Quantification

Question 1

Is quantification an essential step in DNA profiling?

Answer 1

no

Question 2

what is quantification

Answer 2

a measurement of the amount and quality of DNA in you extract is desirable.

allows optimum input for PCR

gives reference sampls

presence of inhibitors

zero=quantity means nothing found but its worth still doing the process as you might find something

Question 3

why’s it important in forensics

Answer 3

difficult to know how well preserved the DNA is

difficult to know how much cellular material has been recovered

Question 4

agarose gel

Answer 4

• Separates the DNA fragments
• Can be used as a basic quantification
• SYBRsafe dye intercalates with DNA double helix
• Visualised under UV light at 260nm
• Shows presence/absence of DNA
• Size of DNA fragments
• Quantification standards can be run to allow concentrations to be estimated
• Compare band intensity

Question 5

in agarose gel how do you know if there is lots of DNA and good quality

Answer 5

The brighter the band under the UV light the more DNA there is present in that band

Good quality DNA will appear as a discrete single band, degraded will appear as a smear

Question 6

disadvantages of agarose gel

Answer 6

• Very subjective
• Not very specific, samples may contain bacterial DNA or RNA
• Poor sensitivity

Question 7

qubit process

Answer 7

• Calibrate system using standards provided
• Dye in reagents specifically intercalates (bind) with dsDNA and increases fluorescence
• At a specific amount of the dye, the amount of fluorescence signal from sample is directly proportional to the conc. Of DNA in the solution
• The Qubit fluorometer can pick up this fluorescence signal and convert it into a DNA concentration measurement using DNA standards of known concentration
• Another step in the laboratory process
• Some simple calculations involved
• dsDNA specific but not human specific

Question 8

is qubit sensitive

Answer 8

yes, (down to 10pg/µl)

Question 9

what is quit

Answer 9

• Fluorescence based quantification

Question 10

what is Real time PCR

Answer 10

fluorescence-based assay

Question 11

what does real time PCR consist of

Answer 11

2 primers and a probe

Question 12

How does real time PCR work

Answer 12

• as the primers are extended during PCR cycles, they eventually meet the probe
• the probe is degraded and quenching of the fluorescence is removed and the florescence is detected
• as more PCR products are generated the fluorescence of the sample increases

Question 13

advantages of real time PCR

Answer 13

• highly sensitive
• human specific
• able to detect inhibition via internal PCR control (IPC)

Question 14

how many methods for quantification?

Answer 14

3