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BIOL 3161 Exam 2
BIOL 3161 Exam 2 Lecture 4
| Question | Answer |
|---|---|
| Machines that automate the chemical reactions for DNA | DNA synthesizers or gene machines |
| What do gene machines make? | Single-stranded DNA of about 50 nucleotides or less (called oligonucleotides) |
| How does the DNA synthesis reaction in the gene machine differ from the biological synthesis? | Does not occur in biological direction – nucleotides are added in the 3’ to 5’ direction |
| What are the chemically synthesized single-stranded DNA used for? | Assembling whole genes, amplifying specific DNA sequences, making mutations in genes, screening DNA libraries, sequencing DNA |
| What is the most commonly used method to chemically synthesize DNA? | Phosphoramidite Method |
| How is chemical synthesis of DNA performed in the creation of a Microarray? | Chemical synthesis is done directly on a chip or glass slide. |
| This is a 2-D array of 1,000’s or 10,000’s of DNA sequences attached to a flat glass surface in a defined location | Microarray |
| This technology can be used to synthesize an entire gene | Microarray (gene chips) |
| Microarrays can be used as _______ ________ | Diagnostic probes |
| What was the first virus synthesized from synthetic oligos? | Poliovirus |
| Disadvantages of Chemical Synthesis of DNA | -less efficient than enzymes (slower and lower yield) -limited to single-stranded sequences <~ 150 bases -cost of synthesis and purification |
| Research Scale Oligo Market | -PCR and sequencing primers -genetic engineering -DNA probes -RNA interference (RNAi) |
| Pharmaceutical Oligo Market | Nucleic acid based drugs potential $ billion/yr markets ($$$$$$) |
| Why is DNA sequencing important? | Function of gene can be determined from sequence also it is essential for DNA cloning |
| Major method of DNA sequencing | Chain termination sequencing, dideoxy sequencing or Sanger sequencing (named after biochemist Frederick Sanger) |
| What is the key principle of Sanger Sequencing? | Dideoxynucleotides (ddNTPs) as DNA chain terminators |
| Why are ddNTPs used in Sanger Sequencing? (what is their role) | ddNTPs lack a 3' -OH group that is required for the formation of a phosphodiester bond. They act as chain terminators since the oxygen has been removed and elongation will not longer occur (no phosphpdiester bond, no elongation) |
| How does a dideocynucleotide (ddNTP) differ from a deoxynucleotide (dNTP)? | ddNTP has no OH group on 3' end (has H instead) dNTP has OH group on 3' end |
| Step 1 of Sanger Sequencing | 1. the ss DNA sample is divided into four separate sequencing reaction tubes |
| Step 2 of Sanger Sequencing | 2. radioactively labeled primer, DNA polymerase, and a mixture of dGTP, dATP, dCTP, and dTTP (dNTPs) added to each tube |
| Step 3 of Sanger Sequencing | 3. chain terminators (ddNTPs) added to all four tubes - one tube is used for each specific nucleotide (ddGTP, ddATP, ddCTP, and ddTTP) |
| Step 4 of Sanger Sequencing | 4. samples are run on a denaturing polyacrylamide gel using one lane per reaction tube |
| Step 5 of Sanger Sequencing | 5. DNA bands are then visualized and the positions of the different bands among the four lanes are used to determine the DNA sequence (read from bottom to top) |
| What is Automated Sequencing? | same method as Sanger method except done by machine using fluorescently labeled chain primers |
| What is Polymerase Chain Reaction (PCR)? | Amplification of small amounts of DNA into larger amount |
| How was PCR developed? | Kary Mullis 1984- based on discovery of DNA polymerases found in thermophiles – Taq polymerase work at 100 °C |
| What are the 4 things needed for a PCR reaction? | Target DNA, Primer(s), Taq polymerase, Nucelotides |
| What do primers do? Why are they powerful? | Primers are short strands of DNA that adhere to target, providing a starting place for replication. |
| What is Taq polymerase | Enzyme in charge of DNA replication |
| What are the steps of PCR | 1. Denaturation 2. Annealing 3. Elongation (Extension) |
| What happens during denaturation? | PCR tube is heated to 95 °C to break double helix and separate into single strands of DNA |
| What happens during annealing? | PCR tube is lowered to 50 °C and the primers then attach to each single strand |
| What happens during elongation? | PCR tube is heated to 70 °C to activate Taq polymerase which adds complementary nucleotides to each single strand sequence |
| Name 5 important considerations for PCR reactions | 1. Contamination 2. Annealing Temp 3. Mg concentration (influences Taq pol) 4. Primer design - must anneal at same temp 5. More is not better! too much Taq pol produces more nonspecific product. Rxn will not work if large amt of DNA used |
| What are the industrial applications of proteins | 1. Medical 2. Food processing 3. Textiles/leather 4. Detergents 5. Paper manufacturing/recycling 6. Adhesives 7. Bioremediation |
| What are the 2 major phases of protein production? | 1. Upstream processing 2. Downstream processing |
