitation establishes that human DNA (not bacterial) has been isolated