Test 2 Chapter 10

Recombinant DNA technologies involve techniques that permit the creation of custom-made DNA molecules that can be introduced back into living organisms. These technologies were first developed in the ______. (a) 1930s. (b) 1950s. (c) 1970s. (d) 1990s.

(c) 1970s.

A double-stranded DNA molecule can be separated into single strands by heating it to 90°C because _______________________. (a) heat disrupts the hydrogen bonds holding the sugar-phosphate backbone together. (b) DNA is negatively charged. (c) heat disrupts hydrogen-bonding between complementary nucleotides. (d) DNA is positively charged.

(c) heat disrupts hydrogen-bonding between complementary nucleotides.

During DNA renaturation, two DNA strands will ________. (a) break the covalent bonds that hold the nucleotides together while maintaining the hydrogen bonds that hold the two strands together. (b) break the hydrogen bonds that hold the two strands together with no effect on the covalent bonds that hold the nucleotides together. (c) re-form a double helix if the two strands have complementary sequences. (d) re-form a double helix if the two strands are identical in sequence

Choice (c) is correct. DNA strands should not renature if their sequences are identical [choice (d)]. The breaking of the hydrogen bonds between two DNA strands is the process of denaturation [choice (b)].

Which of the following techniques is not appropriate if you want to examine the transcriptome of a specific tissue? (a) in situ hybridization (b) production of a cDNA library (c) RNA-Seq (d) microarray analysis

(a) Although in situ hybridization can tell you a great deal about the expression patterns of a particular gene, this technique is not easily adapted to simultaneously monitor the thousands of genes expressed in a particular tissue

Why is an excess of normal deoxyribonucleoside triphosphate molecules (dNTPs) needed during dideoxy sequencing? (a) DNA polymerase uses the dNTPs to synthesize a DNA molecule complementary to the molecule being sequenced. (b) dNTPs are consumed as energy to fuel the sequencing reactions. (c) When dNTP levels are too low, there will be very few chain-termination events. (d) The dNTPs can hybridize to the fragment to be sequenced and serve as primers for DNA polymerase

(a) DNA polymerase uses the dNTPs to synthesize a DNA molecule complementary to the molecule being sequenced.

Figure Q10-20 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 10-20 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? (a) Polymerase will fill in the staggered end to create a blunt end. (b) Polymerase is needed to seal nicks in the DNA backbone. (c) Polymerase will add nucleotides to the end produced by the HaeIII restriction nuclease. (d) Without polymerase, there will not be enough energy for the reaction to proceed.

(a) Polymerase will fill in the staggered end to create a blunt end.

You want to design a DNA probe used for hybridization to isolate a clone from a cDNA library. Which of the following statements about DNA probes is true? (a) The shorter the DNA probe used to probe the library, the greater the number of colonies to which the probe might hybridize. (b) A DNA probe that contains sequences that span two exons is better suited to the purpose than a DNA probe that only contains sequences from one exon. (c) A DNA probe that contains sequences immediately upstream of the DNA that codes for the first methionine in the open reading frame will usually not hybridize to clones in a cDNA library. (d) Hybridization of a DNA probe to the plasmid of interest will permit the detection of the clone of interest; labeling of the DNA probe is not necessary

(a) The shorter the DNA probe, the more likely it is that that particular sequence will show up in the genome at random. cDNA libraries contain sequences represented by exons, so it does not really matter whether or not the probe spans two exons [choice (b)]. mRNAs usually have 5′ untranslated regions that should be represented in a cDNA library, so choice (c) is not true. DNA probes are usually labeled (for example, with radioactivity) for visualization [choice (d)].

Which of the following limits the use of PCR to detect and isolate genes? (a) The sequence at the beginning and end of the DNA to be amplified must be known. (b) It also produces large numbers of copies of sequences beyond the 5′ or 3′ end of the desired sequence. (c) It cannot be used to amplify cDNAs or mRNAs. (d) It will amplify only sequences present in multiple copies in the DNA sample.

