DNA / RNA Extractions Flashcards
What are nucleic acids?
DNA and RNA
Nucleic acids are essential biomolecules that store and transmit genetic information.
Define Genotype.
The sequence of nucleotides in your genetic code inherited from your parents.
Define Phenotype.
An organism’s observable characteristics.
What factors influence phenotype?
- Genotype
- Epigenome
- Environmental effects
What is the first step in any molecular test?
To extract DNA or RNA from the sample and purify it.
List the four basic steps common to all DNA extraction methods.
- Collection of the sample
- Lysis of the cell and nuclear membrane to release the DNA
- Removal of membrane fragments, protein, and RNA
- Concentration of the purified DNA
What anticoagulant is routinely used for whole blood samples?
Ethylenediaminetetraacetic acid (EDTA).
Why is heparin not recommended in DNA extraction?
It can interfere with some results in molecular methods that follow DNA extraction.
What types of specimens is DNA routinely extracted from?
- Whole blood
- Bone marrow
- Buffy coat
- Fresh or frozen tissue
- Tumor
- Skin
- Fetal
- Chorionic villi
- Umbilical cord
- Formalin fixed paraffin embedded tissue (FFPE)
- Saliva
- Amniotic fluid
- Cultured cells
How long can whole blood and bone marrow be stored at room temperature?
4 days.
What is the proper storage condition for fresh tissue?
Good at 4°C for 2 days.
What must be done to amniotic fluid before storage?
Must be centrifuged to obtain the cell pellet.
What is the purpose of using filtered pipette tips in DNA extraction?
To avoid introducing nucleases into the sample and to prevent contamination.
What is the purpose of using proteinase K in DNA extraction?
To digest lysate during the extraction process.
Fill in the blank: DNA is a _______ molecule.
polar
What is the principle behind the phenol/chloroform method?
Phenol dissolves proteins, allowing DNA to be separated.
What is the purpose of using ethanol in DNA extraction?
To precipitate DNA out of solution.
Describe the column extraction method.
DNA from lysate is adsorbed onto the silica membrane in a spin column under specific buffering conditions.
What is the function of RNase enzymes?
To catalyze the degradation of RNA into smaller components.
What is the recommended long-term storage temperature for RNA?
-80°C.
True or False: A 260/280 ratio of 1.8 is considered pure for DNA.
True.
What can affect the 260/280nm reading during nucleic acid quantification?
Any contaminating material.
What is the purpose of using chaotropic salts in magnetic bead extraction?
To facilitate the binding of DNA to the silica surface of the magnetic beads.
What is considered a pure 260/280 ratio for RNA?
2.0
A 260/280 ratio of 2.0 indicates that RNA samples are free from protein contamination.
How does the absorbance at 260nm generally behave compared to 280nm?
Absorbance at 260nm is at a plateau, while absorbance at 280nm is steeply sloped.
What will change the 260/280nm reading?
Any contaminating material.
How can you estimate the concentration of DNA or RNA in a sample?
Using the absorbance value at 260nm with standard concentration formulas.
To calculate the yield of double-stranded DNA in µg/mL, the absorbance at 260nm is multiplied by _______.
50.
To determine the yield of RNA, the absorbance at 260nm is multiplied by _______.
40.
What does a 260/280 ratio less than 1.7 indicate?
Protein contamination, low DNA concentration, or phenol contamination.
What should you do if your 260/280 ratio is less than 1.7?
Try re-extracting DNA.
What does a 260/280 ratio greater than 2.0 indicate?
DNA is contaminated with RNA.
What can be done to address contamination with RNA?
Digest with RNase.
How can you identify purity using a 260/230 ratio?
By dividing the absorbance reading at 260 by the absorbance reading at 230.
What does a 260/230 ratio less than 1.8 indicate?
DNA is contaminated with organic material or carbohydrates.
What should you do if your UV-vis graph curve is not smooth?
Mix and add more buffer to dissolve DNA.
What might cause a low DNA concentration in a sample?
Low white cell count.
What are the advantages of spectrophotometry?
- Simple
- No sample prep
- Provides direct measurement of purity
- Can provide information about contaminants
What are the disadvantages of spectrophotometry?
- Not selective for DNA or RNA
- Limited sensitivity (2.5 ng/μL)
- Cannot determine if DNA is fragmented.
What does fragmented DNA do in terms of absorbance?
Absorbs light at 260nm the same as unfragmented DNA.
What is the basis for fluorescence measurement of nucleic acids?
Use of fluorogenic dyes that bind selectively to DNA or RNA.
What is the sensitivity of fluorescence measurement?
Very sensitive (pg/mL).
How is quantitative real-time PCR used in DNA quantitation?
Determines the amount of amplifiable DNA by measuring a ‘house-keeping’ gene.
What does intact DNA look like on a gel?
A large diffuse band of high molecular weight.
What will intact total RNA show on a denaturing gel?
Sharp, clear 28S and 18S rRNA bands.
