DNA Analysis Flashcards
How many base pairs and nanometers is one turn of a helix.
10 bp and 3.4nm per turn
Difference between prokaryotic and eukaryotic chromosomes
Prokaryotic: circular chromosome, small, only 1 per cell and not housed in the nucleus.
Eukaryotic: Linear chromosome, large, several per cell and housed in the nucleus. Takes form of histones (chromosome spools0.
Central dogma - You should know shit by now
procaryotes do not have any organelles and have genetic information loose in its cytoplasm. Central dogma suggests that the complex nature of eukaryotic cells stems from mutiple prokaryotic cells evolving together as one to perform more complex actions as it is more advantageous.
What does PFGE stand for?
Pulsed-field gel electrophoresis
Methods to separate very large DNAs (up to 10MB)
What are the steps in extraction/precipitation method?
- Disruption of cell walls, such as grinding.
- Lysis of cells in extraction buffer
- Organic extraction such as phenol to allow denaturing proteins and precipitate out organic matter to collect NA in aqueous solution after centrifugation.
- Precipitate nucleic aid by adding alcohol or salt. The pellet is washed with ethanol and then dissolved in buffer again.
What must NA purification always accomplish?
- Disrupt Cells
- Denature proteins and nucleoprotein complexes
- Inactivate endogenous nucleases
- Purify nucleic acid target away from other contents.
What are the steps in absorption chromatography extraction?
- Prepare crude lysate.
- Apply to a filter column and centrifuge to absorb NA in silica membrane.
- Wash away residual contaminants.
- Elute NA and collect that aqueous solution.
What are the steps in any blotting method?
- Gel electrophoresis to separate assay by size.
- Blot to move the sample from the gel to the membrane.
- Immobilize using UV or heat to keep sample stuck on the membrane.
- Block the membrane by coating it with generic RNA.
- Wash
- Probe the sample with a complement sequence to the target.
- Wash and visualize using X-ray or autoradiogram.
What is the molecule target and probe type used for southern, northern and western blots.
Southern: DNA target using a DNA probe.
Northern: RNA target using a DNA probe.
Western: Protein target using an antibody probe.
Describe and draw one temperature cycle of PCR.
- Denaturation at 94-96C
- Annealing at 50-65C.
- Elongation at 72C.
What does RT-qPCR stand for?
Reverse transcription quantitative polyermase chain reaction.
Draw the steps on TaqMan RT-qPCR
