Biology 3594A Lecture Notes - Lecture 18: Woolly Mammoth, Thylacine, Amplicon
13 May 2018
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Greatest concern is contaminations and signal to noise ratio between woolly mammoth and other creatures on the site, plus the DNA of all the workers and stuff
Full clean room, suits, hairnets, booties
Dedicated areas for particular spaces
Same with animal facilities, flow in the room from clean to dirty
Double pairs of gloves, proper ways to put on and take off
Positive controls have amplicon sizes
Negative control: if 8 wells working on something there is a negative control in every well (?) –must be no reaction in those wells. Must throw everything out as soon as
you have a negative that turns out positive
Would have many negatives and positives, more negatives to make sure you don’t suddenly introduce contamination
Often looking for patterns of error (it’s negative up to this point, and then it’s positive)
If you work in a clinical lab, everything is regulated and prescribed
Often lab notebooks are fill-in forms so everyone in the lab follows the protocol
Applies to Tasmanian wolf and human gene sequences as well, not just woolly mammoth
Have to demonstrate you actually have a woolly mammoth, demonstrate the DNA is actually from that animal
It may be a very small portion of something that you want to extract DNA from, want to enrich something that is fragmented, decayed, small
Going to use an Asian elephant
Take a close relative and the mitochondrial genome is small so they can amplify it
Big amplicons (5.5 kb), overlapping so they’ll cover the genome – Asian elephant, reasonable hybridization, control of salt, can use to pull out anything closely related to
an Asian elephant (it will be a woolly mammoth)
We think we have candidate mammoth DNA in our biosample
In such small concentration, so precious, use in situ hybridization to pull it out
“bait” is Asian elephant template DNA, prepared from PCR, known sequence
5.5 kb, Then sonicate it, fragment into little pieces so you have many labelled probes
PCR, soniate, label, bead technologies to pull out mammoth DNA in solution, leave the elephant DNA stuck to the beads (all inthe salt and water concentration?)
Go beyond the hypervariable region (whole genome)
Clades of diff colours, diff SNP differences between mtDNA
Green clade at one time period, then mixture of new clade, then new clade maybe took over
Story: look at time + DNA
Already done a lot of proteomics with collagen –dating their herbivores, dating the grasslands to what they’ve eaten
Now general time periods from radioisotope stuff
This paper uses multiple perspectives and methodologies
Just mtDNA, only 20 samples (small)
Geographically sparse: large geographic area with a small number of samples
For woolly mammoth its’ only Eastern Eurasia, not talking about other parts of the world
mtDNA is matrilineal, small genome diversity
Only 3 clades talking about in paper as a whole
We don’t have any information about male-mediated migration
Do the females really migrate or do the males?
In mice, subordinate males disperse because dominant male and female litter –mice fight. Studying it would follow Y chromosome
Other animals have diff social structures, would want to approach study in a diff way
Woolly mammoth –want Y chromosome DNA studies and nuclear DNA
Doing this in tens of thousands of years, time scale and pictures are coarse, low resolution
Proteomic –tied with diet to the animal. Composition of the biological material tied it to the geographical location
Stable isotope –about dating the landscape, animals that are there. Helped with diet comparisons
Genetic analysis –start with mt, if we get better at preserving the DNA and getting more samples or protecting samples we can move to Y or nuclear
Clades –can look at population dynamics. Genetic diversity: differences between individuals, we can think about extinction because ofa clade with less diversity taking
over? Clades with genotypes, important to track pops
Can track which clade might take over from another
They Identified mt genomes, put it into time and place
Found that there are some region-specific dynamics, clades
This is the national genographic map of the world, with major human migrations being shown
Focus in on Bering strait, moving from Asian over into Alaska
Water is a barrier, but if we were to follow glaciation periods, human beings would be able to just walk across –land bridge that is there
This is an area of great study –if we want to know if all of indigenous persons came from marching over this land bridge
Arguments about how many migrations
Glaciation is dynamic –sometimes you could walk over, other times where it wasn’t possible due to a glacier –gene flow not occurring
Study of global human pop genetics –study of gene flow
If studying mtDNA can talk about matrilineal gene flow, or male Y chromosome
Is this all the same? How did all this divergence come about with diff pops and diff phenotypes and morphologies and languages
This area is rich in fossils too!
Lec 18 Pleistocene Alaskan Genome
May 13, 2018
2:55 PM
3594 Page 1
Document Summary
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