Week 11 Flashcards
Genomic libraries
Collection of DNA stored in cells that theoretically contains a copy of every DNA fragment inside vectors, similar to molecular cloning
Used to find specific DNA sequences, sometimes genome sequencing
To find specific genes, DNA probes are used
- Probes are labeled to be able to visualize
- Identifies clones that contain the complementary DNA
FISH
Fluorescence in situ hybridization
- Detects nucleic acid sequences by binding fluorescent probes, and doesn’t need to match 100%
- Transferred to membrane, cell lysis occurs, incubation with the probes, the probes hybridize to DNA, hybridized DNA is able to be viewed
- Detects COLONIES
Making DNA probes
Probes must be long enough an 80% match
Chemical synthesis, uses PCR
Southern Blot Analysis
- Cut with restriction enzymes, run in gel electrophoresis, transfer to membrane using capillary action, brick and paper towels
- Add probe to membrane
PCR
Polymerase chain reaction
Used in paternity testing, fingerprinting
Can be visualized with gel electrophoresis
Exponential amplification
What do you need for PCR?
Template DNA, primers, dNTPs, DNA polymerase, buffer
How does PCR work?
Machine heats up, denatures DNA template, cools down (anneals: primers hybridize to template), warms up again and DNA polymerase binds and starts replicating, denature again, cools, replicate, but this time stops exactly at the end of the target strand
Sanger Method of DNA sequencing
Chain termination and fluorescent labeling
Used for smaller sequences
Replaces dNTP with ddNTP (doesn’t have OH- at 3’ end, cannot elongate), the chain terminates and is fluorescently labeled
Fragments are then used in capillary electrophoresis
- Separates by size
- Uses a detector to determine sequence
