Nucleic Acid Techniques
Nucleic Acid Techniques
Nucleic Acid Techniques
Nucleic Acid Techniques
Nucleic Acid Techniques
100
What are the 4 steps in nucleic acid extraction?

Pretreatment (dependent on type of specimen)

Lyse cells

Isolate DNA

Precipitate DNA

100
What is the purpose of lysing cells in the nucleic acid extraction process?
To break open, or lyse, the cells in order to get the DNA out
100
Describe the organic method of DNA isolation and how it works to isolate the DNA
High salt, low pH, and organic chemicals are used to remove proteins, lipids, carbs, DNA, or RNA


DNA is left in supernatant after centrifugation


100
If quality of the DNA is <1.8, what does this indicate?
Proteins are still present
100
Calculate quality of DNA:


Absorbance of .200 at 260 with a 1/100 diluted sample=?

If 2 mLs of sample=?

.200 x 50(constant) x 100=1000 ug


1000 x 2 mLs of sample=2000 ug total DNA

200
What is most commonly used to precipitate DNA?
Alcohol
200
Define amplication in terms of nucleic acid testing/extraction
Reproducing the DNA so we can bring it up to a higher level where we can actually detect it
200
Describe the solid phase method of DNA isolation and how it works to isolate the DNA
Cells are lysed, high salt buffer is added, DNA binds to the solid phase 

-DNA sticks/binds to the wall of the tube

200
Does amplification procedures require quality DNA?
No
200
What is the purpose of boiling the cells at 100 C when extracting with chelex?
Lyse the cells

-The lysis products then stick to the beads so they are unable to degrade the DNA

300

This method is an example of what detection means in reference to nucleic acid testing?

Electrophoresis 
300
Between the 3 methods of DNA isolation, which is considered safer than the Organic method but not as effective?
Inorganic
300
What does Ficoll do in terms of pretreatment portion of nucleic acid extraction?
It forms a layer between RBCs and WBCs so you can seperate the RBCs from WBCs
300
What is the purpose of chelating resin beads in DNA isolation?
The negatively charged beads attract metal ions such as Mg2+

- Grabs out stuff we don't want

300

If quality of the DNA is >2.0, what does this indicate?

Indicates RNA may be present
400
Describe the inorganic method of DNA isolation and how it works to isolate the DNA
Similar to organic method 

-Low pH, high salt, DNA left in the supernant and then precipitated out with alcohol

-Doesn't use the organic chemicals

400
Define extraction in terms of nucleic acid testing
Take the DNA from inside the cells to get rid of surrounding proteins
400
What are the 3 types of DNA isolation methods?
Organic, inorganic, solid phase
400
At what light does nucleic acid absorb light at in the spec? At what light does protein absorb light at in the spec?
NA: 260

Protein: 280

400
What is the range for proper, quality DNA?
1.8-2.0
500
What are Tris buffer and proteinase K used for in the lysing portion?
Amplification applications

-Proteinase K digests proteins, lyses cells, and inactivates enzymes

500
What is the purpose of the isolation portion of nucleic acid extraction?
To remove the proteins, lipids, RNA, etc
500
What is Triton and SDS used for in the lysing portion?
Used as a detergent to lyse cells


500
What is the purpose of incubating sample to 56 C when extracting with Chelex?
To soften the membrane and seperate the cells from each other
500
Other than electrophoresis, how can we determine the quality of the DNA that was extracted?
Spectrophotometry
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