(a) To construct primers that will bracket the desired gene, you have to know the sequence at the beginning and end of the DNA to be copied.

You have a circular plasmid that can be cut by the restriction nuclease HindIII, as diagrammed in Figure Q10-4 *See figure 10-4 If you were to cut this circular piece of DNA with HindIII, which of the answers below best predicts what you would get? (a) one linear piece of DNA (b) two circular pieces of DNA (c) two semicircular pieces of DNA (d) two linear pieces of DNA

(a) one linear piece of DNA

During gel electrophoresis, DNA fragments_______________________. (a) travel through a matrix containing a microscopic network of pores. (b) migrate toward a negatively charged electrode. (c) can be visualized without stains or labels. (d) are separated on the basis of their sequence.

(a) travel through a matrix containing a microscopic network of pores.

Figure Q10-64A depicts the restriction map of one segment of the human genome for four restriction nucleases W, X, Y, and Z. Figure Q10-64B depicts the restriction maps of four individual BAC clones that contain segments of human DNA from the region depicted in Figure Q10-64A. *see figure 10-64 From this information, how would you order these BAC clones, from left to right? (a) 1, 2, 3, 4 (b) 2, 1, 4, 3 (c) 3, 4, 2, 1 (d) 4, 1, 3, 2

(b) *see figure 10-64 in answer doc

You have a piece of circular DNA that can be cut by the restriction nucleases XhoI and SmaI, as indicated in Figure Q10-5 *see figure 10-5 If you were to cut this circular piece of DNA with both XhoI and SmaI, how many fragments of DNA would you end up with? (a) 1 (b) 2 (c) 3 (d) 4

(b) 2

Starting with one double-stranded DNA molecule, how many cycles of PCR would you have to perform to produce about 100 double-stranded copies (assuming 100% efficiency per cycle)? (a) 2 (b) 7 (c) 25 (d) 100

(b) After 7 cycles of PCR, you would have 128 molecules of DNA if the reaction were 100% efficient.

Which of the following statements about gel-transfer hybridization (or Southern blotting) is false? (a) This technique involves the transfer of DNA molecules from gel onto nitrocellulose paper or nylon paper. (b) In this technique, single-stranded DNA is separated by electrophoresis. (c) A labeled DNA probe binds to the DNA by hybridization. (d) The DNA that is separated on a gel is not labeled.

(b) During Southern blotting, double-stranded DNA is loaded onto the agarose gel. The DNA becomes denatured (and thus single-stranded) as it gets transferred by the alkali solution from the gel to the nitrocellulose or nylon sheet.

Figure Q10-19 shows the cleavage sites of several restriction .nucleases *see figure 10-19 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? (a) HindIII (b) NcoI (c) MmeI (d) NspV

(b) However, you will not be able to excise the fragment after ligation, because you will destroy both the PciI site and the NcoI site, creating a new site with the sequence 5′-ACATGG-3′ 3′-TGTACC-5

Which of the following statements about PCR is false? (a) PCR uses a DNA polymerase from a thermophilic bacterium. (b) PCR is particularly powerful because after each cycle of replication, there is a linear increase in the amount of DNA available. (c) For PCR, every round of replication is preceded by the denaturation of the double-stranded DNA molecules. (d) The PCR will generate a pool of double-stranded DNA molecules, most of which will have DNA from primers at the 5′ ends.

(b) PCR is particularly powerful because after each cycle of replication, there is a linear increase in the amount of DNA available.

Which of the following statements about restriction nucleases is false? (a) A reproducible set of DNA fragments will be produced every time a restriction nuclease digests a known piece of DNA. (b) Restriction nucleases recognize specific sequences on single-stranded DNA. (c) Some bacteria use restriction nucleases as protection from foreign DNA. (d) Some restriction nucleases cut in a staggered fashion, leaving short, single-stranded regions of DNA at the ends of the cut molecule.

(b) Restriction nucleases recognize specific sequences on single-stranded DNA.

