Download Recombinant DNA Biotech Summary Questions

Block 2 MCB1
Recombinant DNA and Biotechnology
Summary Slide Questions
1. What are restriction enzymes? Restriction fragments?
Restriction enzymes are Type II endonucleases that cut DNA at specific palindromic sequences
of 4-8 bp. Restriction fragments are the pieces of DNA that result, with either staggered or blunt ends.
The site of this is the “restriction site.” The longer the restriction enzyme, the more restriction
fragments you get.
2. How can DNA from different species be pieced together?
You can cut out gene fragments using specific restriction enzymes that are known to cut the gene
you want. Then, the fragments are rejoined to vector DNA. Cut both the gene and the vector with the
same restriction enzyme....the open spot on the vector is called the cloning site.
3. How can a given DNA fragment be characterized?
A given DNA fragment can be identified by the location of restriction sites in a restriction map.
4. How are DNA fragments treated with the same restriction enzyme joined together?
DNA ligase joins them together.
5. How does Southern Blotting work? What is it used for? What happens during the blotting
step?
After conducting a gel electrophoresis on a sample of DNA, the results are blotted to a
nitrocellulose gel (papertowels above it “blot” the solution up though the film, catching the DNA).
Then, the film is treated with radiolabeled DNA probes that will anneal with the fragments of interest.
The resulting autoradiogram will show the length/location of DNA fragments that were complementary
to the probes....DNA-DNA hybridization. This can be used to diagnose mutations that alter restriction
sites.
***This hybridization can be done in fornamide, but different temperatures yield different
fragments. Less fragments at the higher temperature because the hybrids start to denature.
6. How long is a single-stranded probe used in the hybridizations step? What changes if the probe
is shorter or longer?
The single-stranded probe can be between 20-2000 bp....longer the probe, the more mismatches.
(a longer zipper will stay together with more mistakes, but a short zipper would have to be pretty
exact.)
7. How is a region in a polypeptide identified, using probes based on an amino acid sequence?
The polypeptide is identified with the least different codon usage.
8. How does Northern Blotting work? What is it used for? How is it different from Southern
Blotting?
It identifies and sizes mRNAs in a mixture using DNA-RNA hybrization. Similar to Southern
blotting, but does not use restriction enzymes.
9. What are DNA microarrays?
Small glass plates with lots of spots containing a defined DNA sequence. This allows for a
whole lot of fragments to be identified for, not just the few allowed in the other methods. Based on
exome sequencing.
10. How long are the DNA sequences in an expression array? What are they identical to? What
about genomic arrays?
The arrayed DNA sequences are 50 nucleotides long and are identical to selected mRNA, with
Ts for Us. These are then identical to chromosomal DNA sequences.
11. How are different mrNA mixtures compared in expression arrays?
The resulting array compares the expression of using fluorescently-labeled deoxynucleotides in
a reverse transcription step. Add the cDNA to the array, then add the fluoro cDNAs to anneal. Each
spot represents a gene that is expressed.
12. What is expression profiling used for?
This is used as a tool to haracterize diseased tissue, like the staging of tumors. An array test
like Mammaprint can also look for the genes that have been identified for risk of metastisizing of breast
cancer.
13. What is CGH and what is it used for?
Comparative Genomic Hybridization, an ultrasensitive comparison of clones and chromosomes
to look for indels. The resulting graph should balance out around 1, but in areas where its more or less
it implies an indel. This is used for cases with multiple congenital abnormalities that can't be pinned
down to a single mutation or chromosome.
14. What are plasmids? What do they carry? How can plasmid DNA be amplified 1000-fold?
Plasmids are extrachromosomal circular elements of DNA in bacteria which carry antibioticresistance genes.
15. How can an antibiotic resistance gene be used to select for bacteria with an inserted DNA
fragment?
Having a resistance gene in the plasmid allows you to select for the plasmids with an inserted
DNA fragment in that site.
16. How do you get a pure colony of DNA which can be propagated for lots of plasmid DNA?
You can “plate out” bacteria with a plasmid by applying the antibiotics that there are
resisitance genes for. If the DNA you want is in the Ambicillin resistance gene, you can use Tet-R as a
control to narrow it down to the cells you want.
