Final 1-Recombinant DNA Technology Homework

The development of polymerase chain reaction (PCR) was a watershed event in recombinant DNA technology. What are two characteristics of PCR that make it such an important development? -Exponentially more DNA clones can be made in a shorter time frame. -PCR reactions can be performed without any controls. -PCR typically can amplify long segments of DNA (more than 10,000 bp). -PCR is characterized by cost reduction through the use of thermostable Taq polymerase. -Even if there is contamination of the sample with DNA from other individuals DNA product will be obtained properly. -The primers can be synthesized without knowing any information about the nucleotide sequence of the target DNA.

-PCR is characterized by cost reduction through the use of thermostable Taq polymerase. -Exponentially more DNA clones can be made in a shorter time frame.

Order of steps involved in screening a genomic library

1. grow genomic library colonies and transfer to membranes 2. lyse colonies and denature DNA 3. hybridize desired labeled probe 4. identify targeted library colonies

The DNA sequence is repeated here: 5'...Exon 1...AGCTTGGGAGCGGCG GTGAGGCGGGAGGCG...Intron 1...TCCTCTCTCCCCCAG GGCCCACCAGCTCTG...Exon 2...3' Recall that the DNA sequence that corresponds to intron 1 is underlined and bolded. Which of the following DNA probes could you also use to detect this gene in mice? Select all that apply. 5' CGGCGGTGAG 3' 3' GCCGCCACTC 5' 5' CGGCGGGCCC 3' 3' GCCGCCCGGG 5' 5' TCCTCTCTCC 3'

3' GCCGCCCGGG 5' The RNA transcribed from this gene will have the same sense as the coding/nontemplate strand, except that U's will replace T's. Therefore the initial RNA will be: 5'...Exon 1...AGCUUGGGAGCGGCG GUGAGGCGGGAGGCG...Intron 1...UCCUCUCUCCCCCAG GGCCCACCAGCUCUG...Exon 2...3' Following RNA processing, introns will be removed The bolded bases represent the regions of exons 1 and 2 that are spliced together. 5'...Exon 1...AGCUUGGGAGCGGCG GGCCCACCAGCUCUG...Exon 2...3' The splicing together of exon 1 and 2 provides a unique target sequence that is not present in the original genomic sequence. 3' GCCGCCCGGG 5' is the only probe listed that will bind this sequence that is unique to the mRNA.

A probe with the sequence 5'-A-T-G-C-C-A-G-T-3' will serve as a probe for which sequence? 3'-T-G-S-C-C-G-T-A-5' 3'-A-T-G-C-C-A-G-T-5' 3'-T-A-C-G-G-T-C-A-5' 3'-A-C-T-G-G-C-A-T-3'

3'-T-A-C-G-G-T-C-A-5'

BamHI cuts the sequence 5′ G|GATCC 3′. Which of the following sequences would not be recognized by this enzyme?

3′ TCTTAAG 5′

If there are five molecules of DNA containing the target region at the beginning of a PCR reaction, how many copies of the target will be present after three rounds of amplification?

40 The number of target sequences is doubled with each replication cycle.

A restriction enzyme that uses a six (6) base recognition sequence will cut DNA, on average, every ________ bases, if all four nucleotides are present in equal proportions. 1296 500 256 4096 5000

4096

A standard PCR cycle includes three steps: denaturation (95°C), annealing (55°C), and elongation (65°C). Denaturation (95 degrees C) -template DNA Annealing (55 degrees C) -Primers Elongation (65 degrees C) -Taq Polymerase -dNTPS

During the denaturation step of a PCR cycle, the template DNA strands are separated or denatured. Next, during the annealing step, the single-stranded primers hybridize to the denatured DNA template. Finally, during the elongation step, Taq polymerase synthesizes a new strand of DNA from the DNA template in a 5' to 3' direction, starting at the 3' end of the primers, by adding dNTPs to the 3' end of the new DNA strand.

True/False Restriction endonucleases cut DNA at specific recognition sequences and then bond two strands covalently with the same "sticky ends."

