Molecular Cell Biology - Problem Drill 11: Recombinant DNA

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1 Molecular Cell Biology - Problem Drill 11: Recombinant DNA Question No. 1 of Which of the following statements about the sources of DNA used for molecular cloning is correct? Question #1 (A) cdna is complementary DNA, the DNA synthesized from a mature DNA template. (B) cdna is complementary DNA, the DNA synthesized from a mature mrna template. (C) Restriction enzymes synthesize the cdna. (D) Genomic DNA is DNA isolated from organisms, containing a small portion of DNA from the genome of a cell or an organism. cdna is complementary DNA, the DNA synthesized from a mature mrna template. B. Correct! cdna is complementary DNA, the DNA synthesized from a mature mrna template. Restriction enzymes cut the DNA within the molecule. Genomic DNA is DNA isolated from organisms, containing all DNA in the genome of a cell or an organism. To make recombinant DNA, we need DNA source, either genomic DNA or cdna; restriction enzymes, which cut DNA within the molecule; vectors which carry special DNA sequence that can ensure amplification in a host cell; DNA ligase which can put two pieces of DNA molecules together, and finally, host cells, usually E. coli. DNA source are DNA originated from organisms, which can be obtained in two ways: cdna or genomic DNA isolation; or PCR amplification. cdna is complementary DNA, the DNA synthesized from a mature mrna template. cdna is often used to clone genes; genomic DNA is the DNA isolated from organisms, containing all DNA in the genome of a cell or an organism.

2 Question No. 2 of Which of the following statements about polymerase chain reaction (PCR) is correct? Question #2 (A) Taq DNA polymerase adds nucleotides to the 5 end of the primer, to extend the strand. (B) Taq DNA polymerase adds nucleotides to the 3 end of the genomic DNA, to extend the strand. (C) Primers are short single stranded DNA that is complementary to one region of the template DNA. (D) Primers are short single stranded RNA that is complementary to one region of the template DNA. Taq DNA polymerase adds nucleotides to the 3 end of the primer, to extend the strand. Taq DNA polymerase adds nucleotides to the 3 end of the primer, to extend the strand. C. Correct! Primers are short single stranded DNA that is complementary to one region of the template DNA. Primers are short single stranded DNA that is complementary to one region of the template DNA. PCR is short for polymerase chain reaction, it requires template, original double strand DNA from an organism. Primers: short single strand DNA that is complementary to one region of the template DNA; Taq DNA polymerase: add nucleotides to the 3 end of the primer, extend the strand. For amplification: 30 cycles will generate 230 molecules.

3 Question No. 3 of Which of the following statements about restriction enzymes is correct? Question #3 (A) Restriction endonucleases are found in normal cells. (B) Restriction endonucleases are found in viruses. (C) When DNA is cut by a restriction enzyme; it generates either blunt ends or sticky ends of the DNA. (D) When DNA is cut by a restriction enzyme; it leads to the degradation of the blunt ends of the DNA. Restriction endonucleases are found in bacteria. Restriction endonucleases are found in bacteria. C. Correct! When cut by a restriction enzyme, it generates either blunt ends or sticky ends of the DNA. When cut by a restriction enzyme, it generates either blunt ends or sticky ends of the DNA. Restriction endonuclease are found in bacteria, they recognize specific DNA sequences, modify and cut DNA. When cut, it generats either blunt ends or sticky ends of the DNA.

