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Lehninger Principles of Biochemistry

David L. Nelson, Michael M. Cox

Chapter 9

DNA-Based Information Technologies - all with Video Answers

Educators

Chapter Questions

19:08

Problem 1

Engineering Cloned DNA When joining two or more DNA fragments, a researcher can adjust the sequence at the junction in a variety of subtle ways, as seen in the following exercises.
(a) Draw the structure of each end of a linear DNA fragment produced by an EcoRI restriction digest (include those sequences remaining from the EcoRI recognition sequence).
(b) Draw the structure resulting from the reaction of this end sequence with DNA polymerase I and the four deoxynucleoside triphosphates (see Fig. $8-34$ ).
(c) Draw the sequence produced at the junction that arises if two ends with the structure derived in (b) are ligated (see Fig. $25-16$ ).
(d) Draw the structure produced if the structure derived in (a) is treated with a nuclease that degrades only single-stranded DNA.
(e) Draw the sequence of the junction produced if an end with structure (b) is ligated to an end with structure (d).
(f) Draw the structure of the end of a linear DNA fragment that was produced by a PvuII restriction digest (include those sequences remaining from the PvuII recognition sequence).
(g) Draw the sequence of the junction produced if an end with structure (b) is ligated to an end with structure (f).
(h) Suppose you can synthesize a short duplex DNA fragment with any sequence you desire. With this synthetic fragment and the procedures described in (a) through (g), design a protocol that would remove an EcoRI restriction site from a DNA molecule and incorporate a new BamHI restriction site at approximately the same location. (See Fig. 9-2.)
(i) Design four different short synthetic double-stranded DNA fragments that would permit ligation of structure (a) with a DNA fragment produced by a PstI restriction digest. In one of these fragments, design the sequence so that the final junction contains the recognition sequences for both EcoRI and PstI. In the second and third fragments, design the sequence so that the junction contains only the EcoRI and only the PstI recognition sequence, respectively. Design the sequence of the fourth fragment so that neither the EcoRI nor the PstI sequence appears in the junction.

Eric Goldman
Eric Goldman
Numerade Educator
01:58

Problem 2

Selecting for Recombinant Plasmids When 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?

Lottie Adams
Lottie Adams
Numerade Educator
02:30

Problem 3

DNA Cloning The plasmid cloning vector pBR322 (see Fig. 9-3) 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.
(a) In addition to the desired recombinant plasmid, what other types of plasmids might be found among the transformed bacteria that are tetracycline-resistant? How can the types be distinguished?
(b) 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 that in $\mathrm{pBR} 322,$ the PstI and EcoRI restriction sites are about $750 \mathrm{bp}$ apart. The entire plasmid with no cloned insert is 4,361 bp. Size markers in lane 4 have the number of nucleotides noted.

Lottie Adams
Lottie Adams
Numerade Educator
02:10

Problem 4

Restriction Enzymes The partial sequence of one strand of a double-stranded DNA molecule is $5^{\prime}--$ GACGAAGTGCTGCAGAAAGTCCGCGTTATAGGCATGAATTCCTGAGG- $--3^{\prime}$ The cleavage sites for the restriction enzymes EcoRI and PstI are shown below. Write the sequence of both strands of the DNA fragment created when this DNA is cleaved with both EcoRI and PstI. The top strand of your duplex DNA fragment should be derived from the strand sequence given above.

Lottie Adams
Lottie Adams
Numerade Educator
00:48

Problem 5

Designing a Diagnostic Test for a Genetic Disease Huntington disease (HD) is an inherited neurodegenerative disorder, characterized by the gradual, irreversible impairment of psychological, motor, and cognitive functions. Symptoms typically appear in middle age, but onset can occur at almost any age. The course of the disease can last 15 to 20 years. The molecular basis of the disease is becoming better understood. The genetic mutation underlying HD has been traced to a gene encoding a protein $\left(M_{\mathrm{r}} 350,000\right)$ of unknown function. In individuals who will not develop HD, a region of the gene that encodes the amino terminus of the protein has a sequence of CAG codons (for glutamine) that is repeated 6 to 39 times in succession. In individuals with adult-onset HD, this codon is typically repeated 40 to 55 times. In individuals with childhood-onset $\mathrm{HD},$ this codon is repeated more than 70 times. The length of this simple trinucleotide repeat indicates whether an individual will develop $\mathrm{HD}$, and at approximately what age the first symptoms will occur. A small portion of the amino-terminal coding sequence of the 3,143 -codon HD gene is given below. The nucleotide sequence of the DNA is shown in black, the amino acid sequence corresponding to the gene is shown in blue, and the CAG repeat is shaded. Using Figure $27-7$ to translate the genetic code, outline a PCR-based test for HD that could be carried out using a blood sample. Assume the PCR primer must be 25 nucleotides long. By convention, unless otherwise specified, a DNA sequence encoding a protein is displayed with the coding strand - the sequence identical to the mRNA transcribed from the gene (except for $\mathrm{U} \text { replacing } \mathrm{T})$ -on top, such that it is read $5^{\prime}$ to $3^{\prime},$ left to right.

