BTE 101 Quiz Important Points PDF

Important Points for BTE 101 Quiz Lecture 7 1. Recombinant DNA technology involves - (i) the selection of the desired gene for administration into the host. (ii) followed by a selection of the perfect vector with which the gene has to be integrated hence the recombinant DNA...

Important Points for BTE 101 Quiz Lecture 7 1. Recombinant DNA technology involves - (i) the selection of the desired gene for administration into the host. (ii) followed by a selection of the perfect vector with which the gene has to be integrated hence the recombinant DNA is formed. (iii) recombinant DNA then has to be introduced into the host. 2. DNA ligase is isolated commercially from E coli and Bacteriophage. 3. DNA ligase joins the DNA fragments with cloning vector. 4. Restriction Endonuclease enzymes recognize specific sequence called restriction site and cut accordingly. 5. Restriction Endonuclease is isolated from a wide variety of microorganisms. 6. Endonuclease enzyme degrades foreign genomes when they enter inside microbial cells but the host cell's own DNA is protected from its endonuclease by methylation of bases at the restriction site. 7. Terminal Transferase enzyme converts blunt end of DNA fragments into sticky end. 8. If the restriction enzyme cuts DNA forming blunt ends, then efficiency of ligation is very low. 9. Terminal transferase enzyme synthesize short sequence of complementary nucleotide at free ends of DNA, so that blunt end is converted into sticky end. 10. DNA Polymerase - (i) is a complex enzyme which synthesize nucleotide complementary to template strand (ii) it adds nucleotide to free 3’ OH end and help in elongation of strand (iii) it also helps to fill gap in double stranded DNA 11. Alkaline Phosphatase - (i) helps in removal of terminal phosphate group from 5′ end (ii) It prevents self annealing of vector DNA soon after cut open by restriction endonuclease 12. Polynucleotide Kinase adds phosphate group from ATP molecule to terminal 5’ end after dephosphorylation by alkaline phosphatase. 13. There are five basic steps in rDNA technology. 14. Production on an industrial level involves - (i) Culturing the host cells on a suitable medium on a large scale (ii) Extraction of the desired product (iii) Downstream processing of the products 15. Gene splicing is to cut out DNA of one organism at a specific site. 16. Vector – molecule of DNA which is used to carry a foreign gene into a host cell. 17. Transformation - process of introducing free DNA into bacteria 18. Competent cell - a cell that is capable of taking up DNA. 19. Electroporation - The use of an electric shock to disrupt cell walls. 20. Transformed cell - cell with new DNA. 21. Marker gene - a gene that identifies which organisms have been successfully transformed. 22. pBR322 is a plasmid vector. 23. tetr - the gene that gives resistance to the antibiotic Tetracycline. 24. ampr - the gene that gives resistance to the antibiotic Ampicillin. 25. The most common application of recombinant DNA is in basic research. 26. Recombinant proteins are widely used as reagents in laboratory experiments and to generate antibody probes for examining protein synthesis within cells and organisms. 27. Application of recombinant DNA technology in Agriculture For example, manufacture of Bt Cotton to protect the plant against ball worms. 28. Bacteria can make human insulin or human growth hormone. 29. Bacteria can be engineered to “eat” oil spills. 30. Prior to 1982, this insulin was isolated from pancreatic glands of cattle or pigs. 31. In 1982, human insulin produced in genetically engineered bacteria came to market to relieve these patients from the allergenicity of pig and cattle insulin and give the product in a cost effective manner. 32. Recombinant GloFish - First transgenic animal approved by FDA. 33. GloFish are a type of transgenic zebrafish (Danio rerio) that have been modified through the insertion of a green fluorescent protein (gfp) gene. 34. Not all GloFish are green. Rather, there are several gene constructs, each encoding a different colored phenotype, from fluorescent yellow to fluorescent red. 35. Original zebrafish is a native of rivers in India and Bangladesh. 