False Restriction endonucleases cut DNA at specific sequences, but DNA ligase must be used to bond two strands covalently with the same "sticky ends."

What role does DNA ligase perform in a DNA cloning experiment? How does the action of DNA ligase differ from the function of restriction enzymes? -DNA ligase generates recombinant DNA by joining two different DNA molecules, while restriction enzymes convert long stretches of double-stranded DNA into two single-stranded DNA molecules. -DNA ligase has a specific DNA recognition sequence, while restriction enzymes do not. -DNA ligase generates hydrogen bonds between complementary base pairs, while restriction enzymes break those bonds. -DNA ligase generates the covalent bonds of the phosphodiester backbone, while restriction enzymes break those bonds

DNA ligase generates the covalent bonds of the phosphodiester backbone, while restriction enzymes break those bonds.

DNA sequencing by the Sanger method employs which of the following for chain termination? Dinucleotides Ribonucleotides Deoxynucleotides Dideoxynucleotides

Dideoxynucleotides ddNTPs are nucleotide analogs that cause chain termination due to lack of a free 3'-hydroxyl group.

During PCR WHAT ARE THE TWO Primers?

During PCR, a specific sequence of DNA is amplified using short DNA primers. Two primers hybridize to the target sequence in an antiparallel manner. In this example, the forward primer hybridizes to the left side of the bottom strand and the reverse primer binds to right side of the top strand.New bases are always added to the 3' end of the primer, so the GFP gene sequence is to the right of the forward primer and to the left of the reverse primer.

True/False DNA fragments that are 600 bp long will migrate more quickly through a sequencing gel than fragments that are 150 bp long.

False Small DNA fragments have less hindrance in moving through the gel, so they migrate more quickly than larger fragments.

If one wishes to clone a gene using typical restriction endonucleases, how does the restriction endonuclease identify the appropriate cut sites in the genome? The endonuclease recognizes the gene of interest. The endonuclease cannot identify the cut sites. The endonuclease identifies its specific recognition sequence. The endonuclease cuts randomly in the genome.

The endonuclease identifies its specific recognition sequence.

Restriction mapping is used to characterize cloned DNA. What does a restriction map tell the researcher about the cloned DNA? -The number of sites and distance between them for the specific restriction enzyme. -The restricted conditions under which the organism can grow. -The distances between restriction sites for the specific restriction enzyme. -The size of the genome the cloned DNA was isolated from. -The number of restriction sites for the specific restriction enzyme.

The number of sites and distance between them for the specific restriction enzyme.

What is the purpose of raising the temperature to 90-95°C at the beginning of each cycle of PCR? -To extend the primer -To separate the double‑stranded DNA -To renature two single DNA strands -To attach the primer

To separate the double‑stranded DNA The temperature is raised to denature the double‑stranded DNA molecule into single strands.

A knock-out mouse is made with respect to the PFKL gene, and this mouse is now described as which of the following? a loss-of-function PFKL mouse a retention-of-function PFKL mouse a gain-of-function PFKL mouse an enhancement-of-function PFKL mouse

a loss-of-function PFKL mouse

When performing a sequencing reaction using Sanger sequencing, which ddNTPs must be included in the reaction? ddTTP ddCTP ddATP ddGTP all four ddNTPs must be present

all four ddNTPs must be present

In the context of molecular genetics, reverse transcription PCR RT refers to? -assembling an RNA sequence from a DNA sequence -transcribing first, then translating -translating in the 3' to 5' direction -assembling a DNA sequence from an mRNA -making an amino acid sequence from a DNA sequence

assembling a DNA sequence from an mRNA

To ensure that a human gene is translated properly in a bacterial cell, you should clone the _____________ for the gene instead of the genomic DNA fragment that contains the gene.

cDNA Bacteria are able to translate eukaryotic processed mRNAs and produce normal, functioning eukaryotic proteins. cDNAs are complementary copies of mRNAs--processed pre-mRNAs-- and these cDNAs can be cloned into bacterial vectors and expressed in bacteria.

