What is the general reason we quantify DNA
it is important to get the correct amount of DNA into the STR amplification reaction
What will happen if too little DNA is put into a PCR reaction
It would fail completely as little to DNA present
Result in low signal to noise which is extremely difficult to interpret
What happens when too much DNA is input into a PCR reaction
It would fail completely as too much DNA can inhibit PCR
Results in high signal to noise
What is signal
the message you want to detect using the instrument - E.g. nice peaks in an STR profile
What is noise
the background chatter also detected on the instrument E.g. Baseline fluorescence that a machine automatically detects
What are the small peaks found infront of allelic peaks in an STR profile called
Stutter peaks
How can inputting too much DNA into a PCR reaction effect stutter peaks
They will appear as actual peaks due to their size
Name the 3 main methods for quantification
- Agarose gel visualisation
- UV-Visible spectrophotometry
- Real time analysis
What are the benefits of agarose gel visualisation
a simple, quick and cheap way to confirm DNA is present within a sample
What are the disadvantages of agarose gel visualisation
- They have a low accuracy and precision
- Based on visual interpretation
- Do not provide a concentration
- Do not provide information on inhibitors
What is used in agarose gel visualisation to determine quantity
brightness of fluorescence
What are the gels stained with in agarose gel visualisation and why
Intercalating dye - it fluoresces when DNA present is double stranded
Why do we no longer use ethidium bromide as an intercalating dye in Agarose gel visualisation
it is a carcinogen
what is seen via smears in agarose gel visualisation
degradation
Name a common method to understand the concentration of a sample
UV-Visible spectrophotometry
What is a nanodrop
A microvolume spectrophotometer
Why are nanodrops better than regular UV-visible spectrophotometers
- They only require 2ul of a sample (reduces waste)
- Cheap per sample
- Very fast results
- Tells you if inhibitors are present
Why do we not use nanodrops within forensics
Not a sensitive approach and limit of detection is ~2ng/ul - not small enough
How does a UV-Visible spectrophotometer work
By measuring the absorption of ultraviolet and visible light by a sample.
Through its key components — light source, monochromator, sample holder, detector, and data processor — it isolates specific wavelengths, measures transmitted light, and calculates absorbance.
What are the common methods used for real time analysis
- qPCR using a Bio-Rad machine
- TaqMan probes
- HyBeacon probes
How does a Bio-Rad machine work for real time analysis
It reads fluorescence when dye intercalates with DNA
What are the positives of using a Bio-Rad machine for real time analysis
- Only uses 1ul of sample
- Uses PCR and is therefore the most sensitive technique
- Good accuracy and precision
What are the negatives of using a Bio-Rad machine for qPCR
not a lot of reproducibility
What intercalating dye do we use to quantify DNA in qPCR nowadays
SYBR Green
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DNA recovery40
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DNA amplification (how)30
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DNA amplification (Quantification)30
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DNA amplification (Size separation of STR markers)20
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Simple STR profile interpretation25
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Complex STR profile interpretation23
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STR profile analysis - DNA databases & informative stats21
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mtDNA as a lineage marker35
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Y-chromosome lineage markers27
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Applications of SNPs - lineage markers14
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statistical analysis of DNA workshop18
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Why we use DNA to combat wildlife crime32
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How we use DNA to combat wildlife crime40
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DNA and RNA structure and function19
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genetics - mutation, polymorphism and selection30
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historical techniques14
