DNA amplification- quantification:

Question 1

reasons for quantification- too little DNA input into a PCR:

Answer 1

fail completely
-trace- little to no DNA present
-poor storage- decay
-poor CSI training
-need repeating- expensive.
result in low signal to noise- difficult to interpret.

Question 2

reasons for quantification- too much DNA input into the PCR:

Answer 2

fail completely.
-too much DNA can inhibit PCR.
-usually (not always) occurs if quantification step missed.
-need repeating- expensive.
result in high signal to noise- difficult to interpret.

Question 3

methods of quantification: agarose gel visualisation:

Answer 3

-simple way to confirm DNA is present- low accuracy. just shows if DNA is there or not.
-does not give a concentration- low precision. visual interpretation.
-does not provide any information on inhibitors.
-stained with intercalating dye.
-degradation occurs with smears.
-not really used in forensics anymore.

Question 4

methods of quantification: UV-visible spectrophotometry.

Answer 4

common method to understand the concentration of something.
- this system is very easy to use.
-does not waste precious sample. only uses 2ul.
-is cheap per sample.
-not a sensitive approach. cant use on dilution series as no meaningful results weaker than 2ul.
-not used in forensics.

Question 5

methods of quantification: real time analysis

Answer 5

-read fluorescence when dye intercalates with DNA. SYBR green- fluoresces when in contact with double stranded DNA.
- SYBR green can be used to quantify DNA.
-uses 1ul of DNA sample.
-uses PCR so therefore the most sensitive technique.
-good accuracy and precision.
-not a lot of reproducibility.
-unknown DNA is run with DNA of a known concentration that can be diluted to produce a curve for comparison.
-depend on effective primer design.
-cost is low
-used in forensics.

Question 6

real time analysis- taqman:

Answer 6

-Synthetic oligos.
-Specific to wanted DNA region.
-Reporter molecule (fluoresces), quencher- stopes fluorescence when cleaved from primer during annealing. Reporter releases so increases in fluorescence which gets detected.

Question 7

real time analysis- hybeacon:

Answer 7

-Hybeacon probes can be used to quantify DNA.
-More targets give more info.
-Specificity comes from primers and also probes.
-Cost is high.