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 (a) Insulin can be easily produced in large quantities from cells carrying the cloned DNA sequence. (b) The creation of transgenic pigs that expressed insulin was very expensive compared to the cost of creating bacteria that expressed insulin. (c) Insulin made from a bacterial culture and then purified will be free of any possible contaminating viruses that pigs (and any other animals) harbor. Since pigs are more closely related to people than bacteria are, their viruses are more likely to be harmful to people than are viruses that might infect bacteria. (d) The pig protein has slight amino acid differences compared to the human protein, so human insulin produced by bacteria will work better in people

(b) Transgenic pigs were not created for the purification of insulin. Instead, scientists were purifying the endogenous insulin present in the pig pancreas that is made from the normal pig insulin gene

You have a linear piece of DNA that can be cut by the restriction nucleases HindIII and EcoRI, as diagrammed in Figure Q10-9. If you were to cut this linear DNA with HindIII, what type of DNA fragments do you predict you will obtain? *see figure 10-9 (a) three linear pieces of DNA (b) two linear pieces of DNA, only one of which can be cut by EcoRI (c) two linear pieces of DNA, both of which can be cut by EcoRI (d) two linear pieces of DNA, only one of which can be cut by HindIII

(b) two linear pieces of DNA, only one of which can be cut by EcoRI

You have a circular plasmid that has a single EcoRI site in it, as diagrammed in Figure Q10-7, which also shows the cleavage site for EcoRI. Choose the answer below that best represents what the end of the DNA molecule will look like once you cut the plasmid with EcoRI. Note that only the very ends of the DNA molecule are shown in the answers. *see figure 10-7 for answer choices

(c)

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) Sau3AI GATC (b) BamHI GGATCC (c) NotI GCGGCCGC (d) XzaI GAAGGATCCTTC

(c)

Which of the following statements about RNA interference (or RNAi) is false? (a) RNAi is a natural mechanism used to regulate genes. (b) During the process of RNAi, hybridization of a small RNA molecule with the mRNA degrades the mRNA. (c) Because RNAi depends on the introduction of a double-stranded RNA into a cell or an organism, it is not a process that can cause heritable changes in gene expression. (d) In C. elegans, RNAi can be introduced into the animals by feeding them with bacteria that produce the inhibitory RNA molecules

(c) Because RNAi depends on the introduction of a double-stranded RNA into a cell or an organism, it is not a process that can cause heritable changes in gene expression.

Why are dideoxyribonucleoside triphosphates used during DNA sequencing? (a) They cannot be incorporated into DNA by DNA polymerase. (b) They are incorporated into DNA particularly well by DNA polymerases from thermophilic bacteria. (c) Incorporation of a dideoxyribonucleoside triphosphate leads to the termination of replication for that strand. (d) Dideoxyribonucleoside triphosphates are more stable than deoxyribonucleoside triphosphates.

(c) Incorporation of a dideoxyribonucleoside triphosphate leads to the termination of replication for that strand.

Your friend works at the Centers for Disease Control and Prevention and has discovered a brand-new virus that has recently been introduced into the human population. She has just developed a new assay that allows her to detect the virus by using PCR products made from the blood of infected patients. The assay uses primers in the PCR assay that hybridize to sequences in the viral genome. Your friend is distraught because of the result she obtained (see Figure Q10- 45) when she looked at PCR products made using the blood of three patients suffering from the viral disease, using her own blood, and using a leaf from her petunia plant. You advise your friend not to panic, as you believe she is missing an important control. Which one of the choices listed below is the best control for clarifying the results of her assay? Explain your answer *see figure 10-45 (a) a PCR assay using blood from a patient who is newly infected but does not yet show symptoms (b) a PCR assay using blood from a dog (c) a PCR assay using blood from an uninfected person (d) repeating the experiments she has already done with a new tube of polymerase

