DNA Structure and Replication

DNA Structure

• DNA has a double helix shape
• Guanine pairs with Cytosine (G & C)
• Adenine pairs with Thymine (A & T)
• Two hydrogen bonds connect A and T
• Three hydrogen bonds connect G and C
• DNA strands have a sugar-phosphate backbone
• Each strand has a 5' prime end and a 3' prime end.

DNA Replication

• DNA replication creates 2 strands twisted around each other in a double helix
• Each strand includes a sequence of the 4 chemical bases: A, T, C & G
• Determines how each strand is replicated
• Helicase separates the 2 DNA strands by unzipping them and results in the formation of a replication fork
• Separated strands serve as a template for creating a new DNA strand
• Primase starts this process and makes a small piece of RNA called a primer
• The enzyme, DNA polymerase, binds to the primer, marking the starting point for the construction of the new DNA strand
• DNA polymerase adds DNA bases in one direction, from 5' to 3'
• The new leading strand of DNA is made continuously
• Polymerase adds bases in the 5' to 3' direction
• The lagging strand runs in the opposite direction to the leading strand
• DNA polymerase makes Okazaki fragments in small chunks for the lagging strand
• Each fragment is started with an RNA primer
• DNA polymerase adds a short row of DNA bases in the 5' to 3' direction after the RNA primer starts fragment
• Exonuclease removes all RNA primers from both DNA strands
• DNA polymerase adds new DNA bases to fill in gaps
• DNA ligase seals up the DNA fragments in both strands
• Each new DNA molecule contains one old, conserved DNA strand and one new one

Mutations

• Mutations result from radiation and chemical mutagens
• Mutation is the source of all new alleles in a species
• Whether a mutation results in a new allele depends on whether it is a somatic or germline mutation, and whether it occurs in a coding or non-coding region of the DNA.
• A mutagen is an environmental agent that causes mutations by altering the DNA
• Chemical mutagens cause mutations if cells have been exposed to them at a high frequency or for a prolonged period, causing a change in DNA that alters the function of proteins and resulting in impaired cellular processes
• Naturally mutagenic agents are present at normal levels within natural environments
• Natural mutagens can be either non-biological such as certain metals, or biological (e.g. viruses, bacteria, fungi)
• Physical mutagens include heat and ionizing radiation that has enough energy to break chemical bonds in molecules, including DNA.
• A mutation is a permanent change in the genetic material of a cell due to the sequence of nucleotides in DNA being altered
• Criteria distinguish mutations by origin (cause) and the amount of genetic material changed
• Spontaneous mutations are natural errors arising randomly during DNA replication in cells
• Induced mutations are caused by a mutagen
• Point mutations: a base is replaced with another.
• Chromosomal mutations involve changes in chromosome number or structure
• Trisomy: extra chromosome (e.g. Down Syndrome)
• Monosomy: missing chromosome (e.g. Turner Syndrome)
• Changes in chromosome structure:
• Deletion: missing part of a chromosome
• Duplication: extra copies of a gene or chromosome segment are added
• Inversion: chromosome segment reversed
• Translocation: part of a chromosome attached to another
• Frameshift mutations are more significant changes that may affect a single gene or a sequence of genes and arise as a result of a point mutation or a chromosomal mutation.
• Nucleotide base may be substituted, inserted, or deleted which leads to a change in one amino acid
• Effect on phenotype:
  • Silent mutation: no change in the phenotype.
  • Variation: small or large change in the phenotype depending on the type of amino acid substituted Phenotypic changes are most likely to be harmful
• Heritability of mutations depends on whether the mutation occurs in a non-reproductive (somatic) cell or a reproductive (germline) cell
• Point Mutation: Change to a single base pair of DNA
• Point substitution: one base is replaced by another
• Nonsense mutation: changes an amino acid to a stop codon so cuts the proteins short, therefor protein in non-functional
• Missense mutation: amino acid altered at a critical region of the protein, which DOES affect its function

Somatic vs Germline Mutations

• Germline mutations arise in gametes, get inherited, and affect all cells
• Somatic mutations arise in a single body cell (non-sexual), can not be inherited, but only affect patches of tissue derived from the mutated cell
• Phenotype also depends of the presence of particular proteins and the activity of those proteins, as mutations in coding genes have a direct effect on these protein products
• Coding DNA produces proteins that control the function of biological processes
• Non-coding DNA can disrupt normal development or cause serious damage, may regulate gene activity crucial for cell functioning, or carries instructions for making different types of RNA molecules
• Crossing over occurs in meiosis when homologous chromosomes exchange genetic material
• Results in the formation of unique gametes/chromosomes (recombinant chromosome)
• Recombinant chromosome increases genetic diversity in the offspring