| This is the field of study that is dedicated to production and metabolism of proteins | Proteomics |
| Specific methods used to produce proteins in the _______ and _______required for use | quantity and quality |
| What is upstream processing? | creating recombinant DNA containing gene of interest, selecting a host cell, transforming host cells, host cell expressing protein |
| What is downstream processing? | protein is separated from other parts of the cell and isolated from other proteins, purity and activity of protein is verified, stable means of preserving protein is developed |
| Name the steps in upstream processing | 1. Creating recombinant DNA 2. Selecting host cells 3. Transforming host cells 4. Host cell makes protein |
| What factors should you consider in upstream processing? | 1. Quantity of protein produced 2. Quality of protein produced 3. Metabolic load |
| What method is used in transformation of the host cells? | Calcium Chloride or Electroporation |
| How does electroporation work? | uses electroporators (appliances to create electric current) A cell solution plus DNA are pipetted into a cuvet, placed into electroporator and electric current goes thru cuvette which disturbs phospholipid bilayer allowing DNA to pass into cells |
| What factors affect quantity in upstream processing? | 1. Strength of promoter 2. Temp of cultures 3. Copy number 4. Ribosome binding site |
| This influences the amount of protein produced in upstream processing | strength of the promoter |
| How does temp affect the quantity of proteins produced in upstream processing? | Temp influences the copy number of recombinant DNA carrying your gene |
| How does copy number affect protein quantity in upstream processing? | A high copy number contrains host whereas a low copy number is preferred (multiple low copy numbers |
| How does the ribosome binding site affect protein quantity in upstream processing? | recombinant DNA carrying gene of interest is designed in such a way that the mRNA for the gene contains a strong ribosome binding sites (sequences that can obtain secondary structure avoided) |
| What is a disadvantage os using E. coli? | proteins contained in intracellular inclusion bodies, proteins can not be folded or modified in ways needed for mammalian systems, some proteins can be produced only in eukaryotic cells |
| What is a problem encountered in host cell proteases? | sometimes foreign proteins made by host cells are degraded by proteases |
| How do you resolve the problem encountered in host cell proteases? | Fusion proteins- the gene for the protein you are making is fused to one of the host protein-encoding genes (the product is a fusion protein) |
| When are fusion proteins engineered? | When the recombinant DNA molecule is made (step 1) |
| What are the problems with fusion proteins? | Not suitable for clinical use, may affect biological function of target protein, requires more extensive testing before being approved by regulatory agencies |
| What happens in step 1 of Phosphoramidite Method? | 3' terminal nucleoside is added to a spacer molecule which is attached to a support (glass bead) |
| What happens in step 2 of Phosphoramidite Method | Chain is extended in a cyclical cycle |
| What is the cyclical cycle in step 2 of the Phorphoramidite Method? | Deprotection Coupling Capping Stabilization |
| What happens during deprotection in step 2 of Phosphoramidite Method? | DMT blocking group is removed |
| What happens during coupling in step 2 of Phosphoramidite Method? | desired base is added |
| What happens during capping in step 2 of Phosphoramidite Method? | If the wrong base is added the nucleotide is capped with another molecule to prevent the addition of another base. Since the nucleotide is too short its washed away and you start all over again |
| What happens during stablization in step 2 of Phospohoramidite method? | The growing nucleotide is stabilized to prevent cleavage of the phosphodiester bond (oxidation) |
| What happens in step 3 of the Phosphoramidite Mehtod? | Product remains attached to support and unicorporated nucleotides are washed away |
| What happens in step 4 of Phosphoramidite Method? | After synthesis linkage to the support are cleaved to release product |
| What happens in step 5 of the Phosphoramidite Method? | Protecting group removed and full length product is isolated- End result single stranded DNA |
| What is the end product in the Phosphoramidite Method? | Single stranded DNA |
| What happens if you need to synthesize double stranded DNA using the Phosphoramidite Method? | You make two single stranded DNA segments and combine them |
| These microorganisms have a sticky coat of metallothioneins to capture heavy metals like mercury are used in ____________? | Bioremediation |
| Bioremediation uses organisms that have a sticky coat of _________ to capture heavy metals like mercury. | Metallothioneins |
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amandap
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