(c) Primers can sometimes hybridize to unintended sequences and produce unintended products. The appropriate control for your friend's experiment would be DNA from an uninfected person; in that way, she would be able to determine whether the bands present in the PCR from her blood truly correspond to product generated from viral DNA rather than cross-hybridization to DNA sequences in the human genome, because the bands would be absent from a person uninfected by the virus in the former case only. Doing PCR from an infected but asymptomatic person would not be useful [choice (a)], because it would not allow your friend to distinguish whether she is infected. Although doing PCR from dog blood [choice (b)] should not give any viral bands, any nonspecific products from a dog would probably be different from those in your friend. The absence of PCR fragments in the petunia lane suggests that there is no viral contaminant in any of your friend's reagents, so using a new tube of polymerase is not the solution [choice (d)].

You create a recombinant DNA molecule that fuses the coding sequence of green fluorescent protein to the regulatory DNA sequences that control the expression of your favorite genes. Which of the following pieces of information can you NOT gain by examining the expression of this reporter gene? (a) the tissue where the protein encoded by this gene is expressed (b) the cell in which the protein encoded by this gene is expressed (c) the specific location within the cell of the protein encoded by this gene (d) when, during an organism's development, this gene is expressed

(c) The information for localizing proteins within a cell is found on the protein product and not in the regulatory DNA sequences. If you were to fuse your reporter gene to the DNA sequences that encode the protein to produce a GFP fusion protein, then you might determine the specific location of the protein within the cell.

Which of the following statements about genomic DNA libraries is false? (a) The larger the size of the fragments used to make the library, the fewer colonies you will have to examine to find a clone that hybridizes to your probe. (b) The larger the size of the fragments used to make the library, the more difficult it will be to find your gene of interest once you have identified a clone that hybridizes to your probe. (c) The larger the genome of the organism from which a library is derived, the larger the fragments inserted into the vector will tend to be. (d) The smaller the gene you are seeking, the more likely it is that the gene will be found on a single clone

(c) The sizes of the fragments left after a restriction digest do not depend on the total size of the genome; they depend on the sequence of the genome and the frequency with which the restriction enzyme recognition site is found in the genome. Choices (a) and (b) are true: as a limiting case, think of what would happen if a fragment the size of the entire genome were inserted into the bacterial vector. You would have to screen only one colony to find the clone that hybridized to your probe, but it would be very difficult to find out where on the insert your gene of interest lay. Choice (d) is true: the larger the gene you are seeking, the more likely it is that there will be a restriction fragment in the gene (or that the gene will be broken if the DNA was fragmented by random shearing), and hence the less likely it is that the entire gene will be found in one clone.

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) gene regulatory sequences (b) intron sequences (c) sequences of the telomeres (the ends of the chromosomes) (d) amino acid sequences of proteins

(c) The very ends of all of the chromosomes are unlikely to be NotI sites, meaning that the fragments containing the ends of the chromosomes will not be able to insert into the bacterial vector (because they have not been cut by NotI at both ends) and will be lost from the library. All sequences present in genomic DNA (which includes regulatory sequences and introns) should be present in a genomic library. The coding sequence of the gene (and hence the amino acid sequence of the encoded protein) is also present in a genomic clone, although it is interrupted by intron sequences and is therefore somewhat difficult (but not impossible) to determine

PCR was invented in _______. (a) the 1800s. (b) the 1950s. (c) the 1980s. (d) 2009

(c) the 1980s.

You are interested in a single-stranded DNA molecule that contains the following sequence: * figure 10-15 Which molecule can be used as a probe that will hybridize to your sequence of interest? (a) 5′-GATTGCAT-3′ (b) 5′-TACGTTAG-3′ (c) 5′-CTAACGTA-3′ (d) 5′-ATGCAATC-3′

(d) 5′-ATGCAATC-3′

DNA ligase is an enzyme used when making recombinant DNA molecules in the lab. In what normal cellular process is DNA ligase involved? (a) none, it is only found in virally infected cells (b) transcription (c) transformation (d) DNA replication

