Module 5 DNA Extraction

Blood sample extraction method

extracted using EZ1 instrumentation. Small amounts of blood is extracted by using organic.

Semen sample extraction method

extracted using differential extraction method.

Hair sample extraction method

extracted by the organic method.

Teeth and bone extraction method

both are extracted using organic extraction.

What is the purpose of DNA extraction?

To lyse cells to release the DNA molecules.

What is one goal of DNA extraction?

To separate the DNA molecules from other cellular material.

What does DNA extraction aim to isolate?

The DNA from inhibitors that reduce or prevent PCR amplification.

What is a common PCR inhibitor found in blood?

Hemoglobin

What is a common PCR inhibitor found in denim?

Indigo dyes

What is a source of PCR inhibition when amplifying mitochondrial DNA from hair samples?

Melanin

Factors affecting DNA extraction yields

Sample type and condition, extraction methods, inhibitors and contaminants, and handling and storage.

Importance of DNA extraction

To ensure that the DNA is pure and free from contaminations, sufficient quality for amplification, and it is the starting point for analysis. If this step is done poorly, it can affect all subsequent steps.

What are Aerosol Resistant Tips (ART)?

Aerosol Resistant Tips (ART) are specialized pipette tips.

Why are Aerosol Resistant Tips (ART) important?

They help protect pipettes and samples from cross-contamination.

How do Aerosol Resistant Tips (ART) prevent cross-contamination?

They prevent liquids and aerosols from contacting the pipette nose cone.

TE buffer

You will dilute a concentrated DNA sample with TE buffer (Tris-EDTA).

Reagent blank

Used as a control sample to make sure that the instrument is working properly and also to detect contamination.

Action if reagent blank is forgotten

Consult with my supervisor, document the incident, and wait for proper steps with handling the case.

What is the purpose of the digest buffer?

The digest buffer protects nucleic acids by breaking down nucleases, enzymes that can digest DNA/RNA.

What components are included in the digest buffer?

The digest buffer includes SDS, EDTA, NaCl, Tris-HCl, and DI water.

What is the role of Tris-HCl in the digest buffer?

Tris-HCl (tris hydroxymethyl aminomethane) is used to keep a stable pH, as DNA is pH sensitive.

What is the function of 20% SDS in the digest buffer?

20% SDS (Sodium dodecyl Sulfate) is used to destroy the cell membrane (lyse) to release soluble substances in cells and denature proteins.

How does 5M NaCl function in the digest buffer?

5M NaCl helps remove proteins bound to DNA and keeps proteins dissolved in the aqueous layer to prevent precipitation with DNA.

What is the role of EDTA in DNA extraction?

0.5 EDTA (ethylenediaminetetraacetic acid) is a chelating agent that deactivates DNase or RNase enzymes by chelating metal ions.

What is Proteinase K?

An enzyme that destroys proteins.

What is the primary use of Proteinase K?

Efficiently extracting high-quality DNA from a wide range of biological samples.

What does Proteinase K ensure about the extracted DNA?

That the DNA is free from proteins and other contaminants.

What is a spin basket?

A small, perforated container placed inside a microcentrifuge tube.

What is the purpose of a spin basket in centrifugation?

To hold solid samples while allowing the liquid containing DNA to pass through.

What does a spin basket trap during centrifugation?

Solid material.

QIAGEN's extraction robots

The EZ1 system uses pre-filled reagent cartridges for nucleic acid purification, employing magnetic bead-based technology to capture and purify nucleic acids.

Lysis buffer

Breaks down cells and releases their contents during the nucleic acid extraction process.

Phenol

Denatures proteins by disrupting their hydrogen bonds, crucial for removing protein contaminants from DNA; present in the lower organic phase.

Chloroform

Separates the aqueous and organic layers, assists phenol in denaturing proteins, and is part of the lower organic phase.

Isoamyl alcohol

Reduces foaming during the extraction process, important for maintaining a clear interface between the aqueous and organic phases; mixed within the organic phase.

Aqueous Phase

Purified DNA along with any other water-soluble components. (Layer of interest)

Organic Phase

Contains the denatured proteins, lipids, and other contaminants that have been separated from DNA.

Magnetic beads

Used in the EZ1 robot to capture and purify nucleic acids during the extraction process.

Wash steps

Performed by the EZ1 robot to remove contaminants such as proteins, lipids, and other cellular debris from the bound nucleic acids.

Elution buffer

Releases purified nucleic acids from the magnetic beads during the extraction process.

Microcon centrifugal filters

Two purposes are to concentrate and purify solutions of DNA, RNA, protein or other macromolecules.

What does a vaginal swab potentially contain?

A mixture of sperm cells from the perpetrator and epithelial cells from the victim.

What is the purpose of the lysis buffer in differential extraction?

To break down the epithelial cells while leaving the sperm cells largely intact.

What is the purpose of Proteinase K in lysis buffer?

Proteinase K digests the proteins in the epithelial cells.

What role does the detergent SDS play in lysis buffer?

The detergent disrupts the cell membranes, releasing the DNA from the epithelial cells into the solution.

What may a vaginal swab contain?

A mixture of sperm cells from the perpetrator and epithelial cells from the victim.

What role does the detergent (SDS) play in the lysis process?

It disrupts the cell membranes, releasing the DNA from the epithelial cells.

What happens to the epithelial DNA after incubation with the lysis buffer?

It remains in the supernatant, while intact sperm cells form a pellet at the bottom.

What is the purpose of the second lysis step in the Erase Sperm Isolation kit?

To isolate pure sperm DNA from a mixed sample.

What is typically contained in Solution 1 of the Erase Sperm Isolation kit?

Salt

What is the function of Solution 2 in the Erase Sperm Isolation kit?

Nuclease is an enzyme that breaks down nucleic acids DNA or RNA.

What is the role of Dithiothreitol (DTT) solution 3 in the lysis process?

It breaks down the tough disulfide bonds in the sperm cell membrane and nuclear envelope.

What is released into the solution after treating sperm cells with the specialized lysis buffer?

Sperm DNA.

What does DTT stand for and why is it used?

Dithiothreitol which it is used to break down the protein disulfide bridges that make up sperm nuclear membranes. This is used in differential extractions.

During a differential extraction, you have identified both epithelial and sperm cells on the slide. What may have went wrong?

The lysis buffer used during the first step of the differential extraction may not have effectively lysed all the epithelial cells, leaving some intact and still mixed with the sperm cells. This could happen if the incubation time was too short, the temperature was not optimal, or the concentration of the lysis buffer.

What might you expect in terms of final results if the first digest during a differential extraction were carried out too long?

The sperm cells would lyse prematurely leading to a mix (contamination) of the epithelial fraction with sperm DNA and reduced yield of sperm DNA in the sperm fraction leading to a possible mixed DNA profile.