1) The first codon translated by all eukaryotes is a. 2) The corresponding amino acid is.

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1 1) The first codon translated by all eukaryotes is a. 2) The corresponding amino acid is. 3) Translation is terminated when the ribosome encounters. 4) Nucleic acid chains are synthesized in the to direction. 5) The nucleotides in nucleic acids are connected by bonds True/False and why 6) Both Prokaryotes and Eukaryotes use the same stop codons. 7) The single most important feature of Taq DNA polymerase that makes it ideal for use in PCR is its high rate of polymerization of DNA. 8) Restriction enzymes with a four base palindrome will cut the DNA molecule into many more pieces than those that recognize a six base palindrome. 9) If you know the sequence of one strand of a double helix of DNA, you can predict the sequence of the other strand, since both sequences are identical. 10) The linear sequence of information in DNA leads to a linear sequence of amino acids through translation of the RNA message. 11) A colony of bacteria is the visible mass of bacteria all originating from a single cell. Therefore a bacterial colony is a clone. 12) The double helix of the DNA molecule can become two separate single helices if the phosphodiester bonds are broken by heating.

2 13) DNA cloning is the same thing as human cloning. 14) A gene is a piece of DNA that is inherited and which codes for a protein. If there is a change in the DNA sequence this can lead to a change in the protein sequence. 15) If there are 20% guanines in a DNA sequence, there must be 20% adenines, since both are purines. 16) A good plasmid or other cloning vector requires an origin of replication, a selectable marker and a restriction enzyme site to insert new DNA 17) Primer dimers form when PCR mixture includes primers which are complementary and have a free 5 Phosphate ends. 18) Restriction enzymes can leave overhanging sticky ends which can be used for DNA cloning when the single stranded DNA is complementary. 19) DNA with a higher AT content has a higher melting temperature. 20) In the DNA double helix base pairs are usually formed between purines and pyrimidines. 21) A mutation is a mistake that a DNA polymerase has made. 22) The Shine Delgarno Sequence can form base pairs with a rrna sequence. 23) Competent cells are eukaryotic cells that can pick up DNA and insert it into their chromosomes. 24) The genetic code is based on twenty codons.

3 25) Tryptophan and methionine are each encoded by a single codon. 26) Every nucleic acid triplet encodes an amino acid. 27) The third position in the codon is less significant for determining which amino acid is encoded. 28) Different codons can encode the same amino acid. 29) Only 20 different amino acids are found in proteins. 30) High ph is used to hybridize two complementary DNA strands to form a doublestranded molecule. 31) 5' endonucleases cleave nucleotides sequentially from the 5' end of a DNA fragment 32) A G:C base pair is more stable than an A:T base pair. 33) DNA is made up of a sugar-phosphate polymer containing nitrogenous bases. 34) During replication, DNA is synthesized in a 5' to 3' direction. 35) During transcription, RNA is synthesized in a 5' to 3' direction. PCR problem (12 points). 1. The DNA sequence shown below represents the coding or sense strand of a peptide hormone that is thought to affect differentiation of muscle tissue. Only the coding (or sense) strand is shown, as is the custom. Some spaces are added for easier counting. 5 ATG TGC TAC GAA GCA ACG TGT CAT GAG CGC TCC GCT ACT GGG CCC TAG TAA 3

4 a. Write the complementary sequence of the DNA underneath it and mark its 5 and 3 ends. b. To amplify this region, a DNA primer is synthesized with the sequence 5 GGGCCCAGTAGCGGA. This is used for a single cycle of polymerization. How long will the product be? c. A second primer synthetic DNA primer is added to the reaction. Its sequence is 5 TAC GAA GCA ACG TGT. Then, 20 cycles of PCR amplification are carried out using thermostable DNA polymerase. How long do you expect the final PCR product to be? d. Why was a thermostable polymerase used for PCR? e. Why is the hybridization temperature of the primers critical in PCR analysis? f. What are the 6 essential ingredients for a PCR reaction. Why is each needed? To what do the designations 3' and 5' refer to in the DNA?