(d) DNA replication

You have sequenced a fragment of DNA and produced the gel shown in Figure Q10-50. Near the top of the gel, there is a section where there are bands in all four lanes (indicated by the arrow). Which of the following mishaps would account for this phenomenon? Explain your answer *see figure 10-50 (a) You mistakenly added all four dideoxynucleotides to one of the reactions. (b) You forgot to add deoxynucleotides to the reactions. (c) Your primer hybridizes to more than one area of the fragment of DNA you are sequencing. (d) A restriction nuclease cut a fraction of the DNA you are sequencing

(d) If some of the DNA templates you are sequencing are cut at one specific site (as would be the case if a restriction enzyme cut the DNA), the polymerase will stop when it comes to the end of the DNA, giving rise to at least some product of one particular size in all the reaction mixtures. If this were the case, all four lanes will have a band of this particular size. In addition, you would get a normal sequence from the full-length templates, and a normal sequence from those templates in which the polymerase incorporated a dideoxynucleotide before encountering the end. The other options are incorrect: if you added all four dideoxynucleotides to one of the reactions [choice (a)], that lane would have a band at every position because the polymerase would stop at As, Cs, Gs, and Ts instead of at only one type of nucleotide. If you forgot to add deoxynucleotides to the reactions [choice (b)], you would not get any polymerization, and all of your lanes would be blank. If your primer hybridized to more than one part of the fragment of DNA you were sequencing [choice (c)], your gel would look as though two different sequences had been superimposed on each other.

You have been hired to create a cat that will not cause allergic reactions in cat-lovers. Your coworkers have cloned the gene encoding a protein found in cat saliva, expressed the protein in bacteria, and shown that it causes violent allergic reactions in people. But you soon realize that even if you succeed in making a knockout cat lacking this gene, anyone who buys one will easily be able to make more hypoallergenic cats just by breeding them. Which of the following will ensure that people will always have to buy their hypoallergenic cats from you? (a) Inject the modified embryonic stem (ES) cells into embryos that have a genetic defect to prevent the mature adult from reproducing. (b) Implant the injected embryos into a female cat that is sterile as a result of a genetic defect. (c) Sell only the offspring from the first litter of the female cat implanted with the injected embryos. (d) Surgically remove the sexual organs of all the knockouts before you sell them

(d) Neutering all the knockout animals that you sell is the only option of the four listed that will prevent happy pet owners from becoming happy pet breeders. The Page 35 of 36 situation described in choice (a) will not allow you to make any knockout cats because the first litter (which will at best have a few mosaics in which one copy of the gene has been knocked out in the germ cells) will be sterile and you will not be able to mate them. The genotype of the female cat in which you implant the embryos has no effect on the genotype of the embryos, which is why choice (b) is incorrect. Choice (c) is incorrect because the first litter will yield mosaic cats that still have one copy of the allergen-producing gene in their cells and are therefore not hypoallergenic

Second-generation sequencing differs from Sanger sequencing because _____________. (a) second-generation sequencing does not depend on chain-terminator ddNTPs. (b) second-generation sequencing does not require DNA polymerase. (c) for the cost per base sequenced, second-generation sequencing is much more expensive than Sanger sequencing. (d) second-generation sequencing can sequence tens of millions of pieces of DNA at the same time on a single glass slide

(d) Second-generation sequencing is faster and cheaper because millions of reactions can be carried out in parallel on a solid support.

You have a piece of circular DNA that can be cut by the restriction nucleases EcoRI, HindIII, and NotI, as indicated in Figure Q10-6 *see figure 10-6 Which of the following statements is false? (a) One piece of DNA will be obtained when this DNA is cut by NotI. (b) A piece of DNA that cannot be cut by EcoRI will be obtained by cutting this DNA with both NotI and HindIII. (c) Two DNA fragments that cannot be cut by HindIII will be obtained when this DNA is cut by EcoRI and NotI. (d) Two DNA fragments of unequal size will be created when this DNA is cut by both HindIII and EcoRI

(d) Two DNA fragments of unequal size will be created when this DNA is cut by both HindIII and EcoRI

DNA can be introduced into bacteria by a mechanism called ____________. (a) transcription. (b) ligation. (c) replication. (d) transformation.

