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Nucleotides, Nucleic Acids, and Genetic Information

• Chapter 3 is about Nucleotides, Nucleic Acids, and Genetic Information

DNA: The Molecular Basis of Heredity

• The gray wolf is the common ancestor of the different species located in Europe, North America, China, and India

Initial Concepts of Genes and Heredity

• Traits are inherited as discrete units, each acting independently
• Characteristics Gregor Mendel used in his inheritance experiments with pea plants:
• Seed form and cotyledons
• Flower colour
• Pods form and colour
• Stem position of inflorescence and stem size

DNA

• DNA is commonly referred to as the blueprint for an organism
• Blueprint is reversible, recipe is not
• If blueprint is lost its structure is reconstructable

Objectives

• Nucleic acid structure basics
• Genetic information flow: Central Dogma
• Biology overview of nucleic acid function
• Demonstrate that DNA is the genetic material
• Transcription and translation
• Nucleic acid lab techniques including cutting DNA, gel electrophoresis, DNA sequencing, gene cloning, and PCR

Nucleotides

• Nucleotides are involved in nearly all aspects of cellular biochemistry
• Enzyme cofactors
• Energy transfer
• Signaling
• The scientific investigation of inheritance
• A nucleotide is made of a nitrogenous based linked to a sugar and at least one phosphate group
• Two types of Nucleotides: Ribonucleotides and Deoxyribonucleotides

Nucleic Acid Structure: The Basics

• Chains of nucleotides are linked by phosphodiester bridges of 3' and 5' positions of ribose units
• Each monomer is called a nucleotide
• Polymer nomenclature includes monomer, dimer, trimer.......oligomer
• Convention: Nucleotide sequences are written left to right 5' to 3'

Putting Together the DNA Structure Puzzle

• Earlier known facts regarding DNA:
• It was genetic material that stores information
• Made of 4 different deoxyribonucleotides
• Later known facts about DNA of all species:
• Moles of A = moles of T, and moles of G = moles of C
• Moles of A (or T) ≠ moles of C (or G) and is 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

Solving the Puzzle

• In 1953, James Watson and Francis Crick proposed 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

DNA: A Double Helix

• DNA is a double helix that runs in opposite directions
• Bases are in the helix core and occupy the periphery
• Complementary base paring:
• Pyrimidine:Purine
• A to T
• G to C
• ssDNA is rare

RNA: A Single-Stranded Nucleic Acid

• RNA makes antiparallel double helices 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 via the RNA world hypothesis

DNA

• 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
• In 1944, pure DNA isolated from Type S bacteria capable of transformation
• Avery-MacLeod-McCarty experiment

DNA as the Genetic Material: Replication

• DNA Structure Double Helix to Replication

Genes (DNA) Directs Protein Synthesis

• Genes sequence of nucleotides that encodes a polypeptide or RNA
• 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

Transcription and Translation

• DNA serves as a template for replication, RNA synthesized from DNA in transcription
• Protein synthesis from RNA templates is called translation
• Specifies the primary protein sequence
• Delivers the specific amino acid
• Are molecules are components of ribosomes

Omics

• Omics studies biomolecules:
• Complete organism DNA sequence studies gene expression
• Full set of mRNAs transcribed from DNA under a set of circumstances
• Study the full set of proteins produced
• Proteomes and transcriptome vary greatly among tissue types, development, and conditions

DNA Methods

• (rDNA): Any DNA made by artificial (i.e. biochemical or chemical) methods or naturally in cells using recombinant DNA
• Recombinant techniques are critical tools for genomics including:
• Large-scale analysis of entire genomes
• Seeks to understand how biomolecules work together within functioning organisms -"Whole-genome" or "genome-wide" techniques allow monitoring of thousands of gene simultaneously
• Are the labels often applied to the experimental fields that use rDNA

Nucleic Acid Sequencing

• Sequencing a polymer of non-identical monomers translation:

1. Cleave polymer into fragments 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

Restriction Endonucleases

• Restriction Endonucleases include EcoRI and EcoRV
• They 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
• Leaves "sticky" ends that can anneal to other overhangs of the same sequence

Gel Electrophoresis

• Involves charged solutes move in an electric field applied to a solution
• The the direction of movement in the field depends on the charge on the solute
• In presence of drag, larger charged solutes move more slowly than the smaller solutes even if their charge to mass ratios are similar
• The drag is caused by the sieving effect of the gel (agarose or polyacrylamide) and the rigidity of dsDNA

Gel Electrophoresis cont.

• Nucleic acids are negatively charged at neutral pH
• Consequently, they migrate towards the positive pole in an electric field
• The migration rate of linear (double-stranded) nucleic acid is approximately inversely proportional to the log of the length of the nucleic acid
• A fluorescent dye stains the gel, being the fluorescence is more intense when it is bound to the DNA.

DNA Sequencing by Chain-Termination

• DNA sequencing by synthesis
• DNA Polymerase Copies a Template Strand
• Single strand DNA is created by heat
• Terminating with dideoxynucleoside triphosphate
• A short oligonucleotide (primer) is used to initiate replication

DNA Sequencing Data

• "Ladder" of new dye-labeled DNAs produced and separated by size on capillary electrophoresis

Next Generation DNA Sequencing

• Massively Parallel sequencing of many DNA's concurrently, producing 1.2 billion reads of ~100 base pairs each in single experiment
• Responsible for current explosion of genetic and genomic data
• Current cost of sequencing the human genome is <$1000
  • Dante labs $599 (2021)

Human Genome

• Human Genome was finished 2001-2004 with main conclusions:
• About half is various sequence repeats, ~80% is transcribed to RNA
• ~1.2% encodes for protein and contains ~21,000 protein-encoding genes
• Only a fraction of protein encoding genes are unique to vertebrates
• Most humans differ on average by about 1 nucleotide per 1000

Construction of Recombinant DNA Molecules

• Involves molecular cloning of DNA to "Amplify it" via:
• Cutting DNA with restriction endonuclease to generate sticky ends, compatible overhangs.
• molecule is capable of carrying a foreign DNA sequence and replicating in a host cell
• Mixing and allowing overhangs to anneal
• Ligation with DNA ligase and ATP

Molecular Cloning

• Molecular cloning involves different stages, each contributes to the overall process
• Step 1 requires can be DNA from any source including any organism and can be chemically sythesized or PCR DNA
• Vectors are chosen on upon the insert DNA size, an example being bacterial plasmids (15 kb)
• DNA incorporation is made into the host and easy transformation happens via easy (transformation, electroporation)
• The host cell replicates the recombinant DNA along with its own

Polymerase Chain Reaction (PCR)

• Technique to obtain large numbers of copies of a specific DNA sequence
• It proceeds in 3 key steps being:
  • Strand separation
  • Primer annealing
  • DNA synthesis
  • Amplification
• Steps are facilitated by use of a heat-stable DNA polymerase from thermophilic organisms

Cloned Genes Can Be Expressed in other Organisms: Therapeutics

• Use genetic engineering to produce proteins like human insulin for diabetes, human growth for endocrine disorders, erythropoietin for red blood cell production, stimulating factors for white blood cells etc

Cloned Genes Can Be Expressed in other Organisms: Transgenic Organisms

• Animals are often engineered to contain a defective gene or lacking a gene entirely via knockouts
• Genetically modified crops like Golden rice
  • Genes that encode for enzymes necessary to synthesize beta-carotene