Concept explainers
For the sake of simplicity, Fig. 10.4 omitted one step of cDNA library construction. The figure implied that the last step of the process is the ligation of blunt-ended cDNAs into plasmid cloning vectors. Although such ligation reactions can occur, in reality they are highly inefficient. Instead, scientists convert blunt-ended cDNA molecules into sticky-ended molecules using adapters, and then they ligate the cDNAs into vectors with compatible sticky ends.
Adapters are short, partly double-stranded DNA molecules made by hybridization of
Two single-stranded oligonucleotides made in a DNA synthesizer. Suppose that the following two oligonucleotides were synthesized and then mixed together at high concentration and at a temperature that promotes hybridization of complementary DNA sequences:
a. | Draw the hybridized DNA molecules. These are the adapters. |
b. | Suppose you added the adapters and ligase enzyme to blunt-ended cDNAs at a very high molar ratio of adapters to cDNAs, so that each cDNA molecule is ligated to one adapter at each of its ends. Draw a picture of a resulting cDNA molecule. |
c. | The particular adapters discussed in this problem allow the cDNAs to be ligated efficiently into a vector treated with a commonly used restriction enzyme listed in Table 9.1. Name this restriction enzyme. |
Trending nowThis is a popular solution!
Chapter 10 Solutions
Genetics: From Genes to Genomes
- You are using Drosophila as a model system to investigate eye development. You do positional cloning to isolate a mutant gene called eyeless (eyl), which causes flies to develop with reduced sized eyes when homozygous. Using several DNA markers from the candidate interval as probes for colony hybridization to a CDNA library, you obtain a 10 Kb cDNA insert cloned into the unique EcoRI site of a 3.5 Kb plasmid vector. a. Briefly describe how would you use the Drosophila eyl cDNA clone to identify a clone from a mouse genomic DNA library that is homologous to the Drosophila eyl gene? b. You isolate a clone of mouse genomic DNA that appears to be homologous to the Drosophila eyl gene. After DNA sequencing of a small piece of the mouse genomic clone, you use the DNA sequence to search the mouse genome database. You find that there is a SNP locus (n1234) that does NOT cause an RFLP in this genomic clone. You have identified several breeding lines of mice, each having been bred for several…arrow_forwardYou receive purified mRNA that codes for the spike protein of SARS-CoV-2 as well as plasmid pET32(a+). Explain i. how you will prepare SARS-CoV-2 spike cDNA and name the enzymes that you will need ii. how you will clone the cDNA into pET32(a+) and select the recombinant plasmidiii. how you will then produce a recombinant SARS-CoV-2 spike and purify itarrow_forwardTo create a cDNA library, cDNA can be inserted into vectors and cloned. In the analysis of cDNA clones, it is often difficult to find clones that are full length—that is, many clones are shorter than the mature mRNA molecules from which they are derived. Why is this so?arrow_forward
- Besides PUC vectors, there are still other plasmid vectors in the market such as pBR327 and pZERO®-1. Some vectors are designed specifically for TA cloning while some are not. What is TA cloning? Explain in detail. Give two (2) examples of plasmid vectors that can undergo TA cloning. (i) (ii)arrow_forwardBesides the pUC series, there are other plasmid vectors in the market such as pBR327 and pGEM-T. Some vectors are designed specifically for TA cloning while some are not. What is TA cloning? Explain in detail.arrow_forwardYou have isolated a cDNA clone encoding a protein of interest in a higher eukaryote. This cDNA clone is not cleaved by restriction endonuclease EcoRI. When this cDNA is used as a radioactive probe for blot hybridization analysis of EcoRI-digested genomic DNA, three radioactive bands are seen on the resulting Southern blot. Does this result indicate that the genome of the eukaryote in question contains three copies of the gene encoding the protein of interest? Explain.arrow_forward