Which type of DNA library represents the genes expressed by a given cell at a certain time?

cDNA cDNA libraries represent expressed genes from specific cell types under specific conditions. Noncoding sequences are not included.

Immediately after the primers have annealed to the target sequence, _______. -Immediately after the primers have annealed to the target sequence, _______. -the temperature is lowered so that taq polymerase can extend the primers -the temperature is raised to cause denaturation -the annealing temperature is maintained until polymerase has finished extension of the new strands -the temperature is raised so that taq polymerase can extend the primers

the temperature is raised so that taq polymerase can extend the primers The temperature is raised to 70-75∘C, the temperature over which taq polymerase is optimally active.

Characteristics of the Genomic Library

-Can be used to study promoter regions of genes -Is the same regardless of which cell type is used to make the library -Can be used to study splice sites and introns -Likely to contain the entire genome -Contain non-coding DNA

Characteristics of cDNA Library

-Can be used to study the genes expressed at different developmental stages -Rarely contain introns -Originate from mRNA -Dependent on expression levels of genes -Dependent on tissue types

Characteristics of both Genomic and cDNA Libraries

-Can used to study coding regions of genes -Cloned into vectors

Suppose that you would like to make a human protein in bacteria. You clone a fragment of genomic DNA that contains the human gene into a plasmid, transform the plasmid into the bacteria, and isolate colonies containing the recombinant plasmid. Which of the following results would you expect from this experiment? -The bacteria could produce a much longer protein with an incorrect amino acid sequence. -The bacteria could produce a truncated, nonfunctional human protein. -The bacteria could produce no human protein. -The bacteria could produce a normal, functioning human protein

-The bacteria could produce a much longer protein with an incorrect amino acid sequence. -The bacteria could produce a truncated, nonfunctional human protein. Human genes contain introns that are spliced out before the gene is translated. Because bacteria are not able to process eukaryotic genes, the bacteria would not splice out the introns, and would instead translate them. If an intron contained a stop codon, this would lead to a truncated protein. If an intron did not contain a stop codon, the translated protein would be longer than normal and contain an incorrect amino acid sequence as a result of translating the intron.

Which of the following best describes a cloning vector? -A DNA molecule that accepts DNA fragments and degrades them in a host. -The fragment of DNA encoding a gene of interest. -A DNA molecule that accepts DNA fragments and replicates the fragment in a host. -The direction in which DNA is cloned.

A DNA molecule that accepts DNA fragments and replicates the fragment in a host.

In which of the following biochemical reactions is it common to use ddNTPs (dideoxyribonucleoside triphosphates)? -plasmolysis -citric acid cycle -restriction digestion -electron transport -DNA sequencing

DNA sequencing

Which three steps constitute a PCR cycle? -Naturation, annealing, and photolysing -Denaturation, annealing, and extension -Transfection, transformation, and transduction -Inactivation, activation, and transfer

Denaturation, annealing, and extension The double-stranded product is denatured, the primers anneal to the target sequence, and then DNA synthesis (extension) by Taq polymerase proceeds.

True/False. The thermostability of Taq polymerase is required during the annealing phase of PCR.

False. The annealing phase takes place at the lowest temperature of PCR. Taq polymerase is derived from bacteria that live in hot springs, so the enzyme is thermostable, meaning that its enzymatic properties can withstand the high temperatures needed for denaturation.

You cut a gene of interest with HincII and would like to insert it into the multiple cloning site (MCS) of a plasmid. The plasmid's MCS contains the cut sites listed below.

GTA*TAC CAT*ATC GGT*ACC CCG*TGG GTC*AAC CTG*TTG HincII creates blut ends when it cuts DNA. DNA cut with HincII can recombine with any DNA segment that also has a blunt end. Several enzymes leave blunt ends, and these blunt-end cutters can be used to recombine DNA sequences that do not have the same restriction enzyme cut sites.

; express foreign genes introduced in the recombinant DNA

Host Cells

Which of the following elements is not found in a plasmid? Antibiotic resistance Polylinker Lambda arms lacZ gene

Lambda Arms Lambda arms are regions that flank the inserted foreign DNA in phage λ vectors.

