During a CRISPR-Cas9 exp., yeast are transformed with PML104-9RNA1 plasmid and HDR1 template to edit ADE2 gene. Then answer the following questions.. 1.In yeast, what is the template from which the GRNA is transcribed? 2. The HDR fragment must enter the homology directed repair. 3. The Cas9 gene is transcribed from transported from 4. what happens after translation of Cas9? of yeast cell to be used for as template, transcript is of the celi, and then protein is translated. to

During a CRISPR-Cas9 exp., yeast are transformed with PML104-9RNA1 plasmid and HDR1 template to edit ADE2 gene. Then answer the following questions.. 1.In yeast, what is the template from which the GRNA is transcribed? 2. The HDR fragment must enter the homology directed repair. 3. The Cas9 gene is transcribed from transported from 4. what happens after translation of Cas9? of yeast cell to be used for as template, transcript is of the celi, and then protein is translated. to

Human Heredity: Principles and Issues (MindTap Course List)

11th Edition

ISBN:9781305251052

Author:Michael Cummings

Publisher:Michael Cummings

Chapter13: An Introduction To Genetic Technology

Section: Chapter Questions

Problem 20QP: Analyzing Cloned Sequences A base change (A to T) is the mutational event that created the mutant...

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Explain the process of how X-gal screening works with pUC19, you may build a model with boxes and arrows. 2. You are utilizing BamHI (GGATCC) restriction site and HindIII (AAGCTT) restriction site. Within pUC19, BamHI is at position 263, while HindIII is at position 233. (Hint: position is like coordinate on a map). If you manage to insert GTF2H5 in pUC19 vector, what are the sizes of fragments if you digest the pUC19-GTF2H5 (this is after insertion) with the following restriction enzyme combination after gel electrophoresis: BamHI and HindIII There is an NdeI site right in the middle of the GTF2H5 that has been inserted in pUC19, what would be the fragment sizes, if you digest with BamHI and NdeI. HindIII and NdeI BamHI, HindIII, and NdeI. 3. Design an experiment to confirm the presence of insert GTF2H5 in pUC19 vector using the following method (besides restriction analysis above), assuming that you know the sequence of GTF2H5: Southern Blot Polymerase Chain Reaction
Bacteriophage lambda is as one of the routinely used molecular cloning vectors, which alsoserved as a model system for the study of bacteriophage morphogenesis, DNA replication, andgene regulation.a) With the aid of a diagram, generally narrate how a foreign gene can be inserted into alambda insertion vector and subsequently infect an Escherichia coli cell.b) You are cloning a 7.5 kb gene into a lambda gt10 vector, utilizing a restriction site whichspecifically present in the vector. State the restriction site that you can use for this purposeand suggest a screening procedure to indicate successful integration of the gene into thehost genome.
Prior to the publication of the article from which this assay comes from (picture), the Cas9 endonuclease was known to be involved in CRISPR RNA (crRNA) maturation, but the mechanism by which Cas9 participates in targeted DNA disruption was unknown. To test whether and how Cas9 might be able to target DNA cleavage, researchers developed an in vitro assay. One of the first steps was to purify the Cas9 protein from Streptococcus pyogenes and several orthologs. The description of the figure states: SDS-PAGE analysis of purified Cas9 orthologs. Cas9 orthologs were purified as described in Supplementary Materials and Methods. 2.5 ug of each purified Cas9 were analyzed on a 4-20% gradient polyacrylamide gel and stained with SimplyBlue Safe Stain. Answer the following questions based on this information and the figure. a. What is the experimental/research question and state a hypothesis related to this question. b. What is the approach of the figure and provide a description of techniques…
One problem that researchers sometimes encounterwhen editing genomes with CRISPR/Cas9 is that oneor more loci other than the intended target can berecognized by Cas9/sgRNA and cleaved. Part of thereason is that single base pair mismatches betweenthe target site and the sgRNA in the 5′-most half of the 20 bp DNA/RNA hybrid do not prevent Cas9cleavage of the target site. How could scientists usebioinformatics to avoid such off-target effects?
If an enhancer is placed on one plasmid and its corresponding promoter is placed on a second plasmid that is catenated (linked) with the first, initiation is almost as effi cient as when the enhancer and promoter are on the same plasmid. However, initiation does not occur when the two plasmids are unlinked. Explain.
Analyzing Cloned Sequences A base change (A to T) is the mutational event that created the mutant sickle cell anemia allele of beta globin. This mutation destroys an MstII restriction site normally present in the beta globin gene. This difference between the normal allele and the mutant allele can be detected with Southern blotting. Using a labeled beta globin gene as a probe, what differences would you expect to see for a Southern blot of the normal beta globin gene and the mutant sickle cell gene?
Describe one way in which DNA double stranded break repair that occurs because of X-ray damage differs from that caused by Crispr/Cas9 and one way in which they are similar.
The Cas9 enzyme requires a short sequence called a PAM (Protospacer Adjacent Motif) for cleaving the target DNA.This PAM sequence is not found in the CRISPR locus of Streptococcus pyogenes but it is present in viral genomes and we need to make sure it is present adjacent to where the guide RNA binds the yeast DNA. What does this suggest about the function of the PAM?
A plasmid that is both ampicillin and tetracyclineresistant is cleaved with PstI, which cleaves within theampicillin resistance gene. The cut plasmid is ligated withPstI-digested Drosophila DNA to prepare a genomic library,and the mixture is used to transform E. coli K12. Question: How can you explain the presence of colonies thatare resistant to both antibiotics?
You ran the agarose gel but forgot to include the lactose-induced 23S reaction (template - cDNA). Without repeating the gel, what conclusions could you make about gst::egfp mRNA levels after lactose and IPTG induction? What would remain inconclusive? What if the sample you forgot to include was the negative control of lactose-induced 23S reaction (template - RNA)?
For the sake of simplicity, Fig. 10.4 omitted one stepof cDNA library construction. The figure impliedthat the last step of the process is the ligation ofblunt-ended cDNAs into plasmid cloning vectors.Although such ligation reactions can occur, in realitythey are highly inefficient. Instead, scientists convertblunt-ended cDNA molecules into sticky-ended molecules using adapters, and then they ligate thecDNAs into vectors with compatible sticky ends.Adapters are short, partly double-stranded DNAmolecules made by hybridization of two singlestranded oligonucleotides made in a DNA synthesizer.Suppose that the following two oligonucleotides weresynthesized and then mixed together at high concentration and at a temperature that promotes hybridization of complementary DNA sequences:5′ CCCCCG 3′5′ AATTCGGGGG 3′a. Draw the hybridized DNA molecules. These arethe adapters.b. Suppose you added the adapters and ligase enzymeto blunt-ended cDNAs at a very high molar ratio ofadapters to cDNAs, so…
Nonhomologous end-joining (NHEJ) of a doublestrand break almost always results in perfect resealingof the DNA lesion, without the loss or gain of nucleotide pairs. Yet CRISPR/Cas9, which produces doublestrand breaks, is a highly efficient method of makingsmall deletions or insertions at the targeted site. Howcan you resolve this apparent contradiction?