17. What are “inserts?”
characterized by restriction mapping or DNA sequencing.....once you have the insert you want,
you can then subclone them in another vector for expression of a recombinant protein.
18. How do these gene cloning principles apply for bacteriophages, artificial chromosomes, and
yeast?
The same principles apply for these, except the amplification step.
19. What is cloned (subcloned) in another vector for expression of a recombinant protein?
Inserts. ***a Shine-Delgarno sequence is needed in a vector if using bacteria cells.
20. What is cDNA and how is it made?
CDNA libraries are made from mRNA that has been converted to cDNA using reverse
transcriptase and RnaseH. You can take away Rnase H to allow certain cDNA to be synthesized. So,
start with mRNA, then hybridize it....make a DNA copy using reverse trascriptase, then degrade the
RNA with RNase H to be left with the synthesized DNA strand, and use DNA pol to recreate the double
strand.
21. What is the primer for reverse trasncriptase and how does it work?
The primer is oligo dT (a stretch of 20 Ts) that works as a universal primer on all mRNA poly A
tails.
22. what is the difference between cDNA clones and genomic DNA clones?
CDNA clones, since they're made from mRNA, don't contain any of the introns or other premRNA elements. So, when you use it, you're only getting the finished product. Genomic DNA is a copy
of that whole strand, including introns, exons, and intergenic regions. Also, the size of inserts in the
libraries is different....for cDNA, it's either <15 or <23 kbp. For genomic, much larger, at <300 to
<2000 kbp.
23. Why is the cDNA insert fused to another gene fragment in specialized cDNA libraries?
So that the function of the protein coded for by the cDNA insert can be tracked and studied.
24. What are required in vectors used for expression in eukaryotic cells?
Need a eukaryotic promotor and a sequence carrying the polyA tail, and a resistance gene that
works in Eukaryotic cells (neomycin).
25. How is human insulin normally produced? How can it be joined in bacteria?
Produced as larger protein, then undergoes posttranslational proteolytic processing resutling in two
peptides held together by disulfide bridges....but, these can't be produced in bacteria. So, the two
peptides are made separately in bacteria, and then joined by oxidizing the cysteine side chains for the
disulfide bond.
26. What are transgenic animals? How are they created?
Animals that have been genetically engineered by insertion, delection, or replacement.They are
created by microinjection of the gene constructs into the pronucleus of fertizlied eggs.
27. What is the Tet-off system? How does it work?
With the Tet-off system, tissue specific inducible expression of the added gene can be achieved.
The added gene is under the control of an artifical promotor that needs a particular TF. This is
produced from another added gene under the control of the tissue-specific promotor. Th TF can be
dissociated from the promotor by adding tetraccline, which turns off the added gene.
28. What is required for gene knock-outs and replacements? Where is this done? What is required
to get animals of the desired genetics?
Need homologous recombination between the target gene and the modified gene construct. This
can be done in stem cells that are then added to an early embryo. The progeny are then genetic
mosaics, but the next generation have the target gene.
29. Why is the TK gene added at the end of a gene construct?
To get the homologous recombinationg that you're looking for.
30. What gene is used for replacement in knock-outs?
Use the neomycin gene.
31. How is a point mutation introduced in a particular gene?
Site-directed mutagenesis.
32. What are some strategies for gene therapy?
Gene additions or replacements. Like, for SCID, can give retroviral gene therapy.
33. How can DNA be clinically administered?
In liposomes, disabled viruses, or electroporation.
34. What are disabled viruses?
Virusses that lack essential genes coding for viral coat components....these are replaced by the
gene you want to administer.
35. What do microDNA genes code for?
They code for miRNAs that inhibit translation of target mRNAs by binding to the 3' site.
36. What is dicer? What is RISC?
Dicer leads to short dsRNA where one strand is incorporated into RISC, which binds the target
mRNAs.
37. How can degradation of an mRNA be achieved? What accomplishes this?
By introducing short dsRNA that is completely complementary to the 3'UTR of a target mRNA,
you can degrade it. Argonaut makes this cut in the mRNA to degrade.
38. What happens when you add transcription factors to an induced pluripotent stem cell? What is
pluripotency?
Results in complete genetic pregramming.
39. How can induced pluripotent cells be used?
Can replace diseased tissue or for testing new disease models. Or, to live forever.....