Which of the following molecules is not required for a PCR Reaction? -Ligase -Primer -DNA -DNTPs

Ligase Ligase is not required for a PCR reaction. The enzyme used during PCR is a thermostable DNA polymerase.

Restriction enzymes are used in all of the following molecular biology techniques EXCEPT __________. -mapping studies -cloning DNA into vectors -creating DNA libraries -PCR

PCR The polymerase chain reaction does not use restriction enzymes. Instead, it uses DNA primers to amplify selected regions of DNA.

Spectral karyotypes take advantage of which of the following?

Probes can be labeled with different color fluorophores.

Northern blots are used to study what type of molecule? -DNA -RNA and proteins -RNA -Proteins

RNA RNA fragments are separated using gel electrophoresis and blotted onto a nylon membrane. Labeled nucleic acid fragments are used to probe the filter and identify hybridizing sequences.

Which of the following statements about manual Sanger sequencing is true? The DNA sequence is read from the top of the gel to the bottom. The DNA sequence obtained is complementary to the template strand. Each of the four terminating ddNTPs is labeled with a different fluorescent dye. One sequencing reaction is performe

The DNA sequence obtained is complementary to the template strand The DNA fragments produced in sequencing reactions are synthesized by DNA polymerase to be complementary to the template strand.

Sanger sequencing is based on the order in which ddNTPs are added to a growing polynucleotide. Why are ddNTPs integral to the Sanger sequencing method? -They have a 2 hydroxyl that allows for extension of the polynucleotide. -They do not have a 2 hydroxyl, which does not allow the extension of the polynucleotide. -They do not have a 3 hydroxyl, which does not allow the extension of the polynucleotide. -They have a 3 hydroxyl that allows for extension of the polynucleotide.

They do not have a 3 hydroxyl, which does not allow the extension of the polynucleotide.

Which of the following statements about ddNTPs is true?

They have a hydrogen at the 3′ carbon of the sugar

What is the function of restriction endonucleases in bacteria?

They provide a defense mechanism against infection by viruses. Restriction endonucleases recognize and degrade viral DNA, thus preventing viral infections.

True/False? Within a six-base DNA recognition sequence, an enzyme that cuts between the 3rd and 4th bases from the 5' end will generate blunt ends.

True

True/Falase. Phage λ can carry larger DNA fragments than plasmids.

True. Phage vectors can carry DNA fragments of about 20 kb, whereas plasmids can only carry DNA of less than 15 kb.

;are plasmids, bacteriophages, or cosmids that receive, through ligation, a piece or pieces of foreign DNA.

Vectors

The SYBR green dye binds *double-stranded DNA*. The light emitted by SYBR green *increases* as the *amount of DNA* present increases. This is what allows real-time quantification of the DNA.

What is the order of the three main steps in a PCR? denaturation, annealing primers, elongation

A DNA fragment is introduced into the lacZ gene of a plasmid, which also contains a tetracycline resistance gene. What is the appearance of bacteria transformed with this plasmid if they are spread on plates containing tetracycline and Xgal? White colonies that are resistant to tetracycline Blue colonies that are resistant to tetracycline Blue colonies that are sensitive to tetracycline White colonies that are sensitive to tetracycline

White colonies that are resistant to tetracycline The presence of blue colonies means that the plasmid taken up by these bacteria is recombinant, since the lacZ gene was disrupted.

What is a probe in molecular biology? -a type of vector system -an instrument used to manipulate cells in culture -DNA or an RNA molecule used in hybridization reactions -Probes are not used in molecular biology.

a DNA or an RNA molecule used in hybridization reactions A probe is a labeled single strand of DNA or RNA used to locate its complementary sequence. Probes are commonly used in Southern and Northern blots, as well as in library screening.

