DNA and Genetics Lecture Notes - PHSC 208 PDF

Nucleotides, Nucleic Acids, and Genetic Information Chapter 3 DNA-the molecular basis of heredity 2 PHSC 208 Topic 3 2/10/2025 Initial Concepts of Genes and Heredity By: LadyofHats and me. Wikipedi...

Nucleotides, Nucleic Acids, and Genetic Information Chapter 3 DNA-the molecular basis of heredity 2 PHSC 208 Topic 3 2/10/2025 Initial Concepts of Genes and Heredity By: LadyofHats and me. Wikipedia Commons } Mendel determined some traits are inherited as discrete units with each unit acting independently. 3 PHSC 208 Topic 3 2/10/2025  DNA: The blueprint of life? Oliver Burston/Ikon Images/ Getty Images } “DNA is commonly referred to in textbooks of molecular biology as the "blueprint" for an organism. I would rather call it a recipe or like a computer program. The difference between a blueprint and a recipe is that a blueprint is reversible, and a recipe is not. If you have a house and you have lost the blueprint you can reconstruct the blueprint by taking measurements, but if you have got a well prepared dish in a great restaurant you may enjoy the dish and you may dissect it and look at it in every detail but you cannot reconstruct the recipe." } Richard Dawkins 4 PHSC 208 Topic 3 2/10/2025  DNA: The blueprint of life? Richards, Mark By Source, Fair use, Univ. of Wisconsin Extension http://www.computerhistory.org/revo https://en.wikipedia.org/w/index.php? lution/computer-games/16/194/806 curid=1034440 } “DNA is commonly referred to in textbooks of molecular biology as the "blueprint" for an organism. I would rather call it a recipe or like a computer program. The difference between a blueprint and a recipe is that a blueprint is reversible, and a recipe is not. If you have a house and you have lost the blueprint you can reconstruct the blueprint by taking measurements, but if you have got a well prepared dish in a great restaurant you may enjoy the dish and you may dissect it and look at it in every detail but you cannot reconstruct the recipe." } Richard Dawkins 5 PHSC 208 Topic 3 2/10/2025 Objectives } Nucleic acid structure basics } Genetic information flow: Central Dogma } Overview of nucleic acid function in biology } Demonstrating that DNA is the genetic material } Transcription and translation } Nucleic acid lab techniques } Cutting DNA } Gel Electrophoresis } DNA sequencing } Gene cloning } PCR 6 PHSC 208 Topic 3 2/10/2025  Nucleotides § Nucleotides are involved in nearly all aspects of cellular biochemistry } Enzyme cofactors } Energy transfer } Signaling } The scientific investigation of inheritance } DNA and RNA are polymers of nucleotides } Composed of a nitrogenous base linked to a sugar and at least one phosphate group } Purine or pyrimidine base } Ribonucleotides } Ribose } Deoxyribonucleotides } 2’-deoxyribose 7 PHSC 208 Topic 3.1 2/10/2025 Nomenclature of Nucleic Acid Bases, Nucleosides, and Nucleotides 8 PHSC 208 Topic 3.1 2/10/2025 ADP and ATP 9 PHSC 208 Topic 3.1 2/10/2025  Nucleic Acid Structure: The Basics § Chains of nucleotides § Linked by phosphodiester bridges of 3’ and 5’ positions of ribose units § Each monomer is called a “nucleotide residue” § Polymer nomenclature: § Monomer, dimer, trimer…….oligomer § Convention § Nucleotide sequences are written left to right 5’ to 3’ § Example at right: AUCG Figure 3.3 Nucleic Acid Chemical Structure 10 PHSC 208 Topic 3.2 2/10/2025 Putting together the DNA Structure Puzzle Earlier known facts: DNA was genetic material (so it must store information) DNA was made of 4 different deoxyribonucleotides Later known facts: In DNA of all species (Chargaff’s rules): moles of A = moles of T moles of G = moles of C moles of A (or T) ¹ moles of C (or G) and variable by species X-ray diffraction of DNA fibers revealed a cyclical repeating pattern most consistent with a helix. (R. Franklin + Wilkins) Tautomeric preferences of the bases In 1953, this puzzle was solved when James Watson and Francis Crick proposed the double stranded, helical structure for DNA with AT and GC base pairs interacting via hydrogen bonds. 1962 Nobel Prize in Physiology or Medicine with Maurice Wilkins Figure 3-5. X-Ray photograph of DNA by Rosalind Franklin 11 PHSC 208 Topic 3.2b 2/10/2025  DNA: A Double Helix § 2 polynucleotide chains wind around a common axis § Double helix! } Strands are antiparallel } Run in opposite directions } Bases are in the helix core and sugar-phosphates occupy the periphery } Each base is hydrogen bonded to a base on the opposite strand to form a base pair } Complementary base paring } Pyrimidine:Purine } A to T } G to C } ssDNA is rare 12 PHSC 208 Topic 3.2b 2/10/2025  RNA: A single-stranded nucleic acid § Primarily occurs as single strands } Makes antiparallel double helicies intramolecularly } Complementary base paring } Pyrimidine:Purine } A to U G to C } } Varied structures } E.g. stem-loop } Can bind small molecules and catalyze reactions } RNA