Genetic Analysis: An Integrated Approach (2nd Edition)
2nd Edition
ISBN: 9780321948908
Author: Mark F. Sanders, John L. Bowman
Publisher: PEARSON
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Chapter 17, Problem 13P
It is often desirable to insert cDNAs into a cloning vector in such a way that all the cDNA clones will have same orientation with respect to the sequences of the plasmid. This is referred to as directional cloning. Outline how you would directionally clone a cDNA library in the plasmid vector
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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.
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One would be correct in assuming that the restriction sites present in the multiplecloning site of the pUC18 plasmid will
Chapter 17 Solutions
Genetic Analysis: An Integrated Approach (2nd Edition)
Ch. 17 - 15.1 What purpose do the bla and lacZ genes serve...Ch. 17 - The human genome is 3109 bp in length. How many...Ch. 17 - 15.3 Ligase catalyzes a reaction between the...Ch. 17 - You have constructed four different libraries: a...Ch. 17 - Using the genomic libraries in Problem 4, you wish...Ch. 17 - The human genome is 3109bp. You wish to design a...Ch. 17 - 15.7 Using animal models of human diseases can...Ch. 17 - 15.8 Compare methods for constructing homologous...Ch. 17 - 15.9 Chimeric genefusion products can be used for...Ch. 17 - 15.10 Why are diseases of the blood simpler...
Ch. 17 - Injection of double-stranded RNA can lead to gene...Ch. 17 - Compare and contrast methods for making transgenic...Ch. 17 - 15.13 It is often desirable to insert cDNAs into a...Ch. 17 - 15.14 A major advance in the s was the development...Ch. 17 - 15.15 The bacteriophage lambda genome can exist in...Ch. 17 - 15.16 The restriction enzymes Xho and Sal cut...Ch. 17 - 15.17 The bacteriophage has a single-stranded DNA...Ch. 17 - 15.20 You have identified a cDNA clone that...Ch. 17 - You have isolated a genomic clone with an EcoR I...Ch. 17 - 15.18 To further analyze the CRABS CLAW gene (see...Ch. 17 - 15.21 You have isolated another cDNA clone of the...Ch. 17 - 15.22 You have identified five genes in S....Ch. 17 - You have generated three transgenic lines of maize...Ch. 17 - 15.24 Bacterial Pseudomonas species often possess...Ch. 17 - 15.25 Two complaints about some transgenic plants...Ch. 17 - 15.26 In Drosophila, lossoffunction Ultrabithorax...Ch. 17 - Prob. 27PCh. 17 - The highlighted sequence shown below is the one...Ch. 17 - The RAS gene encodes a signaling protein that...Ch. 17 - Vitamin E is the name for a set of chemically...Ch. 17 - 15.31 You have cloned a gene for an enzyme that...
