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What is DNA authentication?
the process of proving that amplified and analyzed DNA is authentic DNA from ancient remains.
What are problems with authentication?
There is no absolute test.
How do you authenticate?
Attempt to exclude all sources of contaiminant DNA.
What is contaminant DNA?
DNA that is the same or similar to the DNA molecule of interest.
What are sources of contaminant DNA?
reference specimens, lab technitions, archaeologists, food eated by people, air in building etc.
What are some contaminant controls?
use disposable gloves
use different pre and post PCR labs
+/- air flow in labs
steralize specimens and lab
What is positive and neegative air pressure?
Positive is used in the aDNA extraction lab. Pushes air out of openings in lab.
Negative is when the air is pulled in, used in post DNA extraction lab.
What contamination controls does the SFU extraction lab have?
positive air pressure
seperate lab from post extraction
How should you treat material to be submitted for aDNA extraction?
store in cool dry place (ziploc in a freezer)
document history of the sample
only submit one half of it
What is a positive control?
When you run the test with nothing in the test tube to see if you are getting contamination.
Minimizes false positives which are when PCR amplification is obtained despite there not being any aDNA to amplify.
What is a negative control?
When you run the test with something you know will work in a test tube to eliminate the possibility of PCR not working.
Minimizes false negatives which is when PCR amplification does not occur despite DNA being there.
Why do false negatives happen?
-presence of PCR inhibitors
-poor primer design
-not enough PCR cycles
-impurity in aDNA sample
Why do false positives happen?
Why is PCR amplification a double edged sword?
-can detect and amplify authentic aDNA
-can amplify contaminant DNA resulting in false positives
Exclusion Contamination Approach Authentication