(d) transformation

For each of the following sentences, fill in the blanks with the best word or phrase selected from the list below. Not all words or phrases will be used; use each word or phrase only once. A nuclease hydrolyzes the __________________ bonds in a nucleic acid. Nucleases that cut DNA only at specific short sequences are known as __________________. DNA composed of sequences from different sources is known as __________________. __________________ can be used to separate DNA fragments of different sizes. Millions of copies of a DNA sequence can be made entirely in vitro by the __________________ technique. DNA sequencing phosphodiester endonucleases polymerase chain reaction exonucleases recombinant DNA gel electrophoresis restriction nucleases hydrogen ribonucleases nucleic acid hybridization

A nuclease hydrolyzes the phosphodiester bonds in a nucleic acid. Nucleases that cut DNA only at specific short sequences are known as restriction nucleases. DNA composed of sequences from different sources is known as recombinant DNA. Gel electrophoresis can be used to separate DNA fragments of different sizes. Millions of copies of a DNA sequence can be made entirely in vitro by the polymerase chain reaction technique

A plasmid ______________. (a) can confer antibiotic resistance to a bacterium. (b) is a single-stranded circular DNA molecule that can undergo horizontal transfer among bacteria. (c) is a tool designed in the lab and never found in naturally occurring bacteria. (d) always becomes part of the bacterial chromosome during transformation.

Choice (a) is correct. Plasmids that are found in naturally occurring bacteria [choice (c)] can confer antibiotic resistance to a bacterium; plasmids in the lab often carry an antibiotic-resistance gene so that scientists can select for bacteria that are carrying their recombinant DNA molecule. Plasmids are made of double-stranded DNA [choice (b)], and typically have their own replication origin, allowing for plasmids to replicate independently of the bacterial chromosome [choice (d)].

Which of the following statements about DNA libraries is true? (a) Production of a DNA library involves the direct insertion of short DNA fragments into bacteria through transformation. (b) By placing the library DNA into bacteria, the bacteria can be used to amplify the desired DNA fragments from the DNA library. (c) Individual bacteria that have taken up most of the library DNA are selected for during the construction of a DNA library. (d) The library DNA within the bacteria will only be replicated when it hybridizes to a DNA probe

Choice (b) is correct. To make a DNA library, the fragmented DNA must first be placed into an appropriate plasmid before it can be placed into the bacteria through transformation [choice (a)]. Individual bacteria in a library should only carry a single DNA fragment from the library [choice (c)]. Although DNA probes are used to identify the bacterial colony that contains the DNA library fragment of interest, this hybridization is done on dead bacteria and is not involved in replication of the desired DNA [choice (d)].

With fully automated Sanger sequencing, all four chain-terminating ddNTPs can be added into a single reaction. This is different from the traditional slab gel Sanger sequencing, where a different reaction had to be carried out for each ddNTP. The mixing of all four ddNTPs can be carried out because (a) the fully automated Sanger sequencing reactions are loaded onto a capillary gel. (b) the fully automated Sanger sequencing reactions utilize ddNTPs each labeled with a different fluorescent tag, which allows all four ddNTPs to be incorporated into a single molecule of DNA. (c) the fully automated Sanger sequencing reactions generate a set of products, each of which carries a single fluorescent tag whose color reveals the identity of the base that is at the end of the product. (d) the fully automated Sanger sequencing reactions do not require DNA polymerase because the bases are read as the DNA is pulled through a tiny pore at the end of the capillary gel.

Choice (c) is correct. Although it is true that fully automated Sanger sequencing requires a capillary gel, this is not why all four ddNTPs can be mixed in a single reaction [choice (a)]. Ideally, a single ddNTP is incorporated into each product; all four ddNTPs should not be incorporated into a single product [choice (b)]. Fully automated Sanger sequencing still requires DNA polymerase, and reads the fluorescent ddNTP at the end of each product [choice (d)].

