DNA

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DNA Genetics in Biotechnology
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What is a DNA?

• A nucleic acid that carries the genetic
  information in the cell and is capable of
  self-replication and synthesis of RNA. DNA
  consists of two long chains of nucleotides
  twisted into a double helix and joined by
  hydrogen bonds between the complementary bases
  adenine and thymine or cytosine and guanine. The
  sequence of nucleotides determines individual
  hereditary characteristics.

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What is a Nucleotide?

• A single molecule of DNA comprised of 2 basic
  parts made from 3 distinct molecules.
  
• Sugar/Phosphate Backbone
• Nitrogenous Base

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Sugar/Phosphate Backbone

• Comprised of deoxyribose sugar and a simple
  phosphate molecule
  
• Forms a strong bond that creates the backbone of
  a DNA strand
  
• EXACTLY THE SAME IN ALL DNA

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Nitrogenous Base

• Bond with complimentary bases in other
  nucleotides to form the rungs of the DNA ladder
  (zip DNA together)
  
• Only 4 types in all DNA-Adenine, Cytosine,
  Guanine, and Thymine
  
• Adenine and Thymine bond only with each other
• Cytosine and Guanine bond only with each other

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DNA form

• DNA nucleotides combine in cells to form long
  strands in the shape of a double helix (looks
  like a twisted ladder)
  

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DNA Form

• Nucleotides bond at two spots
• Sugar/Phosphate molecules form the backbone
  (outside rails)
  
• Nitrogenous bases bond in the middle by hydrogen
  bonds (steps or rungs)
  
• Hydrogen bonds between nitrogenous bases are MOST
  EASILY BROKEN

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DNA Form

• The order of the nucleotides is the determining
  factor in the expression of genes in organisms.

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Characteristics of DNA
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DNA

• Accounts for all genetic variation between
  different individuals and organisms by the use of
  different
  
• Sequences of nitrogenous bases
• Lengths of DNA segments
• Numbers of Chromosomes and amounts of DNA in an
  organism
  
• The amount of DNA in an organism DOES NOT relate
  to the size or complexity of an organism.

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DNA Replication

• The process through which cells copy DNA for
  transmission to daughter cells during cell
  division.
  
• The double helix structure allows DNA to easily
  unzip down the center between nitrogenous bases.
  
• Free floating nucleotides attach to each of the
  separated DNA strands forming 2 new strands of
  DNA, each an exact copy of the original.

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Mutations

• A mutation is an unexpected change in a DNA
  sequence, usually occurring during the
  replication/cell division.
  
• Mutations are common in most organisms
  (especially simple organisms) though only a small
  percentage produce noticeable changes in
  organisms.

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Genetic Hierarchy
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Genetic Hierarchy

• A group of nucleotidesa gene/allele45-150 base
  pairs
  
• A group of genes1 strand of DNA
• Several condensed strands of DNA1 chromosome
• 2 chromosomes1 chromatid pair
• All possible gene forms in a populationGenome

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Gene Mapping

• Mapping the genome of a species allows scientists
  to identify beneficial and harmful genes in a
  population, and is the first step in determining
  the location of specific genes on chromosomes.
• Changes in the genome of a species occur slowly
  in response to environmental changes.

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Transferring of DNA

• DNA is passed to offspring during sexual
  reproduction through single chromosomes.

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Human Genetics

• Almost all humans have 46 chromosomes.
• Individuals with Down Syndrome have one extra
  chromosome.
  
• Humans generally differ from each other by
  approximately 3 million nitrogenous base pairs,
  or 0.1 of the total gene sequence.

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Genetic Disorders
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Genetic Disorders

• Diseases or other problems resulting from errors
  in the transmission of genetic information, or
  the expression of certain negative gene sequences.

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Genetic Disorders

• Most genetic disorders are recessive, and thus
  cannot be predicted without genetic analysis
  
• Recessive disorders are transmitted by
  carriers-parents with one dominant gene (normal)
  and one recessive gene (disorder)
  
• Example-Tt

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Genetic Disorders

• Certain disorders are more common in certain
  populations
  
• Example The occurrence of sickle cell in
  African Americans.

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Common Genetic Disorders

• Inherited Disorders
• Examples Tay-Sachs, Sickle Cell Anemia,
  Hemophilia
  
• Mutations
• Cancer-uncontrolled division of abnormal cells
• Treatment must destroy mutated cells

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Genetic Mutations

• Sudden unexpected changes in the genetic code of
  an organism which appear most often during the
  process of replication
  

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Genetic Mutations

• Often result from increased levels of stress on
  cells just prior to or during cell division
  
• Stresses include-radiation, UV rays,
  environmental, etc.

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Genetic Mutations

• Almost all mutated cells die immediately, or
  never impact living organisms
  
• Most mutations in humans are harmful such as
  cancer
  
• A small fraction of noticeable mutations are
  beneficial, such as Chimeras which are used to
  give us variegated plants.

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Genetic Mutations

• Most mutations occur in developed plants and
  animals, affecting isolated groups of cells.
  
