quantification Flashcards
what is the most important prepratory step in DNA profiling?
how much DNA has been obtained from an extraction
why is quantification important?
- estimate efficiency of extraction methods
- for diluting samples to known concentration for use in PCR
- for best PCR results in relatively narrow range of DNA
why should you dilute DNA samples?
conserving DNA, reducing the possibility of introducing inhibitors which may be present in the extracted solution
why is hair not a good idea for DNA quantification?
there is not a lot of DNA and there are a lot of inhibitors in the hair.
what copurifies when blood is soaked into dirt?
human acids
what does ethidium bromide do in gels?
when running uncut DNA through gel will allow you to estimate the size range and quality of DNA
how is good quality determined by weight?
it will be higher in weight.
what will degraded DNA do in the gel?
it will smear extending from 100-2000bp
what does ethidium bromide do for DNA?
makes DNA glow intercalating between double stranded DNA making it flouresce.
T/F: ethidium bromide only works for dsDNA.
true
what is better than ethidium bromide and why?
sybr green
what does multiple sizes on a smear mean?
it is cutting outside the histone
if DNA stays above ___ then it is high quality DNA.
2000bp
what is the maximal absorption for DNA and RNA?
260nm
what kind of relationship exists between the absorption of light and nucleic acid?
linear
what can A260/280 not do?
differentiate DNA to RNA
the ratio of A260/A280 indicates what?
contamination by protein or phenol
A ratio greater than 1.9 means what?
pure DNA
A ratio greater than 1.8 means what?
pure RNA
what is a ratio indicating lots of protein?
0.6
what is fluorescence?
absorbance of light at one wavelength and emission at a different wavelength
what does picogreen bind to?
preferentially to dsDNA
following contact with wavelengths of light what causes the picogreen to flouresce?
the amount of bound picogreen
stronger picogreen emissions implies what?
more DNA
what is the limitations of picogreen?
not species specific and only dsDNA specific
what does end-point PCR tell you?
how much of a target you have and quantifies whatever the target may be
T/F: end-point specific PCR has an allelic ladder.
false, it has a sample of known amounts of DNA
what does end-point PCR help assess?
functionally available DNA for PCR
how are the DNA inhibitors reflected in end-point specific PCR?
less intense banding on gel
what is real-time PCR?
detection and quantification of DNA with a fluorescent reporter
what is recorded in real-time PCR?
amount of fluorescence emission at each cycle
during what phase is real-time PCR monitored? why?
during exponential phase; this is the first significant increase in amount of PCR product correlating to initial amount of target template.
what is the target gene of quantifier human kit? what is it specific to?
human telomerase reverse transcriptase; humans
T/F: the quantifier human kit is haploid.
false, diploid
what gene is the Quantifier Y kit targetting? what is it specific to?
sex-determining region Y gene; human male DNA
T/F: the quantifier Y kit is haploid.
true
what is the similarities/differences between end-point PCR and real-time PCR?
they are both target specific but RT has a laser shining through wells of PCR tubes tracking increasing fluorescence
Which is more specific: Sybr green with two primers or Taqman with two primers and a fluorescent probe?
taqman
during PCR where does the taqman MGB probe anneal to?
the specific complementary sequence between the forward and reverse primer site
what is the starter dye in Taqman PCR?
reporter dye
what is the NFQ of Taqman PCR?
the nonfluorescent quencher.
the longer the sequence the ___ and ___ specific to the place it will be.
rarer and more
the proximity of the reporter dye to the quencher dye results in what? by what?
suppression of the repporter fluorescence by Förester-type energy transfer
what does the ampliTaq gold DNA polymerase do? and what does that do?
cleaves only probes that are hybridized to the target. this separates the reporter dye from the quencher dye
when the reporter dye is separated from the quencher dye what happens?
an increase in fluorescence.
if the targets sequence is not complementary to a probe in taqman pcr what happens?
it will not fluoresce and amplification is not detected.
why is there no extension of the probe in taqman pcr?
beacuse the 3’ end is blocked
T/F: at 100% efficience there should be triple the amount of DNA at the end of PCR.
false, double.
what does the quantifier trio kit provide?
a simple way to accurately estimate the quantity of human and male DNA with low concentrations
how is quality estmiates achieved in a quantifier trio DNA quantification kit?
the degredation index (DI) will be less than or equal to 1 and over one could indicate degredation
what is an in-class example of real-time PCR?
distinguishing mammoths, to african and asiatic elephants
