Misc Topics Flashcards
What are restriction enzymes?
- Enzymes used to cut DNA at specific sequences, usually palindromic
- Naturally synthesized by bacteria for an immune response against invading viral bacteriophages
– The enzymes destroy the phage’s DNA before it can insert into the bacterial chromosome!
Are bacteria sensitive to their own restriction enzymes?
No, since the sequences their enzymes recognize and cut are silenced via methylation.
What is a palindromic sequence?
A DNA sequence which in 5’-3’ direction reads the same as its complimentary strand read 5’-3’
What are northern, southern, & western blots?
SNOW DROP
- Southern → DNA
- Northern → RNA
- Western → Proteins
What charge do RNA and DNA carry?
-ve charges
What are vectors?
Vectors are carriers of DNA
What is a plasmid vector?
Plasmid → circular DNA molecule capable of autonomous replication.
Usually associated with antibiotic resistance!
What are phage vectors?
VIruses that infect bacteria. Carry a much longer sequence of DNA than plasmids, so preferred for this reason.
Bacteriophage λ has been used as a phage for a long time.
What are genomic libraries?
All DNA (fragmented) from a single host is cloned into a phage.
What is a cDNA library?
All mRNA from a specific host cell is made into complementary DNA (cDNA).
This collection is known as the cDNA library.
The cDNA from a brain cell vs. a liver cell will be very different due to different gene transcriptions!
What is an expression library?
cDNA from a particular cell type in a host is put on a promoter to make proteins.
This is used to make recombinant proteins in large amounts, eg insulin.
What is transfection?
The process whereby you introduce a DNA sequence into a mammalian cell using non-viral methods. Can be done in many different ways:
- Microinjection
- Calcium phosphate precipitation
- Electroporation
- Cationic lipids
- Other non-viral methods
What are the steps in the DNA polymerase chain reaction?
- Denaturation: 90C, 10s
- Annealing: 50-65C, 10s
- Extension: 70C, 30s
Which enzyme catalyzes the PCR process?
Taq polymerase (enzyme resistant to high temps).
What is real-time PCR?
Uses fluorescent probes to
Measures the amount of mRNA
What is restriction fragment length polymorphism (RFLP)?
Differences among people in their DNA sequences at sites recognized by restriction enzymes. Such variation results in different-sized DNA fragments produced by digesting the DNA with a restriction enzyme. RFLPs can be used as genetic markers, which are often used to follow the inheritance of DNA through families.
What is siRNA technology?
It is a double-stranded RNA tech that can selectively silence particular genes.
What is shotgun sequencing?
A computer sequences
Mouse strains have different susceptibilities to radiation-induced lung cancer. Which strain has a low susceptibility?
C3H / H0J
How do mutations differ b/w high and low LET radiation?
High LET → Large Mutations
Low LET → Small Mutations
However, looking at a mutation, you cannot tell whether it came from high or low LET RT.
What is the Chromatin Immunoprecipitation Assay (ChIP) assay used for?
To detect proteins bound to promoter sequences
What are reporter gene assays?
These are genes that researchers attach to a regulatory sequence of another gene of interest..
The reporters are attached to a marker, such as a fluorescent dye, that can identify where the gene promoter is.
What’s the use of propidium iodide?
- DNA-intercalating dye
- Stains cell proportionally to the amount of cellular DNA
What does the TUNEL assay measure?
Apoptosis
What is transduction?
Introduction of nucleic acids or foreign DNA into a cell using viral vectors
What’s electroporation?
Introduction of DNA into a cell by using pulsed, high-voltage electricity that can open pores within the cell membrane
What is transformation?
The process by which normal cells are transformed into malignant cells
What’s Sanger sequencing?
- A DNA replication method, in which the nucleotides that are added to the DNA are observed (the DNA sequence can then be deduced)
- Uses dideoxynucleotide triphosphates (ddATP, ddTTP, ddCTP, ddGTP)
What’s conjugation?
The process where a bacteria transfers a plasmid DNA into another baceterai
What’s conjugation?
The process where a bacteria transfers a plasmid DNA into another bacteria
What is complementation?
Restoring a normal copy of a gene in the setting of biallelic mutations
What’s the MISO PET used for?
Detect tissue hypoxia
What’s the main function of cos sites?
They are essential for packaging DNA in phages
Which enzyme does the clustered regularly interspersed short palindromic repeats (CRISPR) technique?
Cas9
