DNA Extraction

Question 1

In 1869, the first ________________________ was achieved.

Answer 1

DNA isolation

Question 2

In 1958, first ___________ was achieved based on ___________ strategies.

Answer 2

DNA extraction; density gradient centrifugation

Question 3

1950’s: _____________________ was discovered

Answer 3

DNA’s double helix structure

Question 4

When was DNA polymerase discovered?

Answer 4

1956

Question 5

What are two early DNA isolation methods?

Answer 5

cesium chloride/ethidium bromide centrifugation and organic “salting out”

Question 6

How does lysis occur for DNA isolation?

Answer 6

Via salt solution or proteases, or both

Question 7

DNA purification via columns

Answer 7

+ charged materials bind to = charged DNA, washed with salt solutions to remove unbound material, DNA recovered with water or pH 7-8 buffer to break matrix-DNA bond

Question 8

Types of PCR-based HLA assays that use genomic DNA

Answer 8

sequence-specific primer (SSP), sequence-specific oglionucleotide probe hybridization (SSOPH), Sanger sequencing-based typing (SBT), quantitative PCR (qPCR), next generation sequencing (NGS), short tandem repeat (STR) analysis

Question 9

260 nm is the wavelength that _____________. 280 nm is the wavelength that _____________.

Answer 9

DNA absorbs UV light; protein contaminants absorb UV light.

Question 10

Acceptable A260/A280 ratio for purified DNA

Answer 10

between 1.8 and 2.0

Question 11

Protocol: Salting Out Method

Answer 11

1. Disruption and lysis of cell membrane using a buffer containing a detergent (eg. SDS) and proteinase K at 37 C.
2. Unwanted components like proteins are precipitated out using high salt concentrations.
3. After centrifugation, supernatant containing DNA is removed by pipetting into a separate tube.
4. Ethanol is added to supernatant and DNA precipitates out as visible white strands.
5. Following centrifugation to pellet the DNA, ethanol is removed and DNA pellet air dried.
6. Dried pellet is dissolved in Tris-EDTA (TE) or reagent grade water.

Question 12

Protocol: Magnetic Bead Extraction

Answer 12

1. Samples are lysed using a buffer containing a detergent and proteinase K.
2. After cell lysis, magnetic particles are used to selectively purify DNA molecules from the sample lysates.
3. The beads are isolated with a magnet and the liquid removed.
4. The bead/DNA complex is washed with an ethanol buffer solution.
5. DNA is eluted off the magnetic beads using molecular biology grade water.

Question 13

If DNA is stored at -20 C or -80 C, what elution reagent is preferred?

Answer 13

Tris-EDTA

Question 14

DNase-free water can be used as an eluate, but DNA stored in H2O is subject to what?

Answer 14

acid hydrolysis

Question 15

What is Tris-EDTA?

Answer 15

A buffer solution that helps maintain the stability of nucleic acids during sample preparation, electrophoresis, and enzymatic reactions; used for DNA sequencing and PCR applications.

Question 16

What is elution?

Answer 16

A process that uses a solvent to extract one material from other; process of extracting a substance that is adsorbed to another by washing it with a solvent.

Question 17

How can carry-over contamination be avoided during DNA extraction?

Answer 17

physical separation between the pre-amplification and post-amplification areas; use of DNase-free consumables, aerosol barrier pipette tips, laminar-flow hood, dedicated sets of pipettes for extraction and PCR set-up

Question 18

Daily cleaning of all work surfaces should be performed with ____________. Weekly cleaning should be performed with _____________.

Answer 18

70% ethanol; 10% bleach followed by 70% ethanol

Question 19

What is an easy way to detect contamination in the lab?

Answer 19

PCR amplification of a wipe of work surfaces to detect contamination of human genomic and amplified DNA.

Question 20

What can you do if your DNA concentration from a column extraction is too low?

Answer 20

1. Concentrate DNA by alcohol precipitation by adding a volume of cold ethanol that is twice the volume of the suspension. Vortex and centrifuge on highest speed. Pipette out as much alcohol as possible and add water to sample. Vortex and repeat quantitation and purity check.
2. Extract an additional sample with increased volume, or, if WB was used, prepare a buffy coat.
3. Could be indicative of inefficient cell lysis or protein degradation of sample due to insufficient mixing/incubation time. Repeat purification procedure. Make sure sample is thoroughly digested.
4. DNA concentration can be increased by decreasing the volume of elution buffer.

Question 21

What does a high A260/A280 ratio mean?

Answer 21

DNA sample may be contaminated with RNA, which won’t interfere with PCR but could interfere in downstream applications.

Question 22

What does a low A260/A280 ratio mean?

Answer 22

Protein contamination; if ratio is lower than 1.75, PCR will probably be affected

Question 23

Reason for low/poor quality PCR product and/or PCR reaction failure but A260/A280 was within range

Answer 23

Most frequent cause is alcohol contamination.