DNA quantitation Flashcards
LO
- What are the purposes of DNA quantitation
- What are the types of quantitation methods
- What are the steps involved
- Advantages and disadvantages
What is the concentration of DNA that is sufficient enough for use in the commercially available PCR kits?
What happens if there is too much or too little DNA?
- Commercially available PCR kits are optimised for 0.5-1 ng input DNA (older kits up to 2.5 ng)
- **Too little DNA= **potential for stochastic effects such as heterozygous peak imbalance
- **Too much DNA= **over-amplification leading to large peaks and difficulties in DNA profile interpretations
What are some commonly used DNA quantitation methods?
How does the spectrophotometry- nanodrop work?
- Measured nucleic acid concentration by analysing the absorbance of the sample at 260nm
What are the pros and cons to spectrophotometry nanodrop?
How does Agarose Gel electrophoresis work?
- **Separates DNA fragments by size **
When an electric field is applied- the negatively charge DNA migrates through the agarose towards the positive anode - The fluorescent dye (e.g., Ethidium bromide or GelRed) is either present in the gel or is added into the sample
o Intensity of fluorescent is dependent on the amount of dye bound to nucleic acids - Size of band is **compared against a ladder with fragments of known concentration **
- Useful when concentration is too low for spectroscopy and when contaminates are present within the sample
What are the pros and cons to agarose gelelectrophoresis?
How does the Qubit dsDNA HS Assay work?
- Utilises a dye that is highly selective for** dsDNA **(assays also exist for ssDNA, RNA, and proteins)
- Dye emits fluorescent when bound to dsDNA
- The amount of fluorescent is proportional to the amount of DNA-DNA quantity is determined by comparing to known standards- (Qubit fluorometer does this for you)
What are the pros and cons to Qubit dsDNA HS Assay ?
How does the Quantifiler Trio work and what does it use?
- Quantitative PCR (qPCR) assay
- Most sensitive and accurate quantification method
- It is **used for casework samples **or when you suspect the DNA samples are of low quantity and quality
o Can also give you an indication of the sample quality (degradation index)
What does the quantifiler Trio method combine?
- Two separate target-specific human assays; one with a short PCR amplicon and one with a **large PCR amplicon **
o Quantitation of amplifiable human DNA
o Allows for calculation of degradation index - A target specific Human make DNA assay
o Quantitation of amplifiable human male DNA - An internal PCR control (IPC) assay
o To ensure reaction works as expected
o Give an indication of presence of PCR inhibition
NB: amplicon- a segment of chromosomal DNA that undergoes amplification and contains replicated genetic material.
What are the stages to the quantifiler Trio method?
- Probe annealing
- Polymerisation and strand displacement
- Cleavage
- Completion of polymorphism
What occurs during probe annealing?
What occurs during strand displacement and polymerisation?
Whats occurs during cleavage?
What occurs during the completion of polymerisation?
What are the stages of PCR and how they link to the quantifilier Trio method?
Quantifiler Trio- amplification plot
Quantifiler Trio- Cv value
Quantifiler Trio- standards
What are the Quantifiler Trio controls ?
It is very important to have a negative and positive PCR control in order to assess that the experiment has worked correctly
* Inhibition control- each reaction contains an internal positive control (IPC) consisting of synthetic template DNA (not found in nature) and two primers to amplify this template, together with a TagMan probe
* Negative control- the PCR reaction has all reagents apart from the DNA…should give no result
* Positive control- the PCR reaction has all reagents and DNA of a known concentrations…should always give result
What are the pros and cons to the quantifiler trio method?
What DNA input are most commercial PCR kits designed for?
0.5-1ng
What can happen if you put in too much DNA?
Over-amplification
What method would you use for a forensic casework sample and why?
qPCR as gives indication of quality
Which method would be unsuitable to use post-PCR?
Quantifiler, as already using PCR
Does extraction technique have an impact on which quantitation methods you can use?
Yes, ssDNA vs dsDNA. If used Chelex couldn’t use Qubit as former is for ssDNA and the latter is for dsDNA. So cheapest extraction doesn’t work with cheapest quantifiler.
