FRSC-3000: Lecture 2

What is the purpose of presumptive tests?

To indicate whether biological fluids are present on an item of evidence. They can identify sample sources including blood, semen, and saliva.

Presumptive tests should be:

• Simple

• Inexpensive

• Safe

• conservative of materials

• Non-destructive

What are the general steps of DNA extraction?

• Lyse cells to release DNA

• Separate DNA from cellular material

• Isolate DNA for downstream STR typing

• Store DNA at -20 Celsius or -80 Celsius to prevent nuclease activity

List the contaminants which are PCR inhibitors:

• Iron from RBCs

• Ethanol

• Minerals from Bone

• Jeans/indigo dyes

• Melanin

• Proteins

How is nuclease activity prevented in PCR?

• Store sample at low temperature to inhibit hydrolytic cleavage of DNA

• Add EDTA to solution which chelates the free magnesium ions needed for nuclease activity

What is differential extraction?

The separation of non-sperm and sperm fractions based on absence or presence of DTT that breaks open the sperm cell coating.

List the DNA extraction methods:

• Organic (phenol-chlorophorm)

• Solid-phase (vacuum or magnetic bead capture)

• Chelex

• FTA paper

• Differential extraction

What reagents are used to lyse cells and what are their functions?

• Urea: Disrupts hydrogen and hydrophobic bonds holding the cell membrane together

• SDS: An ionic detergent that binds proteins and unfolds them to disrupt DNA-protein complexes

• EDTA: A chelating agent that binds multivalent ions and de-activates nucleases

• Proteinase K: Digests proteins by breaking peptide bonds and is stimulated by detergents (SDS)

What is the function of phenol-chloroform?

Separate proteins by denaturing them and precipitating them, where DNA is more soluble in aqueous solution.

How is the DNA in the aqueous phase of organic extractions precipitated?

An isopropanol or ethanol treatment.

What are the general steps of solid-phase DNA extractions?

• DNA selectively binds to a substrate (silica membrane or silica coated magnetic beads)

• The DNA is retained on the substrate while proteins and cellular debris are washed away

• The DNA is released into solution under low salt conditions or with the addition of an elution buffer (TE)

What are the general steps of a chelex extraction?

• A chelating resin is added directly to a sample of blood or semen

• Magnesium ions are bound by the resin

• The sample is boiled to lyse cells, denature proteins, and release the DNA

• The sample is centrifuged to pellet the cellular debris and the chelex resin

• ssDNA is removed from the supernatant

What are the general steps of an FTA paper extraction?

• A blood sample is added to the FTA paper and allowed to dry

• The cellulose-based paper lyses the cells, binds white blood cells, protects DNA from nuclease activity, and deters bacterial growth

• The punch of paper is washed to remove heme and other PCR inhibitors

• The clean punch can be added directly to PCR

• Chelex or solid-support extractions can be performed on the clean punch

Which extraction procedure yields ssDNA?

Chelex extraction.

Which extraction procedure(s) is amenable to automation?

Solid-phase extraction methods.

Which extraction procedure yields high molecular weight DNA?

Organic extractions (phenol-chloroform).