Genetic Variation

• Genetic variation results from fertilisation, meiosis and mutation
• Fertilisation can increase gene recombination with mixing of maternal and paternal genetic material, resulting in new, unique gene combinations in the offspring
• Interactions in the genes (dominant, recessive), can result in production of traits that neither of the parents had
• Meiosis forms gametes which increases genetic variation
• Meiosis: Crossing over swaps chromosome segments, Random segregation ensures that each gamete receives a random combination of chromosomes leading to offspring with unique genetic makeup
• Mutations introduces new alleles and variations as a result of replication errors, or separation of chromosomes
• New alleles can be passed down to offspring and increases number of alleles for a trait
• Concept of the gene pool includes the total/frequency of all alleles
• New alleles can only be introduced into the gene pool of a species by mutation
• Natural selection: relies on the existence of genetically based phenotypic variation in the population
• Gene flow: changes in allele frequency due to individuals coming in or out of a population increases genetic diversity, and population health
• Genetic drift: changes in allele frequency due to random chance
• Bottleneck/Founder effect: changes in frequency because a small individual creates a larger allele

DNA Profiling and Sequencing

• DNA sequencing determines the precise nucleotide sequence of a gene on a chromosome
• It can be done using manual methods which include the Sanger chain termination method or the Maxam-Gilbert method, or with an automatic method using a DNA sequencer
• DNA profiling is used to identify and compare individuals by characteristics in their DNA using testing of highly variable regions of an individuals DNA which contain Short Tandem Repeats (STRs)
• These DNA profiles can confirm how closely related individuals are, trace inheritance patterns, and solve crimes Polymerase chain reaction (PCR) uses variations in temperature to control the replication process via 3 steps:
• (1) Dentaturation - DNA sample is heated to separate it into two single strands; (2) Annealing - DNA primers attach to the 3' ends of the target sequence; (3) Elongation - Activates the DNA polymerase to start replication process
• Gel electrophoresis is a lab technique used to separate mixtures of DNA based on molecular size where the molecules.
• The smaller the molecule, the faster they move

1. DNA is extracted, 2. PCR isolation and amplification of DNA, 3. DNA added to the gel wells, 4. Electric current applied to the gel, 5. DNA bands are separated by size, 6. DNA bands are stained

• Genetic sequencing using PCR and gel electrophoresis, the exact sequence of nucleotides, in a chormosome or gene is determined

Genetic Testing

• For genetic testing of diseases, PCR is used to generate a DNA profile of parents to detect for recessive alleles and amplified gene product size is analyzed via get electrophoresis to determine if the mutation is present
• Genetic testing for paternity testing uses multiple band matches for restriction fragment analysis and a 2 key step process: (PCR with restriction to amplify multiple regions + DNA fragments tagged with dye +Gel electrophoresis separating fragments)
• In forensic science 100% band match is required In paternity testing, half an individuals genetic information comes from thier mother and half from their father

Karyotypes and FISH

• Karyotypes is standard and detects chromosome abnormalities
• FISHing includes fluorescent probes
• Both for observed chromosomal abnormalities

Polypeptide synthesis

• Main source of genetic material
• Both prokaryotes and eukaryotes have cytoplasm Genetic
• Prokaryotes mostly lack membrane bound organelles and plasmids and have free ribosomes
• Eukaryotes have a nucleus and a wider range of organelles, no plasmids and ribosomes present
• Prokaryotes-singular chromosomes, Eukaryotes-linear (associated with histones) DNA
• Mitochondrial DNA (separate location). Human mtDNA has 37 genes
• Chloroplast genomes have 150

Cell structure

• Genetic material (DNA + packaging)
• Prokaryotes (one linear chromosomes, smaller and limited packaging) vs Eukaryotes (multiple linears chromsomes+complex packing)
• Content and Replication in both structures

Cell Replication

• Chromsomes are homologous pairs
• Chromsaomes model and simplify complex science concepts

Mitosis and Meosis

• Main function for the process and for repair in somatic
• Meisosi-sexual reprocution in somatic
• Chromsomes replicated in a certain way depending on process
• Compare stages of chromsome movement and final cell strucutres

Genetic Variation

• Gregor Mendal
• Gene segregation and combination