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 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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As this is a non-directional cloning,
recombinant plasmids can contain
an insert ligated into the vector in two
different orientations.
Provide 2 diagrams to illustrate the
2 potential recombinant plasmids,
with the inserts ligated in opposite
orientations. Include all RE sites
and distances between sites on the
diagram.
Explain the THREE (3) important regions of this plasmid that enable it to work efficiently as
a cloning vector?
Besides 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.
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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- Describe 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_forwardA shuttle vector is a vector (usually a plasmid) constructed so that it can propagate in two different host species. One of the most common types of shuttle vectors is the yeast shuttle vector. Examples of such vectors derived from yeast are Yeast Episomal Plasmid (YEP), Yeast Integrating Plasmid (YIP) and Yeast Replicating Plasmid (YRP). Why is YEP preferred over YIP and YRP? Give your thoughts on this.arrow_forwardWhen cloning a foreign DNA fragment into a plasmid, it is often useful to insert the fragment at a site that interrupts a selectable marker(such as the tetracycline-resistance gene of pBR322). The loss of function of the interrupted gene can be used to identify clones containing recombinant plasmids with foreign DNA. With a yeast artificial chromosome (YAC) vector, it is not necessary to do this; the researcher can still distinguish vectors that incorporate large foreign DNA fragments from those that do not. How are these recombinant vectors identified?arrow_forward
- 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.arrow_forwardIn biotechnology, gene cloning is a crucial technique used in many genetic modification experiments. A vector is normally required to perform this process. The vector commonly used to transform a bacterial host cell is known as a plasmid. Below is the general plasmid map of pBR322. Elaborate the functions of ori, Ap and TeR present in pBR322. Bul 3759 Prul 3733 PM 3507 BDI 3602 Asel 3537 Bal 3433 Finci 3905 Scal 3844 3787 Bad 3420 Ahdi 3361 Acul 3000 AlwNI 2884 Sspl 4168 Earl 4155 Acul 4048 Xust 3961 Bell 2777 Bc1 2682 ori Aall-Zeal 4284 BeiVI 4209 B 4205 Del 2575 Pail-Att 2473 BAB 2404 EcoRI 4350 Earl 2351 BspQI-Sapl 2350 Ndel 2295 BAP 2291 178-Accl 2244 Cial-Ispit 23 Hindill 29 pBR322 4,361 bp Ball 2225 TthJ11- Part 2217 rop EcoRY 185 Bmtl-Nhel 229 Hamill 375 Sgr 400 Ball 471 Xml 2029 Pull 2064 Dalt 2162 BamB 2122 Banil 485 Bigl 128 Sphl 582 EcoN1 822 Sall-Accl-Hell 651 Pshu 712 BsaBl 1668 Engl 939 BeY1 945 Nrul 972 BAPT 1045 BspMI-BfuM 1063 PAMI 1315 Bumi 1353 Bgl 1650 BapEl 1664…arrow_forwardAlu 1 EcoRI ori pKY Bgl I Tet: Tetracycline resistance gene A. What would be the donor organism to be used in this cloning? B. How would you make sure you only get your gene of interest (EGF) from donor but nothing else? C. How do you put EGF gene into pKY plasmid? What is the best RE to use when inserting the gene into pKY plasmid? What happens if you use the enzyme Bgl I to insert the gene of interest? How does it affect your cloning result? D. How do you make sure you only grow E.coli that have the gene of interest?arrow_forward
- 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.arrow_forwardShown below is a diagram for a plasmid vector you want to use to clone a gene. The diagram shows the location of the recognition sites for four restrictions enzymes, BamHI (B), EdoRI (E), Hindill (H), and Xhol (X). The genes encoding beta-lactamase (AmpR) and beta-galactosidase (lacZ) are indicated. If you were to use this vector, which enzyme should be used to linearize the plasmid in preparation for cloning? E B lacz O Hindi!! BamHI O EcoRI O Xhol H EcoRI and Xhol E -X AmpRarrow_forwardA PCR reaction was performed to amplify the XULA4 gene, which is bp 524-6,480 on a plasmid that is 9,435 bp. After the PCR, the product was digested with XhoI. There are XhoI sites on the plasmid at bp 151, 1,336, and 4,795. Calculate the size(s) that would result when the product is digested with XhoI. Then enter the size of the largest fragment (in bp).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_forwardThe map of plasmid pUC19 is shown below. The restriction site coordinate is the position of the 5’base on the top strand of each site sequence. The restriction enzyme sites are in bold type if there is only one site in pUC19. Please list the fragments in order of size, largest to smallest, which will result from a complete digestion by the restriction enzyme PvuII. Please list the fragments in order of size, largest to smallest, which will result from a complete digestion by the restriction enzyme DrdI.arrow_forwardExamine the structure of the pBR322 plasmid depicted below. Assume total size of the plasmid is 4,361 bp and the blue numbers indicate locations of restriction sites relative to the O point at the top of the plasmid. What size fragments would be generated by the following restriction digestion reactions? 1. Sall 2. Sal 1 + BamH1 3. Sal 1 + EcoR1 4. Sal I + BamH1 + EcoR1 PstI 3607 3000 4000 amp ori HindIII Edit View Insert Format Tools Table EcoRI EcoRV 4359 0 29 185 pBR322 4361 bp 2295 NdeI tet 2000 BamHI 375 651 SalI 1000 Type a short answer in the space provided below.arrow_forward
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