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Where do impurities in aDNA come from?
- Diagenesis (inorganic and organic substances).
- Soils and microbes (which result for 99% of DNA extracted from total remains) stain DNA
Why must impurities be removed from aDNA?
Because they inhibit PCR amplification, more specifically the polymerase enzyme.
What are the basic steps of aDNA extraction?
- 1. Bone Preparation
- 2. Bone powder incubation
- 3. DNA isolation
- 4. Further concentration and purifcation
What is involved in bone preparation?
Select the sample bone, remove surface contaminants, and make fine bone powder.
What is the best kind of bone for aDNA studies and why?
Best to have cortical bone over spongey bone or teeth in order to decontaminate easily.
What do you use to get rid of surface impurities?
- Physically remove contaminants (perhaps with a small drill but this will send bone powder everywhere) or use wet sandpaper.
- Chemically remove it: bleach, works the best.
- UV irradiation:
How does UV irradiation work?
The radiation excites DNA molecules in skin cells, causing aberrant covalent bonds to form between adjacent cytosine bases, producing a dimer. When DNA polymerase comes along to replicate this strand of DNA, it reads the dimer as "AA" and not the original "CC". This causes the DNA replication mechanism to add a "TT" on the growing strand.
How do you make bone into bone powder?
- -mortor and pestle or drill
- -liquid nitrogen grinding mill
Why do you have to make the bones into powder?
To free the DNA trapped in the collagen.
Why do you incubate the bone powder?
You add chemicals to the bone powder to remove bone mineral components.
What chemicals do you use during DNA incubation?
- You can use EDTA which is a very dangerous chemical that takes the calcium out of cell walls, but is very dangerous and also can damage DNA.
- You can use Proteinase K: (PK) which is an enzyme that digests proteins.
- You can use SDS (detergent) to denature proteins.
- PK and SDS are what Dongya uses.
What do you do after you add the chemicals to the bone powder?
You wait 24 hours while the solution sits in a rotating oven or shaking water bath.
How do you isolate/extract/seperate the DNA from the protein, DNA, chemical solution?
- There are three methods:
- -phenol and chloroform solution
- -silica glasses/silica beads
- -chelex beads
What is phenol-chloroform extraction?
- Removes proteins and other chemicals from the mixed solution resulting in a DNA only solution. Like oil and water.
- Good because you get a high DNA yeild
- Bad because DNA is still with other inorganic substances, phenol and chloroform are really toxic, and the process is complicated.
What is the silica based method?
- In high salt solutions, DNA will bind to silica. In low salt solutions, DNA is released from silica.
- Good: DNA is well seperated, fast, non-toxic.
- Bad: less DNA than organic extraction (phenol-chloroform) and PCR is inhibited if silica is not removed.
How do chelex beads work?
- Chelex beads bind to almost all other substances but DNA.
- Good: fast, non-toxic.
- Bad: may not seperate DNA well enough, like phenol-chloroform.
What is DNA concentration?
Concentrate DNA after phenol-chloroform isolation to remove chemicals and proteins.
How do you concentrate DNA?
- chemically: alcohol precipitation
- physically: use centricon microconcentrator.
What is a centricon microconcentrator?
A micro-seive really. just have a seive where things !!! are collected so you have retained DNA after phenol-cholorform seperation. Also used to simply concentrate DNA solution if you need to load a silica spin column which only has capacity for a certain amount of solution.
Tell me about the method Dongya invented?
- Silica based spin column (QIAquick). Very simple.
- Direct purification from proteinase K digests (without any phenol-chloroform
- extractions) using only the QIAquicky column which is a silica based spin column.
- Uses a silica-membrane to purify DNA >100 bp
What do you look for in DNA extraction?
To maximize the potential for success, DNA extraction protocols should be capable of purifying trace amounts of DNAwhile at the same time removing potential inhibitors of PCR. In addition, extraction protocols should be designed to minimize the potential for contamination with modern DNA