Biology 3594A Study Guide - Final Guide: Woolly Mammoth, In Situ Hybridization, Cave Bear
13 May 2018
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Lecture 18 Flashcards
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Biggest concern with aDNA lab
Contamination!
- from other creatures on the site
- and from the DNA of all the technicians
Some precautions that are taken to
prevent contamination in an aDNA lab
•DediatedLa
•Clearoo protools
•Positiepressure airflo la
•LaUV irradiated dail
•Surfaesleaed ith leah
•Allaterials & reagets UV irradiated if possile
•Fulllea roo suits, hairets, asks
•Separatedediated hoods for reaget prep, sampling and
reactions
•Doulepairs of gloes
•Positieetratio otrols – cave bear
•Negatieotrol to assess for otaiatio a egaties
•Multipleotrols used per eperiet
Why we need to enrich the mammoth
mtDNA
- there is candidate mammoth biosample material and we want
to extract the DNA
- need to demonstrate that the DNA is actually that of a woolly
mammoth
- might be fragmented, decayed, small piece of DNA so we
need it to be enriched somehow
In solution capture of mammoth
mtDNA using elephant mtDNA as bait
- why an Asian elephant mt DNA?
Close relative and mt genome is small so they can can amplify it
In solution capture of mammoth
mtDNA using elephant mtDNA as bait
- process
1. Prepare 3 overlapping 5.5 kb elephant mtDNA amplicons -
big amplicons so they'll cover the genome
2. PCR and sonicate amplicons (this will fragment the DNA and
create random ds breaks)
3. Label elephant DNA
- you have many labelled probes for the genome
4. In situ hybridization
- control salt and water concentrations (lower stringency?
Decrease temp, increase salt)
- bead technologies to pull out mammoth mtDNA in solution
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Some precautions that are taken to prevent contamination in an adna lab. Why we need to enrich the mammoth mtdna. In solution capture of mammoth mtdna using elephant mtdna as bait. There is candidate mammoth biosample material and we want to extract the dna. Need to demonstrate that the dna is actually that of a woolly mammoth. Might be fragmented, decayed, small piece of dna so we need it to be enriched somehow. You have many labelled probes for the genome: in situ hybridization. Bead technologies to pull out mammoth mtdna in solution: proteomic - collagen analysis. Tied with diet, and the composition of the biological material ties it to the location: stable isotope - dating, diet. Dating the landscape and the animals that are there. Helps with diet comparisons: genetic analysis - genotyping, clade identity. Mtdna is a good start: clades - many shared derived alleles.