Sana Riaz
Sana Riaz
Numerade Educator
09:13

Problem 6

Using PCR to Detect Circular DNA Molecules In a species of ciliated protist, a segment of genomic DNA is sometimes deleted. The deletion is a genetically programmed reaction associated with cellular mating. A researcher proposes that the DNA is deleted in a type of recombination called site-specific recombination, with the DNA at either end of the segment joined together and the deleted DNA ending up as a circular DNA reaction product. Suggest how the researcher might use the polymerase chain reaction (PCR) to detect the presence of the circular form of the deleted DNA in an extract of the protist.

Sana Riaz
Sana Riaz
Numerade Educator
01:15

Problem 7

Glowing Plants When grown in ordinary garden soil and watered normally, a plant engineered to express green fluorescent protein (see Fig. 9-16) will glow in the dark, whereas a plant engineered to express firefly luciferase (see Fig. $8-36$ ) will not. Explain these observations.

Lottie Adams
Lottie Adams
Numerade Educator
01:51

Problem 8

Mapping a Chromosome Segment A group of overlapping clones, designated A through $F,$ is isolated from one region of a chromosome. Each of the clones is separately cleaved by a restriction enzyme, and the pieces are resolved by agarose gel electrophoresis, with the results shown below. There are nine different restriction fragments in this chromosomal region, with a subset appearing in each clone. Using this information, deduce the order of the restriction fragments in the chromosome.

Lottie Adams
Lottie Adams
Numerade Educator
01:18

Problem 9

Immunofluorescence In the more common protocol for immunofluorescence detection of cellular proteins, an investigator uses two antibodies. The first binds specifically to the protein of interest. The second is labeled with fluorochromes for easy visualization, and it binds to the first antibody. In principle, one could simply label the first antibody and skip one step. Why use two successive antibodies?

Emily Himsel
Emily Himsel
Numerade Educator
03:02

Problem 10

Yeast Two-Hybrid Analysis You are a researcher who has just discovered a new protein in a fungus. Design a yeast two-hybrid experiment to identify the other proteins in the fungal cell with which your protein interacts and explain how this could help you determine the function of your protein.

Lottie Adams
Lottie Adams
Numerade Educator
02:23

Problem 11

Use of Photolithography to Make a DNA Microarray Figure $9-22$ shows the first steps in the process of making a DNA microarray, or DNA chip, using photolithography. Describe the remaining steps needed to obtain the desired sequences (a different fournucleotide sequence on each of the four spots) shown in the first panel of the figure. After each step, give the resulting nucleotide sequence attached at each spot.

Lottie Adams
Lottie Adams
Numerade Educator
01:09

Problem 12

Use of Outgroups in Comparative Genomics A hypthetical protein found in human, orangutan, and chimpanzee has the following sequences (red indicates amino acid residue differences; dashes indicate a deletion - the residues are missing in that sequence):
Human: ATSAAGYDEWEGGKVLIHL - - KLQNRGALLELDIGAV Orangutan: ATSAAGWDEWEGGKVLIHLDGKLQNRGALLELDIGAV Chimpanzee: ATSAAGWDEWEGGKILIHLDGKLQNRGALLELDIGAV
What is the most likely sequence of the protein present in the last common ancestor of human and chimpanzee?

Lottie Adams
Lottie Adams
Numerade Educator
01:10

Problem 13

Human Migrations I Native American populations in North and South America have mitochondrial DNA haplotypes that can be traced to populations in northeast Asia. The Aleut and Eskimo populations in the far northern parts of North America possess a subset of the same haplotypes that link other Native Americans to Asia, and also have several additional haplotypes that can be traced to Asian origins but are not found in native populations in other parts of the Americas. Provide a possible explanation.

Lottie Adams
Lottie Adams
Numerade Educator
01:20

Problem 14

Human Migrations II DNA (haplotypes) originating from the Denisovans can be found in the genomes of Indigenous Australians and Melanesian Islanders. However, the same DNA markers are not found in the genomes of people native to Africa. Explain.

Lottie Adams
Lottie Adams
Numerade Educator
01:29

Problem 15

Finding Disease Genes You are a gene hunter, trying to find the genetic basis for a rare inherited disease. Examination of six pedigrees of families affected by the disease provides inconsistent results. For two of the families, the disease is co-inherited with markers on chromosome $7 .$ For the other four families, the disease is co-inherited with markers on chromosome 12. Explain how this difference might have arisen.

