Exam 10: Analyzing the Structure and Function of Genes

You have purified DNA from your recently deceased goldfish.Which of the following restriction nucleases would you use if you wanted to end up with DNA fragments with an average size of 70 kilobase pairs (kb) after complete digestion of the DNA? The recognition sequence for each enzyme is indicated in the right-hand column.

A DNA library has been constructed by purifying chromosomal DNA from mice, cutting the DNA with the restriction enzyme NotI, and inserting the fragments into the NotI site of a plasmid vector.What information CANNOT be retrieved from this library?

A plasmid

Second-generation sequencing differs from Sanger sequencing because

Why is a heat-stable DNA polymerase from a thermophilic bacterium (the Taq polymerase) used in the polymerase chain reaction rather than a DNA polymerase from E.coli or humans?

To study gene function, scientists use various methods to disrupt the sequence of the gene.In some cases, this is not possible and conditional knockouts must be used instead.Which of the following statements about the Cre-Lox system for creating conditional knockouts is TRUE?

Which of the following statements about restriction nucleases is FALSE?

Figure 10-12 shows the cleavage sites of several restriction nucleases. Figure 10-12 You cut a vector using the PciI restriction nuclease.Which of the following restriction nucleases will generate a fragment that can be ligated into this cut vector with the addition of only ligase and ATP?

Which of the restriction nucleases listed below can potentially cleave a segment of cDNA that encodes the peptide KIGDACF? $\text { The Genetic Code }$ U C A G UUU Phe (F) UCU Ser (S) UAU Tyr (Y) UGU Cys (C) UUC - UCC - UAC - UGC - U UUA Leu (L) UCA -- UAA Stop UGA Stop UUG - UCG - UAG Stop UGG Trp (W) CUU Leu (L) CCU Pro (P) CAU His (H) CGU Arg (R) CUC - CCC - CAC - CGC - C CUA - CCA - CAA Gin (Q) CGA - CUG - CCG - CAG - CGG - AUU Ile (I) ACU Thr (T) AAU Asn (N) AGU Ser (S) AUC - ACC - AAC - AGC - A AUA - ACA - AAA Lys (K) AGA Arg (R) AUG Met (M) ACG - AAG - AGG - GUU Val (V) GCU Ala (A) GAU Asp (D) GGU Gly (G) GUC - GCC - GAC - GGC - G GUA - GCA - GAA Glu (E) GGA - GUG - GCG - GAG - GGG - Table 10-56 EcoRI GAATTC HindIII AAGCTT Nsil ATGCAT

Indicate whether a cDNA library or a genomic DNA library will be more appropriate for use in the following applications. A.You want to study the promoter of gene A. B.Gene A encodes a tRNA and you wish to isolate a piece of DNA containing the full-length sequence of the tRNA. C.You discover that gene A is alternatively spliced and you want to see which predicted alternative splice products the cell actually produces. D.You want to find both gene A and the genes located near gene A on the chromosome. E.You want to express gene A in bacteria to produce lots of protein A.

You want to design a set of PCR primers to specifically amplify a portion of a gene from genomic DNA.Which of the following statements about this set of primers is TRUE?

PCR was invented in

You are studying a protein that contains the peptide sequence RDWKLVI.The part of the DNA encoding this peptide is included in the sequence shown below. $\text { The Genetic Code }$ U C A G UUU Phe (F) UCU Ser (S) UAU Tyr (Y) UGU Cys (C) UUC - UCC - UAC - UGC - U UUA Leu (L) UCA -- UAA Stop UGA Stop UUG - UCG - UAG Stop UGG Trp (W) CUU Leu (L) CCU Pro (P) CAU His (H) CGU Arg (R) CUC - CCC - CAC - CGC - C CUA - CCA - CAA Gin (Q) CGA - CUG - CCG - CAG - CGG - AUU Ile (I) ACU Thr (T) AAU Asn (N) AGU Ser (S) AUC - ACC - AAC - AGC - A AUA - ACA - AAA Lys (K) AGA Arg (R) AUG Met (M) ACG - AAG - AGG - GUU Val (V) GCU Ala (A) GAU Asp (D) GGU Gly (G) GUC - GCC - GAC - GGC - G GUA - GCA - GAA Glu (E) GGA - GUG - GCG - GAG - GGG - Table 10-56 5′-GGCGTGACTGGAAGCTAGTCATC-3′ 3′-CCGCACTGACCTTCGATCAGTAG-5′ This sequence does not contain any HindIII restriction enzyme sites; the target sequence for the HindIII restriction nuclease is shown in Figure 10-65. Figure 10-65A Your goal is to create a HindIII site on this plasmid without changing the coding sequence of the protein.Explain how you would do this.

