Bio-arch

• Diagenesis (inorganic and organic substances).
• Soils and microbes (which result for 99% of DNA extracted from total remains) stain DNA

Because they inhibit PCR amplification, more specifically the polymerase enzyme.

• 1. Bone Preparation
• 2. Bone powder incubation
• 3. DNA isolation
• 4. Further concentration and purifcation

Select the sample bone, remove surface contaminants, and make fine bone powder.

Best to have cortical bone over spongey bone or teeth in order to decontaminate easily.

• 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:

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.

• -mortor and pestle or drill
• -liquid nitrogen grinding mill

To free the DNA trapped in the collagen.

You add chemicals to the bone powder to remove bone mineral components.

• 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.

You wait 24 hours while the solution sits in a rotating oven or shaking water bath.

• There are three methods:
• -phenol and chloroform solution
• -silica glasses/silica beads
• -chelex beads

• 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.

• 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.

• Chelex beads bind to almost all other substances but DNA.
• Good: fast, non-toxic.
• Bad: may not seperate DNA well enough, like phenol-chloroform.

Concentrate DNA after phenol-chloroform isolation to remove chemicals and proteins.

• chemically: alcohol precipitation
• physically: use 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.

• 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