B. Incorrect! Ligation is also a necessary step for cloning.

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1 Genetics - Problem Drill 15: The Techniques in Molecular Genetics No. 1 of Which of the following is not part of the normal process of cloning recombinant DNA in bacteria? (A) Restriction endonuclease digestion of cellular and plasmid DNAs. (B) Production of recombinant DNA using DNA ligase and a mixture of digested cellular and plasmid DNAs. (C) Separation of recombinant DNAs by electrophoresis using the Southern technique to determine where the desired recombinant migrates. (D) Transformation of bacteria by the recombinant DNA plasmids and selection using ampicillin. (E) Probing blots of bacteria clones with radioactive DNA complementary to the target gene. In fact, discovery of restriction enzymes made the recombinant DNA technology possible. Restriction endonuclease digestion is necessary for cloning. Ligation is also a necessary step for cloning. C. Correct! Determining the desired recombinant DNA does not need Southern blotting; normal ethidium bromide staining is sufficient. Transformation is necessary for putting the recombinant DNA into bacteria for amplification. Colony hybridization is often used to determine which clone is the desired one. Cloning follows certain steps and every step is important to understand the whole procedure. (C)Separation of recombinant DNAs by electrophoresis using the Southern technique to determine where the desired recombinant migrates.

2 No. 2 of The "Southern" technique involves. (A) The detection of RNA fragments on membranes by specific radioactive DNA probes. (B) The detection of DNA fragments on membranes by radioactive DNA probes. (C) The detection of protein on membranes using radioactive antibodies. (D) The detection of proteins on membranes using specific radioactive antibodies. (E) The detection of DNA fragments on membranes by specific radioactive antibodies. This is a Northern blot, not Southern. B. Correct! To detect DNA on membranes using DNA probes is a procedure developed by a scientist named Southern. Radiolabeled antibodies are rarely used to detect proteins in a lab. Radiolabeled antibodies are rarely used to detect proteins in a lab. DNA is rarely detected directly using antibodies. Southern blot was developed by a scientist named, Southern; Northern blot (using DNA probe to detect RNA) and Western blot (using antibody to detect protein on the membrane) followed that naming convention and are not named after their inventors. (B)The detection of DNA fragments on membranes by radioactive DNA probes.

3 No. 3 of Chromosomal DNA from a eukaryotic organism is digested with a restriction endonuclease and the resulting fragments cloned into a plasmid vector. Bacteria transformed by these plasmids collectively contain all of the genes of the organism. This culture of bacteria is referred to as a. (A) cdna library (B) Restriction map (C) Genomic map (D) Genomic DNA library (E) Lysogenic phage cdna library are made from cdna, which is reverse-transcribed from mrna. Restriction map is the location and arrangement of restriction enzyme sites. Genomic map is either a restriction map or a genetic map. D. Correct! The DNA source for a genomic DNA library is the chromosomal DNA. Normally, genomic DNA is constructed using a phage vector, but this one is not. Key point: Learning the vocabulary of biotechnology will greatly enhance a student s ability to understand the methods and techniques used in this area of science. (D)genomic DNA library

4 No. 4 of Restriction endonuclease generated DNA fragments separated by gel electrophoresis and blot transferred onto a membrane filter are probed with a radioactive DNA fragment. This procedure is called. (A) Gene Cloning (B) The Southern technique (C) The polymerase chain reaction (D) Recombinant DNA (E) Gene Mapping Gene cloning is the procedure to make recombinant DNA. B. Correct! The Southern Blot technique involves the absorption of DNA fragments of interest onto a membrane filter, which is then probed by radioactive DNA that have complementary sequences to the target gene. PCR involves primers and DNA polymerase, not radioactive DNA fragment. Recombinant DNA involves putting two pieces of DNA together. Gene mapping is a totally different technique that is used in classical genetics. Southern blot is a very important technique in early stages of molecular genetics. (B)The Southern technique

5 No. 5 of The requirement for a vector include all the following except. (A) A replication origin (B) Restriction enzyme sites (C) Ribosome binding sites (D) Selection markers (E) All of the above Replication origin is critical for a vector because it enables the plasmid to replicate in bacterial cells. The restriction enzyme site enables a plasmid to be recombined with another piece of DNA. The ribosome binding site is for protein translation; it is not needed for regular cloning. D. Correct! Selection markers are needed for selecting the positive clones. There is only one correct answer. Vectors are the carriers for foreign DNA to survive and amplify in a host cell. (D)Selection markers

6 No. 6 of Which of the following statements about polymerase chain reaction (PCR) is correct? (A) PCR is used to amplify a small segment of DNA millions of times. (B) PCR is used to amplify a genome millions of times. (C) Step 1 is when primers anneal to the template DNA. (D) A special machine, known as an incubator, is used for PCR cycles. (E) The DNA polymerase used to amplify the DNA comes from the same species as the original sample of nucleic acid. A. Correct! PCR is used to amplify small segments of DNA with primers. PCR is used to amplify small segments of DNA with primers. Step 1 is when the double-stranded DNA is melted to separate the strands. A thermal cycler is used to heat and cool the DNA mixture quickly for short periods of time. A special heat-stable DNA polymerase from thermos, Thermus aquaticus, is typically used. PCR stands for polymerase chain reaction and has been described as one of the most significant inventions in molecular biology. PCR is a method used to amplify a small segment of DNA millions of times. The process allows the sequencing of specific genes or DNA targets. It is fast and inexpensive. PCR is used in forensics, medicine development, diagnostics and research. (A)PCR is used to amplify a small segment of DNA millions of times.