4 Question No. 4 of Which of the following statements about the different types of restriction enzymes is correct? Question #4 (A) Type I: Complex, multisubunit, combination restriction-and-modification enzymes which cut DNA at random far from their recognition sequences. (B) Type I: Complex, multisubunit, combination restriction-and-modification enzymes which cut DNA within the recognition sequences. (C) Type II: Cut DNA at random far from their recognition sequences. (D) Type III: Cut DNA at defined positions close to or within their recognition sequences. The only class used in the laboratory for DNA analysis and gene cloning. A. Correct! Type I: Complex, multisubunit, combination restriction-and-modification enzymes which cut DNA at random far from their recognition sequences. Type I: Complex, multisubunit, combination restriction-and-modification enzymes which cut DNA at random far from their recognition sequences. Type II: Cut DNA at defined positions close to or within their recognition sequences. The only class used in the laboratory for DNA analysis and gene cloning. Type III: Large combination restriction-and-modification enzymes. Require two recognition sequences in opposite orientations. There are three types of restriction enzymes: Type I: Complex, multisubunit, combination restriction-and-modification enzymes that cut DNA at random far from their recognition sequences. Type II: Cut DNA at defined positions close to or within their recognition sequences. The only class used in the laboratory for DNA analysis and gene cloning. Type III: Large combination restriction-and-modification enzymes. Require two recognition sequences in opposite orientations.

5 Question No. 5 of Which of the following statements about vectors is correct? Question #5 (A) All DNA pieces can survive in the cell, and vectors facilitate this. (B) There are two types of vectors, cloning vectors and restriction vectors. (C) There are two types of vectors, cloning vectors and expression vectors. (D) The cloning vector is for reproducing all genomic DNA in the cell. Not all DNA pieces can survive in the cell, vectors are designed to ensure DNA survives and replicates. There are two types of vectors, cloning vectors and expression vectors. C. Correct! There are two types of vectors, cloning vectors and expression vectors. The cloning vector is for reproducing a DNA fragment in the cell. Not all DNA pieces can survive in the cell, vectors are designed to ensure DNA survive and replication. By definition, Vector is an agent that can carry a DNA fragment into a host cell. There are two types of vectors, cloning vector and expression vector. The cloning vector is for reproducing DNA fragment in cell, and expression vector is for gene expression in cells.

6 Question No. 6 of Which of the following statements about the required features of a plasmid vector is correct? Question #6 (A) An ori sequence: allows the transformed E. coli to be identified through antibiotic resistance. (B) An ori sequence: allows the plasmid to replicate in all normal cells. (C) A dominant Selection marker: allow the transformed E. coli distinguish from untransformed cells, often antibiotic resistance genes. (D) A dominant Selection marker: provides the necessary unique sites for the restriction enzyme to cleave the DNA. An ori sequence: allows the plasmid to replicate in E. coli. An ori sequence: allows the plasmid to replicate in E. coli. C. Correct! A dominant Selection marker: allow the transformed E. coli distinguish from untransformed cells, often antibiotic resistance genes. A dominant Selection marker: allow the transformed E. coli distinguish from untransformed cells, often antibiotic resistance genes. A plasmid vector must have three features: An ori sequence: allows the plasmid to replicate in E. coli. A dominant Selection marker: allow the transformed E. coli distinguish from untransformed cells, often antibiotic resistance genes. Unique restriction enzyme sites: allow cloning of the foreign DNA into the vector.

7 Question No. 7 of Which of the following statements about cdna library construction is correct? Question #7 (A) Step 1: isolate cdna, Step 2: making mrna: reverse transcription, Step 3: Clone cdna into vector, Step 4: introduce the clones into cells. (B) Step 1: isolate mrna, Step 2: making cdna: reverse transcription, Step 3: Clone cdna into vector, Step 4: introduce the clones into cells. (C) During Step 3, the mrna is cloned into a vector. The cdna is then ligated into vector to create a pool of recombinant plasmids. (D) During Step 3, the cdna is cloned into a vector. The cdna is then degraded within the vector to create a pool of recombinant plasmids. Step 1: isolate mrna, Step 2: making cdna: reverse transcription, Step 3: Clone cdna into vector, Step 4: introduce the clones into cells. B. Correct! Step 1: isolate mrna, Step 2: making cdna: reverse transcription, Step 3: Clone cdna into vector, Step 4: introduce the clones into cells. During Step 3, the cdna is cloned into a vector. The cdna is then ligated into vector to create a pool of recombinant plasmids. During Step 3, the cdna is cloned into a vector. The cdna is then ligated into vector to create a pool of recombinant plasmids. cdna library construction involves a few steps: Step 1 is to isolate mrna. mrna is extracted from tissue. Step 2 is to make cdna. mrna is mixed with cdna, and reverse transcriptase is added to synthesize cdna from mrna. After that, second strand cdna is synthesized by DNA polymerase. Not all DNA pieces can survive in the cell, vectors are designed to ensure DNA survive and replication. There are two types of vectors, cloning vector and expression vector. The cloning vector is for reproducing DNA fragment in cell, and expression vector is for gene expression in cells. Step 3 is to clone the cdna into a vector. A vector is cut with restriction enzymes, and the polylinkers are added to the end of cdna to create digested enzyme sticky ends. The cdna is then ligated into vector to create a pool of recombinant plasmids.