Lottie Adams
Lottie Adams
Numerade Educator
01:11

Problem 16

HincII: The First Restriction Endonuclease Discovery of the first restriction endonuclease to be of practical use was reported in two papers published in $1970 .$ In the first paper, Smith and Wilcox described the isolation of an enzyme that cleaved doublestranded DNA. They initially demonstrated the enzyme's nuclease activity by measuring the decrease in viscosity of DNA samples treated with the enzyme.
(a) Why does treatment with a nuclease decrease the viscosity of a solution of DNA? The authors determined whether the enzyme was an endonuclease or exonuclease by treating $^{32}$ P-labeled DNA with the enzyme, then adding trichloroacetic acid (TCA). Under the conditions used in their experiment, single nucleotides would be TCA-soluble and oligonucleotides would precipitate.
(b) No TCA-soluble $^{32}$ P-labeled material formed on treatment of the $^{32}$ P-labeled DNA with the nuclease. Based on this finding, is the enzyme an endonuclease or exonuclease? Explain your reasoning.
When a polynucleotide is cleaved, the phosphate usually is not removed but remains attached to the $5^{\prime}$ or $3^{\prime}$ end of the resulting DNA fragment. Smith and Wilcox determined the location of the phosphate on the fragment formed by the nuclease in the following steps:
1. Treat unlabeled DNA with the nuclease.
2. Treat a sample (A) of the product with $\gamma$ - $^{32}$ P-labeled ATP and polynucleotide kinase (which can attach the $\gamma$ -phosphate of ATP to a $5^{\prime}$ OH but not to a $5^{\prime}$ phosphate or to a $\left.3^{\prime} \mathrm{OH} \text { or } 3^{\prime} \text { phosphate }\right) .$ Measure the amount of $^{32} \mathrm{P}$ incorporated into the DNA.
3. Treat another sample (B) of the product of step 1 with alkaline phosphatase (which removes phosphate groups from free $5^{\prime}$ and $3^{\prime}$ ends), followed by polynucleotide kinase and $\gamma$ - $^{32}$ P-labeled ATP. Measure the amount of $^{32}$ p incorporated into the DNA.
(c) Smith and Wilcox found that sample A had 136 counts/min of $^{32} \mathrm{P} ;$ sample $\mathrm{B}$ had
3,740 counts/min. Did the nuclease cleavage leave the phosphate on the $5^{\prime}$ or the $3^{\prime}$ end of the DNA fragments? Explain your reasoning.
(d) Treatment of bacteriophage $T 7$ DNA with the nuclease gave approximately 40 specific fragments of various lengths. How is this result consistent with the enzyme's recognizing a specific sequence in the DNA as opposed to making random double-strand breaks?
At this point, there were two possibilities for the site-specific cleavage: the cleavage occurred either (1) at the site of recognition or (2) near the site of recognition but not within the sequence recognized. To address this issue, Kelly and Smith determined the sequence of the $5^{\prime}$ ends of the DNA fragments generated by the nuclease, in the following steps:
1. Treat phage T7 DNA with the enzyme.
2. Treat the resulting fragments with alkaline phosphatase to remove the $5^{\prime}$ phosphates.
3. Treat the dephosphorylated fragments with polynucleotide kinase and $\gamma$ - 32 p. labeled ATP to label the $5^{\prime}$ ends.
4. Treat the labeled molecules with DNases to break them into a mixture of mono-, di-, and trinucleotides.
5. Determine the sequence of the labeled mono-, di-, and trinucleotides by comparing them with oligonucleotides of known sequence on thin-layer chromatography.
The labeled products were identified as follows: mononucleotides: A and G; dinucleotides:
$\left(5^{\prime}\right) \operatorname{ApA}\left(3^{\prime}\right)$ and $\left(5^{\prime}\right) \operatorname{GpA}\left(3^{\prime}\right) ;$ trinucleotides: $\left(5^{\prime}\right) \operatorname{ApAp} C\left(3^{\prime}\right)$ and $\left(5^{\prime}\right) \operatorname{GpApC}\left(3^{\prime}\right)$
(e) Which model of cleavage is consistent with these results? Explain your reasoning. Kelly and Smith went on to determine the sequence of the $3^{\prime}$ ends of the fragments. They found a mixture of $\left(5^{\prime}\right) \operatorname{Tp} \mathrm{C}\left(3^{\prime}\right)$ and $\left(5^{\prime}\right) \operatorname{Tp} \mathrm{T}\left(3^{\prime}\right) .$ They did not determine the sequence of any trinucleotides at the $3^{\prime}$ end.
(f) Based on these data, what is the recognition sequence for the nuclease, and where in the sequence is the DNA backbone cleaved? Use Table $9-2$ as a model for your answer.

Sana Riaz
Sana Riaz
Numerade Educator