Week 1 Lecture 2: Nucleic Acids, DNA, and RNA Review
Week 1 Lecture 2: Nucleic Acids, DNA, and RNA Review PCB 4024
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This 4 page Class Notes was uploaded by Justin Bartell on Monday September 5, 2016. The Class Notes belongs to PCB 4024 at Florida State University taught by Dr. Stroupe in Fall 2016. Since its upload, it has received 41 views. For similar materials see Molecular Biology in Biology at Florida State University.
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Date Created: 09/05/16
PCB 4024– Week 1 – 02 (Thursday Lecture 9/1/2016) Nucleic Acids, DNA, and RNA Review I. Nucleic Acids: encode genetic information 2 types: DNA and RNA a. Deoxyribonucleic acid (DNA) i. Deoxyribonucleotides are its building blocks ii. A single DNA molecule contains hundreds of genes and millions of nucleotides; The sequence of the nucleotides is the informative part 1. There are about 3 billion bases in the human genome (compared to about 600,000 in a bacteria). 2. Each human (linear) chromosomes have 51245 million base pairs 3. Genes (the bits that code for protein) comprise only about 2% of the genome. 4. There are 46 chromosomes in human somatic cells and 23 in sex cells (22 unique somatic chromosomes + 2 sex chromosomes). iii. Typically double stranded (antiparallel) iv. In eukaryotes, DNA is in nucleus or mitochondria/chloroplasts 1. In mitochondria/chloroplasts DNA is found as a circular plasmid 2. In nucleus, DNA is found as chromosomes v. In prokaryotes DNA is in nucleoid. 1. plasmid b. Ribonucleic Acid (RNA) i. Ribonucleotides are its building blocks ii. To access the info in DNA< a single, complementary RNA molecule is made. The DNA remains unchanged. iii. In eukaryotes, RNA is found in nucleus and cytoplasm. iv. Typically single stranded. II. Polynucleotide Structure a. Nucleotide has 3 parts: i. Nitrogenous base (ATCG in DNA, AUCG in RNA) 1. Pyrimidine6 membered ring (cytosine, thymine, and uracil) 2. Purine6+5membered rings (adenosine, guanine) ii. 5carbon sugar, either ribose or deoxyribose iii. Phosphate b. Nucleoside is the nitrogenous base + sugar. c. Polynucleotide is directional because is building blocks are directional i. Covalent linkage from the 5’ Carbon to the hydroxyl of the 3’ Carbon. III. Central Dogma: DNA –[translation] RNA –[transcription] protein What matters is the sequence of nucleotides in DNA. a. What happens if DNA sequence changes? i. Change could be damaging: results in bad protein product or changes the correct phenotype of cell. ii. Change could be neutral: does not change the phenotype of the cell iii. Change can be productive: results in better protein product or makes the cell/organism more viable. iv. The change may or may not be passed down. IV. DNA is the genetic information a. Genotype (genetic information) vs. Phenotype (physical implementation) b. How did we get to the understanding of genetic inheritance? Which is the genetic information: DNA or protein? [hint: it’s DNA] Several landmark experiments: i. Griffith 1. Live Streptococcus pneumonia could be pathogenic or nonpathogenic. Step 1: kill pathogenic bacteria Step 2: mix with nonpathogenic bacteria Step 3: test nonpathogenic bacteria for pathogenicity (is it now pathogenic?) They did become pathogenic! Conclusion: Something (other than live cells) “transformed” [altered] both the genotype and phenotype of the nonpathogenic pneumonia. ii. Avery 1. Isolated each type of macromolecule (DNA, RNA, and protein) and attempted to transform bacteria with it 2. Only DNA resulted in new phenotypes 3. Conclusion: DNA is genetic material iii. Hershey and Chase 1. Made phage with either radiolabeled sulfur 35S (highlights protein coat) or phosphorus 32P (highlights DNA). 2. Let phage infect bacteria 3. Physically removed nongenetic material (via household blender XD) 4. Centrifuge to isolate infected bacteria 5. Analyzed to find radio labeled genetic info 6. Conclusion: DNA is genetic material iv. Chargaff 1. Measured the composition of bases in different species and found they all differed but found out: 2. “Chargaff’s rule”: concentration of adenosine is equal to the concentration of thymine and concentration of cytosine is equal to the concentration of guanine. a. Aka: [A] = [T] and [C] = [G] c. How does DNA work? First we need to know what it looks like (more experiments): i. Linus Pauling 1. Proposed triple helix model for DNA (incorrect) ii. Maurice Wilkins and Rosalind Franklin 1. Xray diffraction of DNA 2. Tried to calculate the structure of DNA from this diffraction a. Proposed that phosphates were on the outside (correctly). iii. James Watson and Francis Crick 1. Used Franklin’s model to propose double helix iv. Conclusion 1. Purinepyrimidine pairs is the only arrangement for the DNA double helix to get a constant diameter. V. Molecular Techniques a. Gel electrophoresis i. Run DNA from cathode () to anode (+) ii. Smaller stuff moves farther 1. Larger DNA strands at top, smaller DNA strands at bottom iii. Separates on size, shape, and charge b. Autoradiography i. Start with gel electrophoresis ii. Run a gel on a material that has been labeled with something that will react with film (often 32P, recently fluorescent tags) iii. Transfer that information to film or a digital detector c. Nucleic acid hybridization i. Start with gel electrophoresis ii. Run a gel iii. “Blot” – transfer to membrane 1. Now DNA or RNA is on membrane iv. Block (because DNA and RNA strands tend to be “stick” and want to combine) v. Probe with complementary fragment to highlight specific regions [thing on membrane / probe material] 1. Southern Blots – DNA/ DNA (named for Dr. Southern) 2. Northern Blots – RNA/RNA (opposite of southern _) 3. Eastern Blots – protein/antibody 4. Western Blots – protein/ post translational modification tag 5. Southeastern Blots – protein/DNA d. DNase footprinting i. Label DNA ii. Mix DNA with protein of interest (in this case 3 increasing amounts in lanes 24, plus 0 protein in lane 1). iii. Treat DNA:protein with an enzyme that degrades DNA (DNase). iv. Run a gel. v. If the protein binds the DNA, the DNase can’t cut. Otherwise, you see the resulting fragments. vi. Comparing to lane 1 to 23 tells you about where the protein binds and shows a direct interaction between the DNA and protein
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