Which of the following describes a feature found in bacterial expression vectors but not in cloning vectors? (a) origin of replication (b) cleavage sites for restriction nucleases (c) promoter DNA sequences (d) a polyadenylation signal

Choice (c) is correct. Origins of replication [choice (a)] and cleavage sites for restriction nucleases [choice (b)] are found in both cloning vectors and expression vectors. Bacterial mRNAs do not undergo polyadenylation [choice (d)].

PCR involves a heating step, followed by a cooling step, and then DNA synthesis. What is the primary reason for why this cooling step is necessary? (a) Cooling the reaction ensures the integrity of the covalent bonds holding the nucleotides together in the DNA strand. (b) Cooling the reaction gives the DNA polymerase an opportunity to rest from the previous cycle so that it will be ready for the next round of synthesis. (c) Transcription takes place during the cooling step. (d) Cooling the reaction brings the temperature down to a level that is compatible with the short primers forming stable hydrogen bonds with the DNA to be amplified

Choice (d) is correct. During PCR, the reaction is heated to a temperature that will break the hydrogen bonds holding the strands together but should not be harmful to the covalent bonds holding the nucleotides together [choice (a)]. The DNA polymerase used in PCR is a thermostable enzyme that can tolerate the high temperatures used in PCR [choice (b)]. Transcription involves the production of RNA from DNA and does not occur during PCR [choice (c)].

You want to design a DNA probe used for hybridization to isolate a clone from a cDNA library. Which of the following concerns about DNA probe design is the most legitimate? (a) You must be careful when designing your probe to take into account which DNA strand was transcribed in mRNA and choose a probe complementary to the mRNA. (b) You must be careful not to include any DNA sequences in your probe that are upstream (5′) of the AUG start codon. (c) You must make sure that all the DNA sequences in your probe lie within an exon, and do not span two exons. (d) You must make sure that all the DNA sequences in your probe are not located downstream (3′) of the polyadenylation signal.

Choice (d) is correct. mRNAs are converted into cDNAs, which are double-stranded, so it is not necessary to worry about which strand the probe will hybridize with [choice (a)]. The transcriptional and translational start sites of an mRNA molecule differ, so including some sequences 5′ of the AUG start codon is not necessarily a problem [choice (b)]. Because cDNAs are made from mRNAs, the exon-splicing events have already occurred so it shouldn't matter if your probe sequences span two exons or lie within a single exon [choice (c)].

For each of the following sentences, fill in the blanks with the best word or phrase selected from the list below. Not all words or phrases will be used; use each word or phrase only once. During gel __________________, DNA fragments can be loaded into one end of an agarose slab to separate the fragments on the basis of charge. As ____________________ is applied across the agarose slab, the DNA molecules, which have a __________________ charge, will migrate toward the ___________________ electrode. Because _________________ DNA fragments will migrate more quickly, they will be found furthest away from the area of the gel where the DNA fragments were loaded. One method to visualize the DNA on the agarose slab involves staining the DNA with a dye that will __________________ under ultraviolet light. centrifugation negative digested neutral electrophoresis positive fluoresce radioactive gravity sequencing irradiate smaller larger voltage

During gel electrophoresis, DNA fragments can be loaded into one end of an agarose slab to separate the fragments on the basis of charge. As voltage is applied across the agarose slab, the DNA molecules, which have a negative charge, will migrate toward the positive electrode. Because smaller DNA fragments will migrate more quickly, they will be found furthest away from the area of the slab where the DNA fragments were loaded. One method to visualize the DNA on the agarose slab involves staining the DNA with a dye that will fluoresce under ultraviolet light

Which of the restriction nucleases listed below can potentially cleave a segment of cDNA that encodes the peptide KIGDACF? *figure 10-35 and "The Genetic Code" table

figure 10-35 answers The enzyme NsiI cleaves at ATGCAT