world hypothesis 13 PHSC 208 Topic 3.2c 2/10/2025  DNA is the Genetic Material } Reported in 1928 by F. Griffith } Experiment indicated some genetic material from dead Type S bacteria to live type R } “transforming” it to the virulent type } In 1944, pure DNA isolated from Type S bacteria capable of the same transformation! } Avery-MacLeod-McCarty experiment 14 PHSC 208 Topic 3.3 2/10/2025  DNA as the Genetic Material: Replication } Double stranded nature of DNA facilitates its replication Figure 3-11 15 PHSC 208 Topic 3.3 2/10/2025 Genes (DNA) Directs Protein Synthesis The central dogma of molecular biology describes the flow of genetic information among molecules in biology. Transcription Translation Replication DNA RNA Protein } DNA of a gene is transcribed to produce a complementary RNA } That RNA is translated into the corresponding sequence of amino acids to form a protein } Gene: Sequence of nucleotides that encodes a polypeptide or RNA 16 PHSC 208 Topic 3 2/10/2025  Transcription and Translation } Just as DNA serves as a template for replication, RNA is synthesized from DNA in transcription } Protein synthesis from RNA templates is called translation § Messenger RNA (mRNA) specifies the Figure3-12 primary protein sequence § Transfer RNA (tRNA) delivers the specific amino acid § Ribosomal RNA (rRNA) molecules are components of ribosomes Figure3-13 17 PHSC 208 Topic 3 2/10/2025 Omics: Studying these biomolecules on the whole } Complete DNA sequence of an organism is a genome. } Transcriptomics studies gene expression } Full set of mRNAs transcribed from DNA under a set of circumstances } Proteomics is the study of full set of proteins produced } Proteomes and transcriptome vary greatly among tissue types, development, and conditions 18 PHSC 208 Topic 3 2/10/2025 DNA Methods } Recombinant DNA (rDNA): Any DNA made by artificial (i.e. biochemical or chemical) methods or made naturally in cells using recombinant DNA } Recombinant techniques are critical tools for genomics } Genomics-large-scale analysis of entire genomes } Functional genomics seeks to understand how biomolecules work together within functioning organisms } “whole-genome” or “genome-wide” techniques allow monitoring of thousands of gene simultaneously } Molecular biology or molecular genetics are the labels often applied to the experimental fields that use rDNA 19 PHSC 208 Topic 3.5 2/10/2025  Nucleic Acid Sequencing } General approach for sequencing a polymer of nonidentical monomers translation § 1. Cleave polymer into fragments that are small enough to be fully sequenced. § 2. Determine the sequence of residues in each fragment § 3. Determine the order of fragments in original polymer by aligning smaller fragments that have overlapping sequences Figure 3-14 20 PHSC 208 Topic 4 2/10/2025 Restriction Endonucleases } bind DNA at specific sequences and cut both strands of DNA at or near the binding site } part of a bacterial defense system against viruses } Leave “sticky” ends that can anneal to other overhangs of the same sequence Figure 3-15 } useful for recombinant DNA methods 21 PHSC 208 Topic 4a 2/10/2025  Gel Electrophoresis Charged solutes move in an electric field applied to a solution. + - The direction of movement + + in the field depends on the - charge on the solute. - In the presence of drag, the larger charged solutes move more slowly than the smaller solutes even if their charge to mass ratios are similar. The drag caused by the sieving effect of the gel (agarose or polyacrylamide) and the rigidity of dsDNA. 22 PHSC 208 Topic 4b 2/10/2025 Gel Electrophoresis - well electrode buffer + (cont.) gel wells Electric - + Field All nucleic acids are negatively charged at neutral pH, so they migrate towards the positive pole in an electric field The rate of migration of linear (double stranded) nucleic acid is approximately inversely proportional to the log of the length of the nucleic acid. The gel is stained with a fluorescent dye which is even more fluorescent when it is bound to DNA 23 PHSC 208 Topic 4b 2/10/2025  DNA Sequencing by Chain-Termination } Sequencing by synthesis } DNA Polymerase Copies a Template Strand } Single strand DNA is created by heat } A short oligonucleotide (primer) is used to initiate replication } Inclusion of dideoxynucleotides terminates the reaction Figure 3-18 24 PHSC 208 Topic 4c 2/10/2025  DNA Sequencing Data } ”Ladder” of new dye-labeled DNAs produced } Separated by size on capillary electrophoresis Figure 3-20 25 PHSC 208 Topic 4c 2/10/2025  Next Generation DNA Sequencing } Massively Parallel sequencing of many DNA’s concurrently Cost to sequence a human genome as estimated by NHGRI (September 2001 to July 2015) Can produce 1.2 billion reads of ~100 base pairs 100,000,000 } each in single experiment 10,000,000 1,000,000 Responsible for current explosion of genetic Cost per genome (USD) } and genomic data 100,000 } Current cost of the human genome