5 Multiple Choice 1. Which of the following tools of recombinant DNA technology is INCORRECTLY paired with its use? a. Restriction enzyme - production of DNA fragments for gene cloning. b. DNA ligase - enzyme that joins cut ends of DNA by reforming the phosphodiester bonds. c. Ampicillin used in selection of bacteria making beta lactamase that can resist Ampicillin. d. Ethidium Bromide- used to determine the ph of solutions e. Agarose gel electrophoresis used to separate DNA fragments of different sizes. 2. "Gene library" is a term used to describe: a. a computerized listing of known DNA sequences. b. bacteria with plasmids containing DNA fragments representing the majority of the genetic information from a plant or animal. c. a collection of books about recombinant DNA technology. d. a compilation of the amino acid sequences of protein coding genes. e. a store that specializes in the sale of Levis. 3. You have found the DNA sequence that codes for the ability to play "On Wisconsin" on the kazoo while hopping on one leg. The DNA sequence is as follows: 5' CTA GTA AAC TGC ACG TTC CAT 3' What is the complementary pre-mrna sequence? 5' GAU CAU UUG ACG UGC AAG GUA 3' 5' ATG GAA CGT GCA GTT TAC TAG 3' 5' CUA GUA AAC UGC ACG UUC CAU 3' 5' AUG GAA CGU GCA GUU UAC UAG 3' 5' GAT CAT TTG ACG TGC AAG GTA 3' 4. What is the amino acid sequence for the appropriate polypeptide that would be synthesjzed? Asp-His-Leu-Thr-Cys-Lys-Val Met-Tyr-Val-Ala-Arg-Glu-Stop Met-Glu-Arg-Ala-Va1-Tyr-Stop Leu-Val-Asn-Cys-Thr-Phe-His Val-Lys-Cys-Thr-Leu-His-Asp

6 5. Under conditions where methionine must be the first amino acid, what protein would be coded for by the following mrna? 5'-CCUCAUAUGCGCCAUUAUAAGUGACACACA-3' A. pro his met arg his tyr lys cys his thr B. met arg his tyr lys cys his thr C. met arg his tyr lys D. met pro his met arg his tyr lys cys his thr E. arg his ser glu tyr tyr arg leu tyr ser 6. "Gene library" is a term used to describe: A. a computerized listing of known DNA sequences. B. bacteria with plasmids containing DNA fragments representing the majority of the genetic information from a plant or animal. C. a collection of books about recombinant DNA technology. D. a compilation of the amino acid sequences of protein coding genes. E. a store that specializes in the sale of Levis. 7. 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 8. 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 desired gene.

7 9. One of the most significant discoveries which allowed the development of recombinant DNA technology was: A. the discovery of antibiotics used for selecting transformed bacteria. B. the identification and isolation of restriction endonucleases permitting specific DNA cutting. C. the discovery of DNA and RNA polymerase allowing workers to synthesize any DNA sequence. D. the development of the polymerase chain reaction. E. the Southern technique for separation and identification of DNA sequences. 10. Which of the following bases pairs with adenine in RNA? A. Thymine. B. Guanine. C. Uracil. D. Cytosine. E. A and C 11. Show the replicated strands of the following piece of DNA. Old Strand : 5 GGC ATT GCG TTA TGC 3 New Strand: A. 5 CCG TAA CGC AAT ACG 3 B. 3 CCG TAA CGC AAT ACG 5 C. 3 GGC ATT GCG TTA TGC 5 D. 5 GGC ATT GCG TTA TGC3 E. None of the above 12. Show the messenger RNA that would result from the following piece of DNA. DNA Strand: 5 CCG TAA CGC AAT ACG 3 mrna: A. 3 GGC ATT GCG TTA ACG 5 B. 5 CGU AUU GCG UUA CGG 3 C. 5 GGC UTT GCG UUT UGC 3 D. 5 CCG UAA CGC AAU ACG E. None of the Above

8 13. The "Southern" technique involves: A. the detection of RNA fragments on membranes by specific radioactive antibodies. B. the detection of DNA fragments on membranes by a radioactive DNA probe. C. the detection of proteins on membranes using a radioactive DNA probe. D. the detection of proteins on membranes using specific radioactive antibodies. E. the detection of DNA fragments on membranes by specific radioactive antibodies. 14. A Northern involves which of the above? 15. A Western involves which of the above? 16. Which of the following statements is NOT true of Blue-White Cloning a. The blue colonies contain the inserts in their plasmids. b. Special strains of bacteria and plasmids are required. c. Special blue Beta Galactosidase is required in the agar plates along with antibiotics. d. The gene for Beta Galactosidase is interrupted by a multiple cloning site. If a new gene is inserted, the correct enzyme is no longer made. e. All of the above are true for Blue-White cloning. 17. What source of contamination in setting up PCR reactions that is eliminated by barrier tips? A. Mislabeled reagents. B. Aerosols during pipeting. C. Contaminated glassware. D. Contaminated lab clothing. E. Cells shed by lab workers. 18. Which of the following is the correct order of steps in a single PCR cycle? A. Denaturation, annealing, extension. B. Denaturation, extension, annealing. C. Extension, annealing, denaturation. D. Annealing, denaturation, extension. E. Annealing, denaturation, annealing. 18. The lac-z gene, which is found in many plasmid cloning vectors, encodes which of the following proteins? A. DNA polymerase B. Reverse transcriptase C. Beta-lactamase D. Beta-galactosidase E. DNA ligase