7 No. 7 of What is a restriction enzyme? (A) It is an endonuclease that cuts the peptide bond between nucleotides. (B) An enzyme that cuts randomly within a nucleotide sequence. (C) Restriction enzymes that are found in normal eukaryotic cells. (D) Restriction enzymes that are found in bacteria. (E) A special enzyme that cuts only within their recognition sequences. A restriction enzyme is an endonuclease that cuts the phosphodiester bond between nucleotides. A restriction enzyme cuts at specific sites, which facilitates their usefulness. Restriction enzymes are found in bacteria. D. Correct! Restriction enzymes are found in bacteria. A type 1 restriction enzyme cuts far from their recognition sequence and type 2 cuts close to or within their recognition sequence. An endonuclease is an enzyme that cuts the phosphodiester bond within a polynucleotide chain. Restriction enzymes cut at specific sites and these are divided into three categories: Type l, II and III. The restriction endonucleases are found in bacteria and they recognize specific DNA sequences. When the DNA is cut, it creates either blunt ends or sticky ends. (D)Restriction enzymes that are found in bacteria.

8 No. 8 of 10 Instructions: (1) Read the problem and answer choices carefully (2) Work the problems on paper as 8. A plasmid is. (A) An extra-chromosomal DNA without an origin. (B) An extra-chromosomal DNA with an origin. (C) A DNA molecule without a dominant selection marker. (D) A random sequence of DNA with no specific restriction enzyme sites. (E) Chromosomal DNA with more than one origin. A plasmid contains an origin to facilitate replication. B. Correct! Extra-chromosomal DNA with an origin. A plasmid typically contains a selection marker to correctly identify their location in a culture. A plasmid contains unique restriction sites to facilitate cloning of the foreign DNA into the vector. A plasmid DNA sequence is an extra-chromosomal sequence of DNA, which can be replicated independent of the chromosomal DNA. A plasmid: 1. Contains an origin, which allows the plasmid to replicate in E. coli. 2. Contains a dominant selection marker, which allows the transformed E. coli to distinguish from untransformed cells, often antibiotic-resistant genes. 3. Contains unique restriction enzyme sites, which allow cloning of the foreign DNA into the vector. (B)An extra-chromosomal DNA with an origin.

9 No. 9 of The process of making recombinant DNA includes all the following except:. (A) Two samples of double-stranded DNA that are digested with a restriction enzyme, such as Bam l. (B) The generation of sticky ends. (C) Sticky ends are ligated with DNA ligase. (D) After DNA ligase, a nuclease enzyme cleaves randomly in the newly formed DNA. (E) The introduction of foreign DNA into a host DNA sequence. This is true, and it generates the necessary sticky ends. Sticky ends are needed to ligate the DNA. DNA ligase does anneal the sticky ends. D. Correct! After DNA ligase, the recombinant DNA sequence is finished. Foreign DNA is recombined with host DNA to make a recombinant DNA sequence. Making Recombinant DNA: First, it uses two samples of double-stranded DNA to digest them with the enzyme Bam I. This generates DNA with sticky ends. The sticky ends are annealed together using a DNA ligase. The ligated molecule is the new recombinant DNA structure. (D)After DNA ligase, a nuclease enzyme cleaves randomly in the newly formed DNA.

10 No. 10 of Which of the following statement about electrophoresis is true? (A) A sample is loaded onto a gel for passive movement through the gel matrix. (B) DNA has a net negative charge, so it migrates towards the positive pole. (C) A gel electrophoresis apparatus has two positive poles to attract the DNA sample. (D) Samples can only be separated based on charge during electrophoresis. (E) After separation of the samples, the DNA is ready to visualize with no further steps. Gel electrophorsis requires current to move the sample. B. Correct! DNA has a net negative charge, so it migrates towards the positive pole. There is one positive and one negative pole. The gel can act as a molecular sieve and separate the samples based on their size also. In order to visualize the results, a dye or radioactive label must be used to label the DNA. A sample containing DNA is loaded on the end of the gel and the electrical current applied. DNA has a net negative charge, so it migrates toward the positive pole. In addition to migrating toward the + pole, the gel acts as a molecular sieve, which separates the molecules by size. A gel made up of acrylamide and SDS is made. On either end of the gel, two electrodes are attached having a +/- electrical charge. Electrophoresis is a method that is used to separate molecules based on their size and electric charge. (B)DNA has a net negative charge, so it migrates towards the positive pole.

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