DNA Quantification & PCR Techniques: Spectrophotometry, Fluorometry, and qPCR
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What is the primary function of a spectrophotometer?
To measure absorbance of light at specific wavelengths.
What is the primary function of a fluorometer?
To use a light source to excite specific molecules and detect the amount of fluorescence emitted.
What is PicoGreen?
An ultrasensitive fluorescent nucleic acid stain for DNA quantification that exhibits >1,000-fold fluorescence enhancements when bound to dsDNA.
What is the detection range of PicoGreen?
10 pg/mL to 1 μg/μL of DNA.
What is required to determine the concentration of an unknown DNA sample?
DNA concentration standards and a standard curve.
What is a standard curve?
A graph that allows accurate determination of the concentration of a target molecule in an unknown sample.
What is a serial dilution?
A method to provide accurate standards for comparison, beginning with a known concentration and diluting by a known factor.
What is the sensitivity of fluorescence detection compared to UV absorbance?
Fluorescence detection is 10,000-fold more sensitive than UV detection.
What is the detection range of UV absorbance?
12 ng to 15 mg/mL.
What contaminants can interfere with UV absorbance results?
RNA, nucleotides, proteins, salts, phenol, etc.
What is the main advantage of quantitative PCR (qPCR)?
It allows real-time analysis of PCR product accumulation during the PCR cycle.
Who devised the Polymerase Chain Reaction (PCR)?
Kary Mullis in 1983.
What are the three basic steps of the PCR cycle?
Denaturation, annealing, and extension.
What is the role of primers in PCR?
Short synthetic pieces of DNA that define the initiation site for the elongation of the new DNA molecule.
What is the significance of Taq DNA polymerase in PCR?
It is a heat-stable enzyme that catalyzes the synthesis of new DNA molecules.
What is the error rate of Taq DNA polymerase?
1 x 10^-4 to 2 x 10^-5.
What is the purpose of the buffer solution in PCR?
To create optimal conditions for polymerase activity.
What is the function of Mg2+ ions in PCR?
They are a cofactor critical for Taq DNA polymerase activity.
What does qPCR measure during each cycle?
The fluorescence of the PCR product as it accumulates.
What is the difference between conventional PCR and qPCR?
Conventional PCR analyzes the amplicon after the reaction is finished, while qPCR allows real-time detection during the reaction.
What is the relationship between fluorescence increase and amplicon accumulation?
Increase in fluorescence is proportional to the accumulation of amplicons.
What are the two main types of fluorescent chemistries used in PCR?
DNA binding dyes (not sequence specific) and hydrolysis (Taqman) probes (sequence specific).
What are the four phases of PCR amplification?
Lag (Baseline), Exponential, Linear, and Plateau.
What occurs during the Lag phase of PCR?
The signal is too low to be distinguished from background noise.
What characterizes the Exponential phase of PCR?
Exact doubling of PCR product with 100% efficiency and specific, precise measurements.
What happens during the Linear phase of PCR?
High variability occurs as reaction components are being used up, leading to linear amplification.
What is the Plateau phase in PCR?
The reaction components have been consumed, and if left too long, the PCR product can degrade.
What is the Cycle Threshold (CT) in PCR?
An arbitrary threshold set during the exponential phase, indicating the cycle at which fluorescence reaches a predefined level.
How is the amount of starting DNA in an unknown sample determined?
By comparing the CT value of the unknown to a standard curve of known samples.
What is Absolute Quantification in DNA analysis?
It requires generating stable and reliable standards to construct standard curves for comparison.
What is the function of Taqman probes in PCR?
They consist of a reporter dye attached to the 5'-end and a quencher attached to the 3'-end, which emits fluorescence when cleaved during extension.
What is SYBR Green used for in PCR?
A DNA-binding dye that intercalates with double-stranded DNA, increasing fluorescence dramatically as more amplicon is synthesized.
What is the purpose of specialized thermal cyclers in qPCR?
To measure fluorescence during each PCR cycle.
What does the Quantifiler Trio DNA Quantification kit assess?
It provides quantitative and qualitative assessment of total human and male DNA in a single, highly sensitive real-time PCR reaction.
What is an Internal Positive Control (IPC) in PCR?
A short strand of synthetic DNA amplified in the same reaction to assess sample quality and confirm assay functionality.
How can DNA degradation be assessed in qPCR assays?
By comparing the ratio of amplification between short and long PCR amplicon targets.
What does a Degradation Index (DI) indicate?
It compares the concentration of small autosomal targets to large autosomal targets to assess DNA quality.
How can a mixture of male and female DNA be detected in qPCR?
By calculating the ratio of autosomal DNA to Y-chromosomal DNA detected.
What happens as the ratio of female DNA increases in a mixture?
It becomes more difficult to detect the male contributor in an STR profile.
What is the purpose of DNA quantification?