36. Advantages of rDNA technology - (i) Provide substantial quantity (ii) No need for natural or organic factors (iii) Tailor made product that you can control (iv) Unlimited utilizations (v) Cheap (vi) Resistant to natural inhibitors 37. Concerns regarding rDNA - (i) May affect natural immune system of the body (ii) Can destroy natural ecosystem that relies on organic cycle (iii) Prone to cause mutation that could have harmful effects (iv) Manufacturing of biological weapons such as botulism & anthrax to target humans with specific genotype. Additional: (i) Step 5 of Recombinant DNA Technology (ii) Q. What is the difference between competent and transformed cell? A: Competent cells are the cells that are prepared to take up DNA/G.O.I, introducing DNA/G.O.I has not occurred yet. On the other hand, Transformed cells are the cells that have successfully taken up and expressed the foreign DNA/G.O.I. Lecture 8 DNA Extraction 1. DNA can be extracted from any nucleated cell. 2. The first isolation of DNA was done by Friedrich Miescher in 1869. 3. Goal of DNA extraction : to obtain a relatively purified form which can be used for further investigation a.e: PCR, sequencing, cloning etc. 4. Most DNA extraction protocols consist of two parts - (i) a technique to lyse the cell gently and solubilize the DNA (ii) enzymatic or chemical methods to remove contaminating proteins, RNA or macromolecules. 5. In plants, the nucleus is protected within a nuclear membrane which is surrounded by a cell membrane and a cell wall. 6. Four steps are used to remove and purify the DNA from the rest of the cell - (i) lysis (ii) precipitation (iii) wash (iv) re-suspension 7. Cells are lysed using detergent that disrupts the plasma membrane. 8. Cell contents are treated with Protease and RNAase to destroy protein and RNA. 9. After centrifugation cell debris pelleted at the bottom and DNA is at supernatant. 10. Supernatant containing DNA is transferred to a clean tube and precipitated and washed with ethanol. 11. Possible samples for DNA extraction - Blood, Tissue. 12. Lysis of cells using detergent + enzyme. 13. Precipitation of nucleic acids with isopropanol. 14. Washing DNA pellets with room temperature 70% ethanol. 15. DNA concentration can be determined by measuring the absorbance at 260 nm (A260) in a spectrophotometer. 16. The ratio of absorbance at 260 and 280 nm wavelength is used to assess DNA purity. A ratio of ∼1.8 is generally accepted as “pure” for DNA. If the ratio is appreciably lower (≤1.6), it may indicate the presence of proteins, phenol, or other contaminants that absorb strongly at or near 280 nm. Gel Electrophoresis 1. Gel electrophoresis separates molecules on the basis of their charge and size. 2. The charged macromolecules migrate across a span of gel because they are placed in an electrical field. 3. The gel acts as a sieve to retard the passage of molecules according to their size and shape. 4. The basic principle is that DNA, RNA, and proteins can all be separated by means of an electric field. 5. The movement of charged molecules is called migration. 6. Molecules migrate towards the opposite charge.  7. In agarose gel electrophoresis, DNA and RNA can be separated on the basis of size by running the DNA/RNA through an agarose gel. 8. In gel electrophoresis, the buffer provides ions that carry a current through the gel, and to maintain a constant pH. There are a variety of buffers, and the most common buffers for DNA separation are Tris acetate EDTA (TAE) and tris borate EDTA (TBE) buffer. 9. Gel electrophoresis is a method of separating DNA in a gelatin-like material using an electrical field. 10. DNA is negatively charged. 11. Size of a DNA fragment affects how far it travels - (i) small pieces travel further. (ii) larger pieces travel slower and lag behind. 12. Electrophoresis is the movement of molecules by an electric current. 13. A phosphate group on a DNA polymer gives it net negative charge. 14. The molecule(s) to be separated should have charge(s). 15. When charged molecules are applied to a macromolecular matrix such as agarose or polyacrylamide, their migration under the pull of the current is impeded, depending on their sizes, charges and spaces in the gel. 16. For uniformly charged molecules the charge-to-mass ratio of molecules of different sizes will remain nearly constant. 