8 Question No. 8 of Which of the following statements about electrophoresis of DNA is correct? Question #8 (A) During Gel Electrophoresis, the negatively charged DNA molecule, and is moved by electric current through a matrix of agarose. (B) During Gel Electrophoresis, the positively charged DNA molecule, and is moved by electric current through a matrix of agarose. (C) The rate of migration is inversely proportional to size, the smaller fragments moving more quickly. (D) The rate of migration is proportional to size, the smaller fragments moving more slowly. A. Correct! During Gel Electrophoresis, the negatively charged DNA molecule, and is moved by electric current through a matrix of agarose. During Gel Electrophoresis, the negatively charged DNA molecule, and is moved by electric current through a matrix of agarose. The rate of migration is proportional to size, the smaller fragments moving more quickly. The rate of migration is proportional to size, the smaller fragments moving more quickly. Gel Electrophoresis: DNA is a negatively charged molecule, and is moved by electric current through a matrix of agarose. The rate of migration is proportional to size, the smaller fragments moving more quickly. DNA is visualized by staining with ethidium bromide fluoresce under UV light

9 Question No. 9 of Which of the following statements about Southern Blotting is correct? Question #9 (A) First DNA is separated on a gel, and then it is blotted onto a membrane. (B) First RNA is separated on a gel, and then it is blotted onto a membrane. (C) After blotting onto a membrane, it is hybridized with a restriction enzyme. (D) After restriction enzyme cleavage it is hybridized with a restriction enzyme. A. Correct! First DNA is separated on a gel, and then it is blotted onto a membrane. First DNA is separated on a gel, and then it is blotted onto a membrane. After blotting onto a membrane, it is hybridized with a radiolabelled probe. After blotting onto a membrane, it is hybridized with a radiolabelled probe. Southern blot is named after Edward M. Southern who developed this procedure. The purpose is to locate a particular sequence of DNA. First DNA is separated on a gel, then it is blotted to a membrane and hybridized with a radiolabelled probe, the membrane is then exposed to an X-ray film and the specific DNA is identified.

10 Question No. 10 of Which of the following statements about Northern Blotting is correct? Question #10 (A) Northern Blot: is a method to analyze gene transcripts, the first step is to isolate cloned DNA. (B) Northern Blot: is a method to analyze gene transcripts, the first step is to isolate total RNA. (C) The purpose of a Northern Blot is to compare the gene DNA accumulation in different conditions. (D) The purpose of a Northern Blot is to compare the mrna from normal cells and bacteria. Northern Blot: is a method to analyze gene transcripts, the first step is to isolate total RNA. B. Correct! Northern Blot: is a method to analyze gene transcripts, the first step is to isolate total RNA. The purpose of a Northern Blot is to compare the gene mrna accumulation in different conditions. The purpose of a Northern Blot is to compare the gene mrna accumulation in different conditions. Northern blot is similar to Southern blot, but it is a method to analyze gene transcripts. First total cellular RNA is isolated and separated on a gel, the RNA is blotted to a membrane and probed with a radioactive DNA probe to detect the mrna accumulation in different conditions.

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