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Concept explainers
To analyze:
a. Question asked to describe the approaches that can be used to sequence each of these clones.
b. Assume that there is a high possibility of diseasecausing mutations in this gene which are within exons or at intron–exon boundaries. If this assumption is correct, question asked to outline the method to identify the mutations in patients by using least amount of sequencing.
Introduction:
CFTR channels are present on epithelial cells of various organs (livers, lungs, pancreas, digestive track, reproductive track); it maintains water and salt balance over the surface of the epithelial cell. Due to mutation in CFTR, mucous accumulates and hardens the surface of the epithelial cell. Cystic fibrosis transmembrane conductance regulator(CFTR) is the transmembrane Cl- (Chlorine) channel. The gene of CFTR is present on chromosome
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Chapter 16 Solutions
Genetic Analysis: An Integrated Approach (2nd Edition)
- DNA polymerase I (Pol I) of E. coli consists of three functional parts (domains): an N-terminal domain with 59 to 39 exonuclease activity required for removal of the RNA primer, a central domain responsible for 39 to 59 exonuclease proofreading, and a C-terminal domain with polymerase activity. Pol I is thought to simultaneously remove RNA primers and fill in the gaps that result (figure 13.14). A group of proteins known as RNaseH also have 59 to 39 exonuclease activity and can thus remove RNA primers. However, they lack the other two functions observed for Pol I. Predict the ability of the following mutants to replicate DNA: (1) a strain with a mutant gene encoding Pol I such that it no longer has polymerase activity (but retains both types of nuclease activities); (2) a strain without RNaseH proteins; (3) a strain with a mutant gene encoding Pol I such that it no longer has 59 to 39 exonuclease activity (but retains 39 to 59 nuclease and polymerase activities); (4) a strain with the…arrow_forwardI recently isolated the human enzyme called fucosidase and prepared anantibody to it. Now I want to isolate a cDNA clone coding for this enzyme from a human cDNAlibrary. A friend of mine in the lab next door has informed me that he had recently isolated acDNA coding for dog fucosidase that I can use if I desire. In addition, I just read an article whichreported the sequence of the first 20 amino acids of human fucosidase. Which of the probeslisted below do you think I could use to screen my library to identify the cDNA clone containinghuman fucosidase?arrow_forwardAfter Drosophila DNA has been treated with a restrictionenzyme, the fragments are inserted into plasmids and selected as clones in E. coli. With the use of this “shotgun”technique, every DNA sequence of Drosophila in a librarycan be recovered.a. How would you identify a clone that contains DNAencoding the protein actin, whose amino acid sequenceis known?b. How would you identify a clone encoding a specifictRNAarrow_forward
- What 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_forwardThe 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 enzymearrow_forwardYou isolate a mouse Tau-gene-containing DNA fragment from the chicken and hybridize it to the freshly-made and isolated hnRNA (primary transcript) from the nucleus of the mouse cells transcribed from the Tau gene (immediately after it was produced), allowing no time for processing of the hnRNA. Describe what you see when you look at the DNA/RNA hybrid molecule under the electron microscope.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_forwardThe 3′-exonuclease activity of E. coli DNA polymerase I was found to show no discrimination between correctly and incorrectly base-paired nucleotides at the 3′-terminus; properly and improperly base-paired nucleotides are cleaved at equal rates there. How can this observation be reconciled with the fact that the 3′-exonuclease activity increases the accuracy with which template DNA is copied?arrow_forwardWhen the cDNA was sequenced by the Sanger method utilizing ddCTP, the following products were obtained: Tetranucleotide Hexanucleotide Nonanucleotide Decanucleotide Dodenucleotide Octadecanucleotide Nonadecanucleotide 21-nucleotide 6c. What is the sequence of the bases in the mRNA coding for the peptide above? Thearrow_forward
- recombinant human insulin, produced by bacteria carrying a cloned insulin gene, is now the major form of insulin used to treat diabetes. It is know that the human insulin gene encodes an mRNA which is only 333 nucleotides long, but the entire gene spreads more than 4000 nucleotides. There are 3 exons and 2 introns. 1. What technique can you use inorder to get a functional insulin coding sequence cloned into bacteria and how does this technique work? 2. The technique used in 1, you would need to start with cells cells from the pancreas, why are these the only cells that would work ?arrow_forwardrecombinant human insulin, produced by bacteria carrying a cloned insulin gene, is now the major form of insulin used to treat diabetes. It is know that the human insulin gene encodes an mRNA which is only 333 nucleotides long, but the entire gene spreads more than 4000 nucleotides. There are 3 exons and 2 introns. 1. If we were to to clone this gene directly from the nuclear DNA, bacteria would not be able to express the insulin protein. Why is this true?arrow_forwardHuman genomic libraries used for DNA sequencing are often made from fragments obtained by cleaving human DNA with Haeiii in such a way that the DNA is only partially digested; that is, not all the possible HaeIII sites have been cleaved. What is a possible reason for doing this?arrow_forward
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