To determine how much DNA has been extracted from samples.
What is the optimal amount of DNA required for standard DNA profiling?
0.5-1.0 ng of DNA.
What can high levels of DNA create during quantification?
Artifact peaks, which are undesirable.
What happens with low levels of DNA during quantification?
They can produce allele dropout.
Name three common methods of DNA quantification.
Spectrophotometry, fluorometric assays, and quantitative real-time PCR (qPCR).
How does spectrophotometry measure DNA concentration?
By measuring the absorbance of light at specific wavelengths.
At which wavelengths are nucleic acids typically measured in spectrophotometry?
260 nm (nucleic acids), 280 nm (proteins), and 230 nm (contaminants like guanidine and phenol).
What is the function of fluorometers in DNA quantification?
They use a light source to excite specific molecules and detect emitted fluorescence.
What is PicoGreen and how does it work?
A fluorescent nucleic acid dye that exhibits enhanced fluorescence when bound to double-stranded DNA (dsDNA).
What concentration range can PicoGreen detect?
From 10 pg/mL to 1 mg/mL of DNA.
What is a standard curve in DNA quantification?
A graph that allows accurate determination of the concentration of a target molecule in an unknown sample.
What is the purpose of a serial dilution in DNA quantification?
To provide accurate standards for comparison by diluting a known concentration.
Who devised the polymerase chain reaction (PCR) and when?
Kary Mullis in 1983.
What is the main purpose of PCR?
To rapidly amplify specific fragments of DNA for study and analysis.
What are the three basic steps of a PCR cycle?
Denaturation, annealing, and extension.
What role do primers play in PCR?
They specify the starting point of PCR on each strand and flank the gene sequence of interest.
What is qPCR and how does it differ from conventional PCR?
Quantitative PCR (qPCR) allows detection of the amplicon as it accumulates during the PCR cycle, unlike conventional PCR which analyzes the amplicon after the reaction has finished.
What is the significance of using thermostable DNA polymerase in PCR?
It catalyzes the synthesis of new DNA molecules and has a very low error rate.
What can high primer concentrations lead to in PCR?
Off-target amplification and primer dimer formation.
What is the 'Goldilocks' principle in PCR optimization?
Finding the right primer concentration that maximizes efficiency without causing issues.
What is the relationship between PCR amplicon synthesis and initial DNA concentration?
The amount of PCR amplicon synthesized during the exponential phase of PCR correlates with the initial concentration of extracted DNA.
How is fluorescence measured in qPCR?
Fluorescence is measured during each cycle of PCR, and the increase in fluorescence is proportional to the accumulation of amplicons.
What are the four phases of qPCR amplification?
The four phases are lag (baseline), exponential, linear, and plateau.
What occurs during the lag phase of qPCR?
In the lag phase, the signal is too low to be distinguished from background noise.
What characterizes the exponential phase of qPCR?
The exponential phase has 100% efficiency with exact doubling of PCR product.
What happens during the linear phase of qPCR?
In the linear phase, reaction components are being consumed, leading to highly variable amplification.
What occurs in the plateau phase of qPCR?
In the plateau phase, the reaction components have been consumed, and if left too long, the PCR product can degrade.
What is the cycle threshold (CT) value in qPCR?
CT is the cycle in PCR when the measured fluorescence in the sample reaches a predefined threshold.
How is a standard curve used in qPCR?
A standard curve is built from known samples to compare CT values of unknown samples to determine the amount of starting DNA.
What is absolute quantitation in qPCR?
Absolute quantitation requires generating stable and reliable standards for the construction of a standard curve.
What is the function of TaqMan probes in qPCR?
TaqMan probes use a reporter dye and a quencher; when the probe is intact, fluorescence is quenched, and during extension, the probe is cleaved, releasing fluorescence.
What is SYBR Green in qPCR?
SYBR Green is a dye that intercalates with double-stranded DNA, dramatically increasing fluorescence as more amplicon is synthesized.
What type of thermocycler is used for qPCR?
Specialized thermocyclers are required to measure fluorescence during each PCR cycle.
What is the purpose of the Quantifiler Trio DNA Quantification Kit?
The kit provides quantitative and qualitative assessment of total human male DNA in a single, highly sensitive real-time PCR reaction.
What is the role of the Internal Positive Control (IPC) in qPCR?
IPC assesses the quality of the sample and confirms that assay components are functioning properly.
What does a failure of IPC amplification indicate?
If IPC amplifies but the sample does not, it indicates low starting template; if both fail, it indicates PCR inhibition.
How is DNA degradation assessed in qPCR?
DNA degradation is assessed by comparing the ratio of long PCR amplicon to small PCR amplicon.
What does the mixture ratio in qPCR indicate?
The mixture ratio of male DNA to female DNA helps determine the extent of the mixture and whether to proceed with autosomal STR or Y-STR analysis.