A scientist is troubleshooting the synthesis of a cDNA library. The scientist performs both a Northern and a Southern blot. The Northern blot demonstrated the presence of RNA while the Southern blot indicated that no cDNA was present in the sample. What is likely to be the cause of the failed synthesis of the cDNA library? -proper primers -too many dNTPs -defective reverse transcriptase -temperature too cold for annealing

defective reverse transcriptase

Nucleic acid blotting is widely used in recombinant DNA technology. In a Southern blot, one generally ________. -ligates DNA with DNA ligase -cleaves RNA with restriction endonucleases -examines amino acid substitutions with radioactive probes -hybridizes filter-bound DNA with a DNA probe -hybridizes filter-bound RNA with a DNA probe

hybridizes filter-bound DNA with a DNA probe

What is the main purpose of a DNA probe? -cuts DNA targets -hybridizes to a target sequence -binds to proteins -extend the growing polynucleotide

hybridizes to a target sequence

X-Gal is included in the growth medium on which cells transformed with bacterial plasmids are grown. The reason X-Gal is included is to _______. -minimize the chances that a vector will re-circularize without incorporating a fragment of foreign DNA -eliminate bacteria that do not contain recombinant plasmid -identify bacteria that contain a recombinant plasmid -eliminate bacteria that do not contain plasmid DNA

identify bacteria that contain a recombinant plasmid Colonies produced from cells containing a recombinant plasmid are white, whereas colonies from cells containing a nonrecombinant plasmid are blue.

Recombinant DNA technologies are methods used to do which of the following? -join together DNA molecules from the same source -manipulate DNA for the study of specific sequences -induce homologous recombination inside a cell -integrate DNA in computers to make them faster -combine chromosomes to make them easier to study

manipulate DNA for the study of specific sequences

Recognition sequences for restriction enzymes possess the unique quality of being the same when read 5 to 3 on either strand. What is this property called? origin sequence recombination sequence consensus sequence gene sequence palindromic sequence

palindromic sequence

DNA ligase ________. -cuts the DNA to produce sticky or blunt ends -adds bases into a growing DNA molecule -removes bases from a DNA molecule -reconnects the bases together between the DNA strands -reconnects the phosphodiester linkage between bases on the same strand of DNA

reconnects the phosphodiester linkage between bases on the same strand of DNA

; cut DNA at specific sites.

restriction enzymes

Agarose gels separate DNA fragments based on what property? -size of the fragment -amount of agarose in the DNA -charge of the DNA molecule -amount of adenine bases in the sequence

size of the fragment

What is the function of a ddNTP in DNA sequencing?--methylation of guanine -termination of DNA synthesis -provide a 3 hydroxyl for continued elongation -enhancing the processivity of the polymerase

termination of DNA synthesis

During the hybridization/annealing phase of a PCR reaction, the primers bind to the target DNA sequence at a specific temperature. What effect would an increase in the GC content of the primer have on the optimal temperature for annealing the primer to a target DNA sequence?

the annealing temperature would increase

There are multiple cloning vector types in modern recombinant DNA technology ranging from plasmids to viral vectors. Which vector type is most useful when cloning an insert of approximately 500kb? -viral vector -yeast artificial chromosome -bacterial artificial chromosome -human artificial chromosome -bacterial plasmid

yeast artificial chromosome

What are the four processes common to most cloning experiments and order?

1. Cutting DNA with restriction endonuclease 2. Ligating DNA fragments 3. Transforming bacteria 4. Plating bacteria on selective medium

First, you would like to use FISH to confirm the position of the gene on chromosome 7 as well as the location of this part of the chromosome in an interphase nucleus. You decide to design a probe to hybridize to the genomic DNA containing this gene. Which of the following DNA probes could you use to detect this gene in mice? Select all that apply. 5' TTGGGAGCGG 3' 3' AACCCTCGCC 5' 5' AACCCTCGCC 3' 3' TTGGGAGCGG 5'

5' TTGGGAGCGG 3' 3' AACCCTCGCC 5' Although only the coding/nontemplate strand is given, the gene is present in the genome as double-stranded DNA. Therefore a probe can be made to hybridize to either strand. The probe must be complementary and antiparallel.

CTY*RAC GAR*YTG The lambda phage genome is 48.5 kb in size and has 50% GC content. Approximately how many times would you expect HincII to cut lambda phage DNA?