Why is an excess of normal deoxyribonucleoside triphosphate molecules (dNTPs) needed during dideoxy sequencing?

Figure 10-13 depicts a strategy by which a DNA fragment produced by cutting with the EcoRI restriction nuclease can be joined to a DNA fragment produced by cutting DNA with the HaeIII restriction nuclease. Figure 10-13 Note that cutting DNA with EcoRI produces a staggered end, whereas cutting DNA with HaeIII produces a blunt end.Why must polymerase be added in this reaction?

Assume that defects in a hypothetical gene X have been linked to a disease.Two copies of a defective gene X will predispose a child to the disease, while a single copy of the gene seems to produce no symptoms.Because early treatment can counteract the effects of the disease, a program of voluntary genetic testing is being performed with prospective parents.Caril Ann is pregnant with Charles's child.You obtain DNA samples from Charles, Caril Ann, and the fetus.You conduct PCR from these DNA samples using two different primer sets that will detect two commonly found deletions in gene X that are associated with the disease.The gene and location of these primer sets are shown in Figure 10-61A.Your results are shown in Figure 10-61B. (A) (B) Figure 10-61 A.Which of the three individuals have defects in gene X? B.Which individuals have a single defective gene and which have two defective copies of the gene? C.Indicate the location of each individual's defects on gene X.

What is the main reason for using a cDNA library rather than a genomic library to isolate a human gene from which you wish to make large quantities of the human protein in bacteria?

You have created a piece of recombinant DNA by placing a cDNA from a gene you believe is important for the differentiation of liver cells (called LC1) onto an expression plasmid that contains all the sequences necessary for propagation of this DNA in bacteria and for the production of the LC1 protein in bacteria.A picture of this plasmid is shown in Figure 10-66A, with the segment of the DNA containing the LC1 gene depicted as a gray rectangle; the promoter sequence is depicted as a white rectangle.The LC1 protein is phosphorylated on serine 54; the nucleotide sequence of the portion of the DNA that encodes this region is shown below the diagram.All HindIII and SalI restriction sites have also been mapped on the plasmid; the recognition sequences for these restriction nucleases are shown in Figure 10-66B. $\text { The Genetic Code }$ U C A G UUU Phe (F) UCU Ser (S) UAU Tyr (Y) UGU Cys (C) UUC - UCC - UAC - UGC - U UUA Leu (L) UCA -- UAA Stop UGA Stop UUG - UCG - UAG Stop UGG Trp (W) CUU Leu (L) CCU Pro (P) CAU His (H) CGU Arg (R) CUC - CCC - CAC - CGC - C CUA - CCA - CAA Gin (Q) CGA - CUG - CCG - CAG - CGG - AUU Ile (I) ACU Thr (T) AAU Asn (N) AGU Ser (S) AUC - ACC - AAC - AGC - A AUA - ACA - AAA Lys (K) AGA Arg (R) AUG Met (M) ACG - AAG - AGG - GUU Val (V) GCU Ala (A) GAU Asp (D) GGU Gly (G) GUC - GCC - GAC - GGC - G GUA - GCA - GAA Glu (E) GGA - GUG - GCG - GAG - GGG - Table 10-56 (A) (B) Figure 10-66 A.Given the information above, write out the amino acids 52 to 57, encoded by the nucleotide sequence shown above.Be sure to number the amino acids appropriately.(Hint: Remember, serine is amino acid number 54.) B.You want to create a mutant version of the LC1 gene that replaces the serine 54 found on this peptide with a glycine.You want to do this by changing only one nucleotide, and you also want to destroy the HindIII recognition sequence with this change.Write out a 21-nucleotide DNA sequence that can accommodate these changes.Be sure to (i) write out the DNA and label the 5′ and 3′ ends, (ii) underline the mutated HindIII recognition site, and (iii) circle any change made to the original sequence.

Insulin is a small protein that regulates blood sugar level and is given to patients who suffer from diabetes.Many years ago, diabetics were given insulin that had been purified from pig pancreas.Once recombinant DNA techniques became available, the DNA encoding insulin could be placed into an expression vector and insulin could be produced in bacteria.Which of the following is NOT a reason why purifying insulin from bacteria is a better way to produce insulin for diabetics than using insulin purified from a pig pancreas?

Which of the following limits the use of PCR to detect and isolate genes?