17. These will therefore migrate at speeds inversely related to their size. 18. The concentration of the agarose dictates the size of the spaces in the gel and determines the size of DNA that can be resolved. 19. Small pieces of DNA (50–500 bp) are resolved on more concentrated agarose gels, e.g., ~2% – 3%. 20. Larger fragments of DNA (2000–50,000) are best resolved in lower agarose concentrations, e.g., 0.8% – 1%. 21. Ethidium bromide is also used during DNA fragment separation by agarose gel electrophoresis. It is added to the running buffer or in agarose gel. Ethidium binds by intercalating between DNA base pairs.When the agarose gel is illuminated using UV light, DNA bands become visible. PCR 1. Kary Mullis invented PCR between 1983 and 1985. Received nobel prize in 1993. 2. Polymerase chain reaction (PCR) is a common laboratory technique used to make many copies (millions or billions!) of a particular region of DNA under laboratory conditions. 3. Each reaction cycle doubles the amount of DNA – a standard PCR sequence of 30 cycles creates over 1 billion copies (230). 4. PCR relies on a thermostable DNA polymerase, Taq polymerase, and requires DNA primers designed specifically for the DNA region of interest. 5. It is routinely used in DNA cloning, medical diagnostics, and forensic analysis of DNA. 6. A PCR amplification reaction may fail due to the presence of co-extracted inhibitors, highly degraded DNA, insufficient DNA quantity, or a combination of all of these factors. 7. An ideal test should accurately reflect both the quality and the quantity of the DNA template present in an extracted sample. 8. A PCR requires following components: a)Template DNA/ Target DNA (1 μg) b)At least two primers (0.1-0.5 μM) c)DNA polymerase (most often Taq polymerase) - 1-2.5 units d)Magnesium chloride (0.5-2.5 mM) e)Deoxynucleotide triphosphates (dNTPs) - 20-200 μM f)Buffer (pH 8.3-8.8) g)(Not always) dye 9. A primer is a short strand of nucleic acid that serves as a starting point for DNA replication. 10. These primers are usually short, chemically synthesized oligonucleotides. 11. Replication starts at the 3'-end of the primer and Taq polymerase incorporates nucleotides complementary to the template strand. 12. Initial Denaturation temperature - 94 C 13. Denaturation 94 C 14. Annealing 65 C 15. Extension 72 C 16. Final Extension 72 C 17. Hold 4 C 18. Denaturation – when the double-stranded template DNA is heated to separate it into two single strands. 19. Annealing – when the temperature is lowered to enable the DNA primers to attach to the template DNA. 20. Elongation – when the temperature is raised and the new strand of DNA is made by the Taq polymerase enzyme. 21. Denaturation is the first step in PCR. 22. The Hydrogen bonds between the two strands are broken down and the two strands are separated. 23. Annealing is the process of allowing two sequences of DNA to form hydrogen bonds. 24. The annealing of the target sequences and primers is done by cooling the DNA to 55° - 65° C. 25. Usually the time taken to anneal is 45 seconds. 26. Elongation - (i) Taq polymerase binds to the template DNA and starts adding nucleotides that are complementary to the first strand. (ii) This happens at 72°C as it is the optimum temperature for Taq Polymerase. 27. Denaturation: 94°- 95°C 28. Primer Annealing: 55°- 65°C 29. Elongation of DNA: 72° 30. Number of Cycles: 25-40 31. At 30 cycles there are 1,073,741,764 target copies (~1×109). 32. Write down the applications of PCR. 33. Advantages of PCR. Blotting 1. Blotting is the technique for transferring DNA, RNA and proteins onto a membrane so they can be separated and detected, and often follows the use of a gel electrophoresis. 2. Southern Blot is used to detect DNA. 3. Northern Blot is used to detect RNA. 4. Western blot is used to detect PROTEIN. 5. Advantage of southern blot : can detect a wide variety of mutations. 6. Advantages of western blot: (i) Good sensitivity (ii) Can evaluate for several target proteins 7. Write down the general disadvantages of blotting.