Genetic Analysis: An Integrated Approach (3rd Edition)
3rd Edition
ISBN: 9780134605173
Author: Mark F. Sanders, John L. Bowman
Publisher: PEARSON
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Chapter 15, Problem 3P
Ligase catalyzes a reaction between the
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Ligation is an essential step in the cloning process. It refers to the joining of the gene of
interest to the vector using DNA ligase.
(i)
Determine FOUR (4) control groups which are important to determine the success of
this step and also to troubleshoot if any problem should occur.
In an experiment, 100 ng vector was added into ligation reaction with 50 ng of insert
(500 bp). The desired vector: insert ratio of 1: 3 was used. Determine the size of the
(ii)
vector.
The human hexokinase enzyme has the same function as the bacterial hexokinase enzyme but is somewhat different in its amino acid sequence. You have obtained a mutant bacterial strain in which the gene for hexokinase is missing. If you introduce into your mutant strain a DNA plasmid engineered to contain the DNA coding sequence of the human hexokinase gene, what must you also include?
a)The human hexokinase promoter
b)The bacterial hexokinase promoter
c)Both the human and bacterial promoters
d)You cannot engineer a bacteria to produce a human enzyme
The plasmid cloning vector pBR322 is cleaved with the restriction endonuclease PstI. An isolated DNA fragment from a eukaryotic genome (also produced by PstI cleavage) is added to the prepared vector and ligated. The mixture of ligated DNAs is then used to transform bacteria, and plasmid-containing bacteria are selected by growth in the presence of tetracycline.
The cloned DNA fragment is 1,000 bp long and has an EcoRI site 250 bp from one end. Three different recombinant plasmids are cleaved with EcoRI and analyzed by gel electrophoresis, giving the patterns shown below.
What does each pattern say about the cloned DNA?
Note: pBR322, the PstI and EcoRI restriction sites are about 750 bp apart. The entire plasmid with no cloned insert is 4,361 bp. Size markers in lane 4 have the number of nucleotides noted.
Chapter 15 Solutions
Genetic Analysis: An Integrated Approach (3rd Edition)
Ch. 15 - 15.1 What purpose do the bla and lacZ genes serve...Ch. 15 - The human genome is 3109 bp in length. How many...Ch. 15 - 15.3 Ligase catalyzes a reaction between the...Ch. 15 - You have constructed four different libraries: a...Ch. 15 - Using the genomic libraries in Problem 4, you wish...Ch. 15 - The human genome is 3109bp. You wish to design a...Ch. 15 - 15.7 Using animal models of human diseases can...Ch. 15 - 15.8 Compare methods for constructing homologous...Ch. 15 - 15.9 Chimeric genefusion products can be used for...Ch. 15 - 15.10 Why are diseases of the blood simpler...
Ch. 15 - Injection of double-stranded RNA can lead to gene...Ch. 15 - Compare and contrast methods for making transgenic...Ch. 15 - 15.13 It is often desirable to insert cDNAs into a...Ch. 15 - 15.14 A major advance in the s was the development...Ch. 15 - 15.15 The bacteriophage lambda genome can exist in...Ch. 15 - 15.16 The restriction enzymes Xho and Sal cut...Ch. 15 - 15.17 The bacteriophage has a single-stranded DNA...Ch. 15 - 15.18 To further analyze the CRABS CLAW gene (see...Ch. 15 - You have isolated a genomic clone with an EcoR I...Ch. 15 - 15.20 You have identified a cDNA clone that...Ch. 15 - 15.21 You have isolated another cDNA clone of the...Ch. 15 - 15.22 You have identified five genes in S....Ch. 15 - You have generated three transgenic lines of maize...Ch. 15 - 15.24 Bacterial Pseudomonas species often possess...Ch. 15 - 15.25 Two complaints about some transgenic plants...Ch. 15 - 15.26 In Drosophila, lossoffunction Ultrabithorax...Ch. 15 - Prob. 27PCh. 15 - The highlighted sequence shown below is the one...Ch. 15 - Vitamin E is the name for a set of chemically...Ch. 15 - The RAS gene encodes a signaling protein that...Ch. 15 - 15.31 You have cloned a gene for an enzyme that...Ch. 15 - 15.32 About of occurrences of nonautoimmune type...Ch. 15 - Describe how having the Cas 9 gene at a genomic...Ch. 15 - 15.34 Would a gene drive system spread rapidly...
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- With the use of well-illustrated diagrams, reconstruct the entire cloning process by explaining different stages of the cloning process that involves the following: d. 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_forwardFigure 2 illustrates the important elements of a cosmid.a) Briefly describe the importance of origin of replication in a cosmid and where are thecos sites derived from.b) The cloning capacity of a cosmid is up to 44 kilo base pairs. State TWO classes of DNAcloning vectors that have higher cloning capacity.arrow_forwardDuring your experiment you analysed only a few of the recombinant clones for the presence of the highly repeated Aluelements. If you wanted to screen for a single-copy gene, you would need to screen a much larger genomic library. Assuming, that you already know the amino acid sequence of unicorn (a species with a similar physiology to humans) insulin, how would you construct a probe which would enable you to use nucleic acid hybridisation to screen a unicorn genomic DNA library for the insulin gene? Hint: you have access to any molecular biology reagents and equipment you might need, such as vectors, enzymes, and DNA sequencers.arrow_forward
- Name any two cloning vectors. Describe the features required to facilitate cloning into a vector.arrow_forwardDescribe the cloning strategies that are mediated by homopolymers tailing, linkers and adaptors. How can adaptors be employed for the creation of a novel restriction enzyme site for the introduction of the insert DNA?arrow_forwarda) Briefly describe the importance of origin of replication in a cosmid and where are the cos sites derived from.b) The cloning capacity of a cosmid is up to 44 kilo base pairs. State TWO classes of DNAcloning vectors that have higher cloning capacity.arrow_forward
- How is telomerase related to aging? How can telomerase-related aging beaddressed therapeutically? What is a potential danger of telomerase activation?arrow_forwardin the cloning vector, what would be the plausible impacts if a mutation at the Ori site renders it non- functional?arrow_forwardDraw roughly the comparative electrophoretic mobilities of close circular DNA, open circular DNA and super coiled DNA, all having the same molecular weight. Describe the process of cloning a DNA fragment into the EcoR1 and AluI sites of the vector pUC18. How would you screen for clones that contain an insert?arrow_forward
- When performing cloning experiments, it is not always necessary to treat sources of DNA with the same restriction enzyme. For example, DNA treated with EcoRI can be combined with DNA from a treatment using FunII. Explain why this is possible.arrow_forwardWhat is linker scanning mutagenesis?arrow_forwardA) Outline the experimental procedure for cloning a eukaryotic gene and expressing it in E. coli. Focus on the essential steps starting with eukaryotic gene amplification to transformation of E. coli cells B) Explain how insertional inactivation can help you identify the colonies that carry the plasmid with your eukaryotic gene of interest C) Plasmids containing antibiotic resistance genes are widely used in gene cloning and other molecular biology techniques. What would happen if the eukaryotic gene was inserted into an antibiotic resistance gene on the plasmid?arrow_forward
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