Hall 17 - Molecular Techniques in RadBio Flashcards
What do restriction endonucleases do?
Cut DNA at a predictable site within or adjacent to a palindromic recognition sequence, creating sticky ends.
What are vectors?
Self-replicating DNA molecules that carry foreign DNA into host cell
Plasmids = circular DNA, abx resistance, 10 kb
Bacterial artificial chromosome (BAC) = low copy number plasmid, genome-sequencing, 300 kb
Viruses = reverse transcribe genes into DNA
How are genomic libraries created?
Cut up genomic DNA (restriction endonuclease) > ligate into vector > introduce into bacteria or yeast
How are cDNA libraries created?
Isolate mRNAs > reverse transcribe into cDNAs > ligate into vector
What are the temperatures for polymerase chain reaction (PCR)?
Heat to 94 C to denature
Cool to 50 C to anneal
Heat to 72 C for DNA polymerase
Each cycle ~7 min and doubles the number of DNA molecules
What is a southern blot?
DNA separated by size then probed with labeled DNA
Restriction fragment length polymorphisms (RFLPs) occur when differences in DNA (ex, SNPs) result in restriction fragments of different sizes
What is comparative genome hybridization (CGH)?
DNA from tumor digested with restriction endonuclease > amplified > labeled with fluorescent dye Cy5 (red)
Control cells are labeled with Cy3 (green)
> > mix two samples and hybridize to DNA microarray > ratio of red to green (to detect copy number changes)
What is CRISPR?
A gene knockout strategy
= clustered regularly interspaced short palindromic repeats
Serve as genetic memory of past viral infection (Cas9 is antiviral)
Can generate targeted DSBs in DNA
Cas 9 binds to guide RNA (CRISPR RNA complementary to DNA target + trans-activating crRNA as a constant dsRNA region)
PAM also confers specificity
Cas9 complexes cleave DNA > DSB > NHEJ or homology-directed repair (HDR) with exogenous donor DNA template
What is a northern blot?
RNA analysis
How does qRT-PCR work?
RNA reverse transcribed > cDNA > amplified with fluorescent specific marker for gene of interest > fluorescence change is measured after each cycle
*More sensitive and dynamic than northern blot
What are the key advantages of RNAseq?
Next-generation sequencing by creating a de novo transcriptome, providing transcript structure, providing quantitative transcript number
What is an electrophoretic mobility shift assay (EMSA) used to detect?
Protein-DNA interaction (complexes migrate slower than free DNA)
What is chromatin immunoprecipitation (ChIP) used for?
Protein-DNA interaction (gene transcripts*)
Determines protein interactions with a target promoter or DNA element in the native chromatin environment
What is a western blot?
Proteins analyzed by charge and size separation
What does immunoprecipitation detect?
Protein-protein interactions (run on a western blot)
