LEC EXAM #3 CHP. 12 Flashcards
Restriction enzymes:
Enzymes that cuts DNA at specific sites
Recombinant DNA
Takes a human gene and recombines it with bacterial DNA using a restriction enzyme
Vector:
Plasmids that have a MCS (multiple cloning site) that has restriction enzymes that cut enzymes to allow insertion of gene of interest
Genomic libraries:
Goal?
-46 chromosomes-> treat with restriction enzyme-> cuts into fragments-> put inside plasmid-> gets inserted into bacteria cell->
GOAL: make bacteria make multiple copies of fragments that are used for DNA sequencing
cDNA libraries:
Isolates RNA> cuts it up-> use reverse transcriptase to converts RNA to cDNA-> cDNA gets inserted into plasmids-> bacteria transformed with vectors-> make multiple copies
Electroporation:
Current/charge in cells that causes disruption/holes in the phospholipid bilayer that helps you get plasmids into bacterial cells
Why is cDNA more specific than genomic libraries?
You know exactly what genes are being expressed because you make mRNA to make proteins
Microinjection:
How we inject into human cells
DNA probes:
Molecular structure that can be attached to a piece of DNA that makes it visible
(Ex: Fluorescent dye or radioactive)
Purpose of DNA probes: (2)
- Used to distinguish DNA by adding a probe
- Visualization of specific sequence of DNA
Gel electrophoresis structure:
- Gel agarose which is long strands that when cooled layer on top of each other
- Place well comb into agarose to form holes/mesh structure
- Put dye in DNA and load them into wells
- Buffer/current applied to the outside-> negative charge on one end and positive charge on the other end
Goal of gel electrophoresis:
Separates DNA by size
Result of gel electrophoresis:
- Smaller pieces of DNA move through the mesh faster to the positive charge
- Larger pieces of DNA take longer
RFLP mapping abbreviation:
Restriction fragment length polymorphism
RFLP mapping:
Looks at DNA that’s been cut up/digested by restriction enzymes and separates into size by gel electrophoresis
Southern blot:
-DNA is separated by size via gel electrophoresis then tested to see if truly similar in DNA sequence and fragment size
-Transferred to nylon membrane
-Complimentary probe is added to DNA sequence of interest
-Dark spot on the x-ray film allows you to visualize the piece of DNA and see where its located
(probe is radioactive)
DNA microarray process:
Used to isolate mRNA of human cells-> reverse transcriptase-> makes complementary strand cDNA-> red or green fluorescent probes added to complimentary DNA-> combine targets-> hybridize to microarray tray with complimentary pieces of DNA sticking up
Purpose of DNA microarray process:
Used to compare human cells to cancerous cells to see what cells are being expressed
Amount of hybridization=
amount of gene expression
SDS-page
Similar to gel electrophoresis but separates proteins by size instead of DNA
How to treat SDS-page?
With detergent that has a negative charge
What is the gel/linear fiber made of in SDS-page?
Acrylamide
PCR steps:
- Heat up strand-> opens DNA
- DNA primers anneal and bind to complimentary piece of DNA
- Lower temp down so TAQ DNA polymerase can function
- DNA polymerase fills in DNA
At 72C:
PCR
Allows primers to bind and fills in DNA
TAQ polymerase:
- made from DNA
- Comes from archae that live in thermal vents (thermophilus aquadicus)
- Able to heat up and bring back down in temp without denaturing it
What does TAQ polymerase allow you to do?
Repeat making DNA
Di-deoxynucleotides:
- Used for DNA sequencing to generate DNA fragments of of different lengths
- Stop replication sequencing where it picks up a di-deoxy
Can you add onto a dideoxynucleotide?
No, because it’s not a free 3 prime hydroxyl
Every di-deoxy:
- Has a fluorescent label/probe attached to it that comes from DNA primers
- Does not have OH group
GFP:
- Green fluorescent protein produced by jellyfish
- Allows you to track location of protein
mRNA bound by:
miRNA (micro RNA)
siRNA: (small interfering DNA)
miRNA and siRNA BOTH:
- Made by human cells
- Regulate/stop gene expression
RISC complex in an imperfect match:
Sits on mRNA and miRNA segment and blocks translation from occurring (temporary)
RISC complex in a perfect match:
Stops dsRNA-> cleavages mRNA (permanent)
Gene therapy:
- Genetic mutation that was inherited
- Goal is to bring in a good non-mutated copy of gene and find a virus that infects the target tissue
Process of gene therapy:
Takes viral DNA and cuts it with restriction enzymes-> insert new unmodified gene ->insert into known virus-> virus infects target tissue of patient-> inserts DNA into host cell
Plasmid contains:
Multiple cloning sites where you can insert your gene of interest
Why is RISC complex temporary in an imperfect match?
Because it doesn’t hydrogen bond-> not a double stranded RNA structure
Ex of vector:
Bacteriophage or plasmid
