Biology
5th Edition
ISBN: 9781260487947
Author: BROOKER
Publisher: MCG
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Chapter 21, Problem 3TY
Summary Introduction
Introduction: Genome is defined as the complete genetic material that is present in an organism. It consists of the coding as well as the non-coding parts of DNA. The study of the genome utilizes an important technique by which multiple and exact copies of a gene can be produced. This technique is termed as gene cloning. The gene cloning technique contains an important agent known as a recombinant vector. This vector is used to transfer the “gene of interest” to the host organism.
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You are trying to clone a gene, You have successfully isolated it from the genomic DNA of an organism using the Hindill restriction enzyme. You then take a
plasmid with a single EcoRI restriction site and cleave it with EcoRI. You combine these two fragments and treat them with DNA ligase. Answer the two questions
below.
a. Does the cloning reaction succeed as described? If so, what is the product obtained?
b. Explain your answer above,
After transformation you were asked to grow bacterial cells transformed with plasmid on a plate that had X-gal and ampicillin. X-Gal is often used as in indicator dye, which turns blue when metabolized by B-galactosidase protein and used to test if cloning experiments have worked. [Note look at the vector diagrams carefully]
Briefly explain how you would find the bacterial cells that are transformed with the plasmid with the YFG inserted.
A. A plasmid is shown with the locations of various restriction enzyme sites labeled. If you cut the plasmid with Xhol and Xbal, which lane of the agarose gel represents the DNA fragments you would expect from the digestion?
B. If you now decide to cut the plasmid with EcoRI, how many fragments will be produced and what will their sizes be?
C. When running DNA samples on agarose gel, an electric field is applied. Towards which electrode will the DNA migrate and why?
Chapter 21 Solutions
Biology
Ch. 21.1 - Prob. 1CSCh. 21.1 - Prob. 1CCCh. 21.1 - Prob. 2CCCh. 21.1 - Prob. 3CCCh. 21.2 - Prob. 1CCCh. 21.2 - Prob. 2CCCh. 21.2 - Prob. 3CCCh. 21.3 - Prob. 1EQCh. 21.3 - Prob. 2EQCh. 21.3 - Prob. 3EQ
Ch. 21.4 - Prob. 1CCCh. 21.4 - Prob. 1CSCh. 21.5 - Prob. 1CSCh. 21.5 - Repetitive Sequences and Transposable Elements...Ch. 21 - Prob. 1TYCh. 21 - DNA ligase is needed in a cloning experiment a. to...Ch. 21 - Prob. 3TYCh. 21 - Why is Taq polymerase used in PCR rather than...Ch. 21 - Lets suppose you want to clone a gene that has...Ch. 21 - In the CRISPR-Cas technology for editing genes,...Ch. 21 - Prob. 7TYCh. 21 - The enzyme that helps short segments of DNA move...Ch. 21 - Prob. 9TYCh. 21 - Which of the following was not a goal of the Human...Ch. 21 - Prob. 1CQCh. 21 - Briefly describe whether or not each of the...Ch. 21 - Prob. 3CQCh. 21 - Identify and discuss three important advances that...Ch. 21 - Prob. 2COQ
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- You are performing a PCR reaction but unbeknownst to you, there is a significant pool of dUTP in the nucleotide mix (along with dCTP, dTTP, dATP, and dGTP). How might this affect your PCR product? a. If the PCR product was ligated into a plasmid and put into a cell, a totally different mRNA would be made from the insert compared to an insert made with T's. b. If the pool of dTTP ran out before the pool of dUTP, DNA replication could no longer occur. c. During the reaction, uracils incorporated into the product would cause the PCR product to degrade as it is being made. d. Uracil would be incorporated into the product and would lessen the affinity of any DNA binding proteins that might bind to the product in subsequent experiments. e. Nothing would happen since polymerases can't use dUTP to make DNA.arrow_forwardIn Biotechnology, gene cloning is a very important technique. A vector is normally required to perform this process. The vector commonly used to transform a bacterial host cell is the plasmid. (i) State the THREE (3) important regions of the plasmid. Elaborate your answer.arrow_forwardWith the use of well-illustrated diagrams, reconstruct the entire cloning process by explaining different stages of the cloning process that involves the following:a. Isolation of target DNA fragments (often referred to as inserts)b. Ligation of inserts into the plasmid, creating recombinant molecules c. Transformation of recombinant plasmids into bacteria or other suitable host for propagationd. Screening/selection of hosts containing the intended recombinant plasmid. For this stage(d), discuss the importance of a second marker that can be used for screening of genomic DNA for colonies containing the pka-1 under the principle of insertional inactivation. This should be properly explained using all the attributes of the plasmid described above.arrow_forward
