Clinical use of recombinant DNA technology (L19) Flashcards
In prokaryotes, how do the cells prevent site-specific endonucleases from cleaving the DNA site when cleavage isn’t wanted?
Site-specific methylases
Describe the process of a Southern blot
DNA is electrophoresed, and separated by size (smaller DNA particles migrate farthest). DNA is denatured (by heat) and transferred to nitrocellulose paper by “blotting” and hybridized w/ a cDNA probe and viewed w/ autoradiograph.
What are you testing in Souther, Northern, and Western blots?
You use the __ test; when looking specifically for a piece of ___.
- Southern; DNA
- Northern; RNA
- Western; protein
Why does a western blot use 2 antibodies?
One is to bind to the protein, the other is a radioactively labeled Ab that binds to the primary Ab.
How would you make a DNA gene fragment that has cohesive ends?
Use the DNA fragment and attach it to a “linker” using ligase, and then add a restriction enzyme that cleaves the linker to make cohesive ends.
EcoR1 and Sal1 are endonucleases that will produce a DNA fragment that disrupts the tetracycline resistance gene (Sal1 cleaves in this gene). Describe an experiment that would allow you to see if your desired gene was implanted into bacterial cells.
Make your desired DNA fragment have cohesive ends for EcoR1 and Sal1, and then inject it into E. coli.
Plate that E.coli in a Amp+/Tet- plate and plate those cells in a Amp+/Tet+ plate to see if cells do not grow meaning your desired gene was implanted.
Basically A+/T- then A+/T+
How would you use a lambda phage carry foreign DNA?
Remove a non-vital middle section by restriction digestion, and introduce foreign DNA. Only the DNA that has the foreign DNA will be packaged but if it doesn’t, the DNA portion will be too small to be packaged.
What is special about M13 phage DNA?
When it inserts its DNA into E.coli, it is single stranded. So you must add a primer complementary to the site preceding the polylinker so that it can be synthesized in E.coli by DNAPI
Why is a Yeast Artificial Chromosome a good vector?
Because you can clone DNA up to 1000kb in it.
Why make a genomic library?
How do you do it?
This is so that you can sequence DNA sequences that are not present in mRNA (eg promoters, introns, splice sites, etc).
Splice an entire genome and use lambda phage as a vector for each little piece (there will be millions of phages needed), and then infect E. coli w/ the phages, plate it for plaques and transfer it to nitocellulose. Then you can use a labeled cDNA to look for what you want.
How would you isolate Eukaryotic mRNA to build a cDNA library?
Use Oligo-dT column affinitiy chromatography.
Run RNA thru a column containing microbeads that have oligo-dT strands on them that bind to mRNA polyA tails. The tRNA and rRNA wil be washed out of the column and you can use a low-salt buffer to elute mRNA and isolate it.
After isolating mRNA, how would you make it a cDNA duplex?
- Use reverse transcriptase (w/ dNTPs) to make a double stranded mRNA/DNA hybrid.
- Digest the mRNA w/ alkaline solution, AND attach a oligo(dG) to 3’ end of cDNA.
- Use an Oligo(dC) primer and DNAP (w/ dNTPs) to synthesize a ds cDNA.
NOTE: there is an oligo T primer that stays attached to the mRNA poly A tail when it is eluted before step 1. So your cDNA will have oligoG at 3’ end and oligoT at 5’ end.
How would you screen for cDNA clones using protein expression?
- Add cDNA into plasmid vector and transform it into E.coli
- Grow the E.coli in a plate, and transfer the colonies to a replica plate where you lyse bacteria to expose proteins
- Transfer the proteins to nitrocellulose sheet
- Add radiolabeled Ab specific for protein of interest
- Autoradiogram will detect a spot with the bacterial colong expressing the gene of interest
How would you screen for cDNA clones using hybridization?
- Add cDNA into plasmid vector and transform it into E.coli
- Grow the E.coli in a plate, and press nitrocellulose paper onto the plate
- Treat the paper w/ alkali to disrupt cells and then denature the DNA
- Add a radiactive DNA probe to bind to the desired DNA segment
- Autoradiogram recognizes colonies that contain the DNA probe.
In the Sanger Dideoxy sequencing method, what are the ingredient you would need?
4 tubes, each w/ all dNTPS, each with one of the 4 ddNTPs, a primer, and replication enzymes
What are the 3 steps of PCR, to be repeated again and again;
How are the target sequence and parent sequence amplified?
- Add excess primers, heat to separate DNA strands
- Cool to anneal primers
- Synthesize new DNA;
The target sequence is ampified exponentially and the parent sequence is amplified arithmetically
Describe casette mutagenesis
DNA is cleaved at a pair of restriction sites by 2 diff restriction endonucleases. A synthetic oligonucleotide (w/ new gene) is then ligated to the cleaved DNA. Now you have a plasmid w/ a new gene.
What is forward and reverse genetics?
Forward: DNA>Protein
Reverse: Protein>DNA
Gene knockouts are typically done via
homologous recombination
Describe the process of zygotic microinjection
- Inject foreign DNA w/ desired gene mutation into male pronucleus of fertilized mouse egg
- Transfer injected eggs into foster mother
- About 10-30% offspring should carry foreign DNA where it is present in all tissues (AKA transgenic mouse)
- Breed transgenic mouse to continue DNA in the germ line
Describe the process of making a “knockout animal”
for black fur from albino mice.
NOTE: this is an example but a knockout animal can be made this way for any gene.
- Culture embryonic stem cells (ES) from black furred mouse
- Insert ES cells into blastocyst cavity of early embryo
- Surgically transfer embryo into pseudopregnant mouse
- Offspring will be chimeric mouse w/ black and white fur
- Breed chrimeric offspring to get an all black offspring w/ the altered gene.
NOTE: This uses homologous recombination for altered gene to get in there in the first place