48 You can calculate the number of cut sites by determining the probability that the specific cut site sequence will be found in the genome. The probability that the first base, C, will be found in a particular position in the genome is ¼ because there are 4 possible bases (A, T, C, and G). The probability that a particular base will be a purine (represented as R in the cut site sequence) is the probability that the base will be an A (1/4) plus the probability that the base will be a G (1/4), which equals ½.You can use the multiplication rule to calculate the probability that all of the bases in the cut site sequence will occur in order in the genome:1/4 x 1/4 x 1/2 x 1/2 x 1/4 x 1/4 = 0.001Then, to determine the number of cut sites expected in the lambda genome, you need to multiply this probability by the size of the genome:48,500 x 0.001 = 48

Which of the following DNA sequences is one strand of a restriction enzyme recognition sequence? -5' GGATCC 3' -5' GGGGGG 3' -5' GGGTTT 3' -5' AAACCC 3'

5' GGATCC 3' The 5' → 3' sequence of the complementary strand would be the same as the 5' → 3' sequence of this strand; i.e., this sequence is symmetrical about the midpoint.

The PCR (polymerase chain reaction) protocol that is currently used in laboratories was facilitated by the discovery of a bacterium called Thermus aquaticus in a hot spring inside Yellowstone National Park, in Wyoming. This organism contains a heat-stable form of DNA polymerase known as Taq polymerase, which continues to function even after it has been heated to 95oC. Why would such a heat-stable polymerase be beneficial in PCR? -Each cycle includes a "hot" saturation phase (95°C), which allows the primers to anneal to the target DNA. -Each cycle includes a "hot" denaturation phase (95°C), which serves to sterilize the culture. -Each cycle includes a "hot" denaturation phase (95°C), which activates the Taq polymerase. -Each cycle includes a "hot" denaturation phase (95°C), which separates the hydrogen bonds that hold the strands of the template DNA together. -More than one of the above are correct.

Each cycle includes a "hot" denaturation phase (95°C), which separates the hydrogen bonds that hold the strands of the template DNA together.

The cDNA library uses *reverse transcriptase* to copy *mRNA* present in the cell to *DNA*. If cellular conditions change over time, the genes being expressed* could change as well resulting in a different cDNA library being generated.

Explain the three major steps in the polymerase chain reaction. -The first step is *denaturation* of the target DNA molecule by exposure to* high temperature*. -The second step is *annealing* of *primers* to the target DNA. -The third step is the *synthesis* of the target DNA sequence by a(n) *thermostable* DNA polymerase.

Which features make yeast artificial chromosomes (YACs) an excellent cloning tool? -The YAC allows for the expression of bacterial genes. -YACs are large but low copy number plasmids that can accept DNA inserts in the 100- to 300-kb range. -In their linear form, YACs contain telomeres at each end for stability, an origin of replication, and they can be used to clone up to 2-Mb pairs of DNA. A YAC also contains a yeast centromere along with selectable markers and a number of restriction sites. This allows for the insertion of up to 2-Mb pairs of DNA. -Eukaryotic genes are relatively small. Cloning into a YAC allows for the function and the structure of these genes to be studied.

In their linear form, YACs contain telomeres at each end for stability, an origin of replication, and they can be used to clone up to 2-Mb pairs of DNA. A YAC also contains a yeast centromere along with selectable markers and a number of restriction sites. This allows for the insertion of up to 2-Mb pairs of DNA. Like natural chromosomes, YACs contain telomere and centromere sequences and can be used to clone very large fragments of DNA. YACs have been very useful in genome projects.

The role of the primers in PCR is _______. -to denature the template DNA and define the target region -to define the target region and provide a 3' end that can be extended by taq polymerase -the annealing temperature is maintained until polymerase has finished extension of the new strands -solely to define the target region

to define the target region and provide a 3' end that can be extended by taq polymerase Correct. Primers bind to end of the target DNA strands, then taq polymerase synthesizes a new strand using the target DNA as a template.