- You have two samples you have to sequence: one is a cloned plasmid that you want to verify the sequence of, and another is the cDNA library of the transcriptome of a cell. Which method would you use for each sample and why?arrow_forwarda. What is the purpose of molecular cloning?b. What purpose do selectable markers serve in vectors?c. What is the purpose of the origin of replication in aplasmid vector?d. Why do cloning vectors have polylinkers?arrow_forwardYou are studying a new plasmid, and you digest the plasmid with three restriction enzymes: Eco RI (E), HindlII (H), and Xbal (X). You digest the plasmid DNA with each of the following combinations of enzymes and observe the results on an agarose gel. You are provided a partial plasmid map as shown below to the right. E H E+H E+X H+X Kb +4.3 +2.8 +2.5 - +2.0 -- -1.8 -1.5 12 +1.0 +0.8 +0.5 - a. What is the size of this plasmid in base pairs? b. What is the distance in base pairs between E1 and H? c. What is the distance in base pairs between E1 and X? d. What is the distance in base pairs between E2 and H? e. What is the distance in base pairs between E2 and X?arrow_forward
- On the gel shown below are four DNA samples. Samples A to C are taken from tissues of landslide victims that are being identified, while sample D came from a hair sample brought by a mother looking for the remains of her son. (see img) i. If similar band patterns in a gel are created using the same restriction enzyme, what does that tell you about the DNA sequence of the samples? ii. In sample C, only two fragments were created. How many restriction sites (regions where enzymes cut) are present in sample C?arrow_forwardScientists modified the tumor-inducing (Ti) plasmid, a naturally occurring plant plasmid found in Agrobacterium tumefaciens, to create a tool used to introduce any gene of interest into plant cells. They created a binary system because a single Ti plasmid is too large to be easily manipulated. One part of the system is a disarmed plasmid, and the second part is a transformation vector. The following sentences describe the function of key DNA elements in the system. Virulence region Genes for conjugative transfer Disarmed Ti plasmid (T-region removed) ori Kan selectable marker plant selectable marker constitutive promoter T-region conjugative transfer virulence Kan (bacterial selectable marker) Amp selectable marker Plant selectable marker (e.g., herbicide resistance) Constitutive promoter T-DNA left border Place the terms in the appropriate blanks to complete the sentences. Not all terms will be used. 3' transcriptional terminator MCS (inserted gene of interest) T-region Transformation…arrow_forwardYou are researching a gene that is 500bp long called rad. You know the gene is between two EcoRI restriction sites. You treat genomic DNA with EcoR1 and you treat your pUC-19 plasmid with EcoRI and ligate the genomic fragments into your vector. You use blue/while selection and pick four colonies and you perform an insert check (labeled A-D below). After growing cultures, you isolate the plasmids and treat them with EcoRI. You then run them on a gel. Which colonies are white? ABC D 0 O a. A, B and C b. B and C O c. B and C O d. All are white O e. A and C Ladder 2kb 1kb 200bparrow_forward
- Meselson and Stahl grew bacteria in medium containing heavy nitrogen. After many celldivisions, the bacteria were removed, washed and allowed to continue growth in mediumcontaining normal (light) nitrogen.a. In this experiment, what part of the DNA gets labeled with heavy nitrogen?arrow_forwardYou are studying a new plasmid, and you digest the plasmid with three restriction enzymes: Eco RI (E), Hindlll (H), and Xbal (X). You digest the plasmid DNA with each of the following combinations of enzymes and observe the results on an agarose gel. You are provided a partial plasmid map as shown below to the right. +18 a. What is the size of this plasmid in base pairs? b. What is the distance in base pairs between E1 and H? c. What is the distance in base pairs between E1 and X? d. What is the distance in base pairs between E2 and H? e. What is the distance in base pairs between E2 and X?arrow_forwardDescribe the possible outcome of a PCR experiment in which (a) there is a single-stranded break in the target DNA sequence, which is present in only one copy in the starting sample, and (b) there is a doublestranded break in the target DNA sequence, which is present in only one copy in the starting sample.arrow_forward
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