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- Below is a map of the E. coli cloning vector after the insertion of pka-1. Ori denotes the origin of replication; amp denotes the ampicillin resistance gene. HindIII, BamHI, SalI, and EcoRI designate restriction enzyme sites. There are no other restriction enzyme sites found on this vector. The numerals denote the number of base pairs between different locations on the plasmid. For instance, there are 400 base pairs between the HindIII and BamHI site, and there are 3000 base pairs in the entire cloning vector (following the integration of pka-1). With the use of well-illustrated diagrams, reconstruct the entire cloning process by explaining different stages of the cloning process that involves the following: Isolation of target DNA fragments (often referred to as inserts) Ligation of inserts into the plasmid, creating recombinant molecules Transformation of recombinant plasmids into bacteria or other suitable host for propagation Screening/selection of hosts containing the intended…arrow_forwardDescribe the process of cloning a DNA fragment into theBamHI and PstI sites of the vector pUC18. How would youscreen for clones that contain an insert? and explain the process(steps) by drawingarrow_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
- Your cloning vector has restriction recognition sites for two restriction endonucleases, EcorI and BamHI. However, the DNA to be manipulated does not have recognition sites for these two restriction endonucleases. How would you construct a recombinant DNA for the given DNA?arrow_forwardThe total size of the plasmid is 2686 bp. There is a PstI recognition site at position 439, HindIII recognition site at position 447, and ScaI recognition site at 2179. If restriction enzymes ScaI and HindIII are used to cut this plasmid, what would be produced? a. 2 fragments: 954bp and 1732bp b. none of the choices apply c. 1 linear fragment of 2686bp d. 3 fragments: 447bp, 507bp and 1732bparrow_forwardWhat advantages do cDNA libraries provide over genomic DNA libraries? Describe cloning applications where the use of a genomic library is necessary to provide information that a cDNA library cannot.arrow_forward
- As a part of undergrad lab project, you have to clone Your Favorite Gene into plasmid cloning vector (4740 bp) pUC18. Restriction maps for both YFG and cloning vector are provided. The plasmid has a bacterial origin of replication (ori), an ampicillin resistance gene (AmpR) that degrades ampicillin antibiotic. It also has LacZ gene that encodes the β-gal enzyme, which converts white X-gal substrate into a blue product. pLac is a bacterial promoter. Restriction Enzyme Recognition Site : A slash (/) represents the cut site for each restriction enzyme. Enzyme 1 5’- G/TCGA C -3’ 3’- C AGCT/G-5’ Enzyme 2 5’-C/TCGA G-3’ 3’G AGCT/ C5’ Enzyme 3 5’- GTC / GAC- 3’ 3’- CAG / CTG- 5’ Enzyme 4 5’-C AATT /G-3’ 3’-G/ TTAA C-5’ Enzyme 5 5’C AATT/G-3’ 3’G/TTAA C-5’ Enzyme 6 5’G/GATC C-3’ 3’C CTAG/ G-5’ Enzyme 7 5’G/GATC C-3’ 3’C CTAG/ G-5’ 1a) The table below shows restriction enzyme pairs used by various teams to digest the YFG and clone it in the…arrow_forwardA molecular geneticist hopes to find a gene in human liver cells that codes for an important blood-clotting protein. He knows that the nucleotide sequence of a small part of the gene is CTCGACTCACA. Briefly explain how to obtain the desired gene. Briefly describe how to clone the desired gene into a cloning plasmid.arrow_forwardAssume that a circular plasmid is 3200 base pairs in length and has restriction sites for HindIII restriction enzyme at the following locations: 400, 700, 1400, 2600. Give the expected sizes of the restriction fragments following complete digestion.arrow_forward
- You are attempting to clone a 3 kb gene from the bacterial sp Microbacterium foliarum into the EcoRI site of the 6.0 kb plasmid shown. If you restrict the plasmid with EcoR1, how many bands will you obtain on agarose gel electrophoresis? Compare it with a plasmid in which no gene has been inserted. Draw a representative agarose gel showing the bands, and the direction of current flow as well point out the positive and negative electrodesarrow_forwardKnowing that you are using HindIII and EcoRI to cut your plasmids, and that those two enzymes cut within the MCS, use the map of pUC19 provided below to compute: What will be the sizes of the 2 restriction fragments if NO insert is present in pUC19? What will be the sizes of the 2 restriction fragments if the approximately 317 bp RT-PCR product (insert from WT satC dimer) was ligated successfully into the SmaI site? What will be the sizes of the 2 restriction fragments if TWO approximately 317 bp RT-PCR products (2 ligated inserts from WT satC dimer) were ligated into the SmaI site?arrow_forwardIf you use the pUC18 vector to clone in the MCS region, predict the following: a) Do bacteria that are blue in color have a cloned insert? b)Do bacteria that are white in color have a cloned insert? c) If you were to grow these cells on Chloramphenicol (an antibiotic), would the bacteria with the pUC plasmid grow? Why or Why not?arrow_forward
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