9 19. Which of the following is the optimal temperature for DNA synthesis by Taq DNA polymerase? A. 30 o C B. 37 o C C. 55 o C D. 72 o C E. 95 o C 20. If you performed five rounds of PCR on a single (single stranded) DNA template, how many DNA strands (single stranded) total would be present? A. 8 B. 16 C. 32 D. 64 E Restriction Enzymes A. Are only found in a single species of bacteria. B. Are useful in the lab because they are extremely stable. C. Cut prokaryotic DNA only at the sequences that have been specifically methylated. D. Recognize specific DNA sequences and cut the DNA at that location. E. All of the above 22. A plasmid is: A. A circular DNA molecule which can replicate independently B. A virus that infects bacteria C. A fragment of a chromosome that is transferred during bacterial conjugation. D. A molecule of DNA found inside a virus particle. E. A piece of DNA containing a multiple cloning site. Translation Problems What is a Bacillus licheniformis colony? Why is it important to use a single colony to start an experiment? Shown here is the double-stranded DNA sequence coding for human hemoglobin. Below the 2 strands is the one letter abbreviation for the amino acids of the hemoglobin protein.

10 a) Which strand is the template for transcription, the top or the bottom strand? b) What three nucleotide sequences are used as stop codons? What is the stop codon above? Cloning Questions

11 Enzyme Name Pst I Sma I EcoR I Sca I BamH I Recognition site CTGCA/G G/ACGTC CCC/GGG GGG/CCC G/TTAAC CAATT/G AGT/ACT TCA/TGA G/GATTC CCTAA/G You have some restriction enzymes in your freezer. (3 pts) Name one with a 5 overhang. Name one with a 3 overhang? Name one with a blunt end?

12 You are running a gel with the following plasmids cut with enzymes. Draw in the band(s) where they should run on the gel in the lanes specified. (10 pts) 1) pkan uncut 2) pkan cut with EcoR I 3) pkan cut with BamH I and Hind III 4) psoup cut with BamH I and Hind III 5) pkan cut with EcoR I, Sma I and Hind III Here is a map of pgem 7zf(+). Please answer the following questions about this plasmid. What is a polylinker and why is one useful in DNA Cloning? (2 points) How many pieces of DNA will you get if you cut this plasmid with the enzymes Xmn I and Sca I? What sizes are these pieces of DNA? (3 points)

13 What happens to the bacterial colony if there is an insert of DNA into the polylinker? Why? (2 points) What happens to the bacterial colony if there isn t an insert of DNA into the polylinker? Why? (2 points) What are the three essential features of a plasmid and give an example of each from this plasmid (6 points) The plasmid vector pet83 contains an ampicillin resistance gene (Amp) and a kanamycin resistance gene (Kan). The plasmid is cut at the BamHI site shown below, and successfully ligated with a compatible human BamHI fragment. The recombinant plasmid is then transformed into E. coli. The transformation mixture is then spread on petri plates with various antibiotics and allowed to grow. Part II (10 points) a) What safety characteristics are important in the E.coli cells used as competent cells in your plasmid transformation. (2 points)?

14 The Genetic Code Table 5' Base 3' Base U C A G _ _ _ U UUU =Phe UCU UAU =Tyr UGU =Cys U UUC_ UCC =Ser UAC_ UGC_ C UUA =Leu UCA UAA =Stop UGA =Stop A UUG_ UCG UAG_ UGG =Trp G _ C CUU CCU CAU =His CGU U CUC =Leu CCC =Pro CAC_ CGC =Arg C CUA CCA CAA =Gin CGA A CUG CCG CAG_ CGG G _ _ _ A AUU ACU AAU =Asn AGU =Ser U AUC =Ile ACC =Thr AAC_ AGC_ C AUA_ ACA AAA =Lys AGA =Arg A AUG =Met ACG AAG_ AGG_ G G GUU GCU GAU =Asp GGU U GUC =Val GCC =Ala GAC_ GGC =Gly C GUA GCA GAA =Glu GGA A GUG GCG GAG GGG G