Extraction

Chapter 2; DNA Extraction

Explain how an organic phenol-chloroform extraction isolates DNA and removes impurities. What are three disadvantages of using this method?

adding sodium dodecylsulfate (SDS) and proteinase K to the sample to break open the cell membranes and to break down the proteins protecting DNA molecules. After this step, a phenol/chloroform mixture is added to separate the proteins from the DNA. The DNA will now be in the aqueous portion of the mixture. The final step will be to centrifuge, in which the unwanted proteins and debris will be separated away from the aqueous phase and leave the double-stranded DNA molecules can be transferred for analysis.

Three disadvantages

• the use of hazardous chemicals

• it is time-consuming

• contamination can occur due to transferring between multiple tubes.

Explain how a Chelex extraction isolates DNA and removes impurities

The Chelex extraction involves boiling a sample in a 5% Chelex suspension, in which cells are broken and DNA is released. Boiling temperatures of 100°C denatures the DNA in addition to disrupting cell membranes and destroying cell proteins. After this, the Chelex resin and cellular debris is pulled to the bottom of the tube through centrifuging. The supernatant is removed and can now be used for quantitation and amplification

Why is a Chelex extraction only useful when the isolated DNA is intended for PCR-based testing?

denaturing double-stranded DNA and yielding single-stranded DNA, which can only be used for PCR testing

What does Chelex resin do?

remove metal ions from solutions, particularly in DNA extraction and PCR preparation. It works by binding to divalent metal ions like Mg2+, which are crucial cofactors for DNases (enzymes that degrade DNA). By removing these ions, Chelex resin effectively inhibits DNase activity, protecting the DNA from degradation and preventing PCR inhibitors from interfering with downstream reactions

What is FTA paper and how is it used as a medium for DNA extraction?

FTA paper is an absorbent cellulose-based paper that contains four chemical substances to protect DNA molecules from nuclease degradation and preserves the paper from bacterial growth. It is used as a medium for DNA extraction by lysing the cells that meet the paper and can be used to further wash the sample. First, a spot of blood is added to the paper and allowed to dry. The cells are lysed upon contact with the paper and DNA from the white blood cells is immobilized within the matrix of the paper. A small punch of the stain is then placed into a tube to be washed with FTA Purification Reagent

Describe how solid phase extraction is accomplished in QIAGEN chemistries.

Solid phase extraction is accomplished in QIAGEN chemistries using the QIAmp spin columns and EZ1 instrument. Using the spin columns, the nucleic acids are absorbed to silica on small glass beads, in the presence of high concentrations of chaotropic salts. These salts disrupt hydrogen-bonding networks in liquid water and make denatured proteins and nucleic acids more thermodynamically stable than their structured counterparts. If the solution is more acidic than pH 7.5, DNA is absorbed to the silica and impurities can be washed away. To elute the DNA from the silica material, an alkaline and low salt solvent is used. Two advantages of this method are that it can be performed in a single tube and is automatable.

What are two advantages and two disadvantages of solid phase extraction?

Advantage

• Can be performed in a single tube

• Automatable

Disadvantage

• Magnetic bead approach is dependent on DNA binding to beads

• Lower DNA yield compared to other extraction methods

What commercial kit does our laboratory use to accomplish solid phase extraction? What is the solid phase medium in this kit? Why is carrier RNA used with this kit?

QIAsymphony DNA Investigator kit

solid phase medium in this kit is silica-based magnetic particles

Carrier RNA is used to enhance the efficiency and yield of DNA extraction by stabilizing DNA during the process.

Explain how a differential extraction separates epithelial cells from sperm cells in sexual assault samples

A portion of the mixed stain is first incubated with SDS/proteinase K at 37°C to lyse female epithelial cells; the resulting supernatant is kept as the “female fraction.” The remaining sperm cells are then lysed using SDS/proteinase K with DTT.

1.       ? In our laboratory’s differential extraction procedure (FB.DNA.4053), how many washes are typically done and what chemicals are used at each wash step?

three washes done with sterile water and a fourth final wash with Buffer ATL

What % of bleach and ethanol is used to clean the hoods/bench/etc?

10% bleach, 95% ethanol

What folder/box do known samples go into?

S for someone

What folder/box do evidence samples go into?

R for rare

What are the goals of extraction?

(1) Lyse cells to release DNA mol

(2) Separate DNA mol. from other cellular materials

(3) Isolate DNA into a formal compatible w/downstream applications including PCR amp