- You are trying to study the effects of Drug A on the expression of Gene X in a tumor sample (lane 3). You perform an RT- PCR reaction using a set of prímers to amplify a region of Gene X that is about 600bp in length. The forward and reverse primers are on 2 separate exons. You run your target CDNA fragment on an agarose gel (lane 3). Lanes 1 and 2 are hypothetical control lanes to test for 9DNA contamination. 1 3 1000 bp 600 bp Based on the gel image, which of the following statements is correct? 1. The samples loaded in lanes 1 and 2 were not reverse transcribed prior to PCR. 2. Results from lane 2 suggest that 9DNA contamination is present. 3. The upper band in lane 2 represents 9DNA. 4. The fact that lane 1 is blank is indicative that Drug A doesn't lead to expression of Gene X. O A. 1, 2, and 3 O B. 1 and 3 O C. 2 and 4 O D. 4 only O E. All of 1, 2, 3 and 4 are correctarrow_forwardWhy are restriction endonucleases considered a bacteria’s “innate immune system”? Why is CRISPR-Cas9 considered a bacteria’s “adaptive immune system”? What does CRISPR stand for? What is the difference between crRNA and tracrRNA? Why are both needed for Cas9 to function? What does PAM stand for? Where is it found? What is the difference between Non-homologous End Joining (NHEJ) and Homology Directed Repair (HDR)? What is the Guide RNA (gRNA) a chimera of? Why use a gRNA? What new things are researchers doing with CRISPR-Cas9? Reflecting on what you now know about CRISPR-Cas9, what are your thoughts on it’s use in humans and other organisms? What should we be allowed to do? Not do? Are viruses living? Why or why not? What does obligate intracellular parasite mean? What does every virus have? What is the difference between capsomers and…arrow_forwardA certain cDNA of size 2 kb hybridized to eight genomicfragments of total size 30 kb and contained two shortESTs. The ESTs were also found in two of the genomicfragments each of size 2 kb. Sketch a possible explanation for these resultsarrow_forward
- The plasmids from the pUC series are created in the University of California. They carry a lacz gene that plays a significant role in the screening process after transformation. (i) Name the screening process utilizing the lacZ gene. (ii) Elaborate how this gene plays a crucial role in the screening step as mentioned above.arrow_forwardYou want to express the protein of a newly discovered human gene using E. coli, so you clone the human cDNA for the gene with an additional C-terminal purification tag into a plasmid. You then transform that plasmid into E. coli, grow up 1L of cells while inducing the promoter on the plasmid controlling the production of the protein from the gene. You then want to purify the protein using the C-terminal purification tag. What would be the best way of monitoring the purification process? Why did you choose this method? Please keep brief - 2 sentences/dot points max.arrow_forwardYou were provided with the genetic sequence for one of these genes and asked to design a cloning strategy to generate a dsRNA expression construct to knock down that gene by RNAi . 1. What two restriction sites are you going to use to clone your PCR product into the pL4440 plasmid? What are their DNA sequence? 2. How would you go about cloning this amplified DNA into pL4440? Using your knowledge of cloning list 5 important aspects of the method.arrow_forward
- Human Anatomy & Physiology (11th Edition)BiologyISBN:9780134580999Author:Elaine N. Marieb, Katja N. HoehnPublisher:PEARSONBiology 2eBiologyISBN:9781947172517Author:Matthew Douglas, Jung Choi, Mary Ann ClarkPublisher:OpenStaxAnatomy & PhysiologyBiologyISBN:9781259398629Author:McKinley, Michael P., O'loughlin, Valerie Dean, Bidle, Theresa StouterPublisher:Mcgraw Hill Education,
- Molecular Biology of the Cell (Sixth Edition)BiologyISBN:9780815344322Author:Bruce Alberts, Alexander D. Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter WalterPublisher:W. W. Norton & CompanyLaboratory Manual For Human Anatomy & PhysiologyBiologyISBN:9781260159363Author:Martin, Terry R., Prentice-craver, CynthiaPublisher:McGraw-Hill Publishing Co.Inquiry Into Life (16th Edition)BiologyISBN:9781260231700Author:Sylvia S. Mader, Michael WindelspechtPublisher:McGraw Hill Education