• Mutations are most devastating when the occur in
  the early development of organisms. (STEM CELL
  STAGE)

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Types of Mutations

• Point mutation
• A mutation that changes DNA at a single point,
  substituting one nucleotide pair.
  
• Frameshift
• Nucleotides are inserted or deleted, altering the
  entire DNA sequence after the mutation

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Mitosis and Meiosis
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What is Mitosis?

• The process of cell division in all diploid
  cells
  
• Constantly occurs in cells throughout plants and
  animals at all times
  
• Muscle cells
• Skin cells
• Stem cells
• Cambium cells
• Results in two diploid daughter cells

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Stages of Mitosis

• Interphase
• Prophase
• Metaphase
• Anaphase
• Telephase
• Cytokinesis

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Interphase

• The period of cell growth and function prior to
  the beginning of true mitosis, in which the cells
  store energy for cellular division
  
• The cell replicates DNA and produces chromatid
  pairs
  
• This is the longest period in the life of a cell

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Prophase

• The first true stage of mitosis
• The nuclear membrane dissolves, centromeres form,
  and centrioles move toward opposite ends of the
  cell

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Metaphase

• The second and shortest stage of mitosis
• Chromatids align in the center of the cell and
  spindle fibers attach to centromeres from
  centrioles

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Anaphase

• The third stage of mitosis
• Chromatids are separated and pulled towards
  opposite ends of the cell by spindle fibers
  
• Errors in the transmission of genetic information
  are most likely to occur at this stage

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Telephase

• The final and longest stage of mitosis
• Chromosomes reach opposite ends of the cell, and
  new nuclear membranes form for each new daughter
  cell

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Cytokinesis

• The actual division of daughter cells at the end
  of mitosis
  
• A cleavage furrow forms pinching apart cells in
  animals
  
• In plant cells, a cell plate forms between
  daughter cells, dividing cells and forming the
  new section of the cell wall.

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Cytokinesis
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What is Meiosis?

• The specialized form of cell division that occurs
  only in haploid cells
  
• Sperm
• Egg
• Pollen
• Ovum
• Very similar in process to mitosis, except with
  two cycles, producing 4 haploid daughter cells
  (23 chromosomes each)

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Spermatogenesis

• Production of male sex cells through meiosis
• Produces 4 sperm

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Oogenesis

• Production of female sex cells through meiosis
• Usually produces 1 viable egg-other 3 abort

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Stages of Meiosis

• Interphase
• Meiosis I
• Meiosis II
• The stages of Meiosis I and Meiosis II are
  identical to the stages of Mitosis, but with
  different cells for a different purpose

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Interphase

• Same as mitosis
• Period of growth and function

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Meiosis I

• Prophase I
• Metaphase I
• Anaphase I
• Telephase I
• Cytokinesis
• Reduction process-changes cells from diploid to
  haploid

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Meiosis II

• Prophase II
• Prophase II is responsible for aligning
  chromosomes for the final division
  
• Metaphase II
• Anaphase II
• Telephase II
• Cytokinesis

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DNA Extraction and Analysis
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DNA Extraction

• The process of isolating nucleic acids (DNA) from
  organic material.
  
• DNA can be extracted from almost any intact
  cellular tissue (more cells make it easier)
  
• Skin, blood, saliva, semen, mucus, muscle tissue,
  bone marrow, etc.
  
• DNA cannot be extracted from hair, unless skin is
  attached at the bottom
  
• Mitochondrial DNA can often be extracted long
  after nuclear DNA has degraded.

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Simple DNA Extraction

• For observation only, not feasible for analyzing
  DNA
  
• Works well with fruit (Example Strawberries)

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Simple DNA Extraction

• Step 1
• Physically break apart plant material, usually
  fruits
  
• Step 2
• Use a detergent to break apart the cell membrane
• Step 3
• Treat with ethyl alcohol to isolate DNA from
  remaining proteins and sugars
  
• Step 4
• Spool using a glass rod to view a large clump of
  nucleic acids (DNA)

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Advanced DNA Extraction

• The organism to be tested is chosen, and a sample
  is taken from which DNA can be extracted.
  
• Detergents are used in simple DNA extraction
  procedures to break down cell membranes, blending
  the contents of the cell.

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Advanced DNA Extraction

• The DNA sample is treated with enzymes to isolate
  nucleic acids, usually both DNA and RNA
  
• Enzymes dissolve proteins, sugars, and other
  materials
  
• Examples protease, amylase, etc (enzymes end
  with the suffix ase)
  
• A second enzyme may be applied to cut DNA into
  gene segments for analysis

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Restriction Digests and Enzymes

• Restriction enzymes are used to cut extracted DNA
  into smaller gene sequences.
  
• Make analysis easier during the process of gel
  electrophoresis.
  
• Enables scientists to isolate specific genes with
  specific enzymes for use in genetic engineering.

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Restriction Digests and Enzymes

• Cuts the gene from the chromosome making a sort
  of gene soup after the removal of proteins
  
• Leaves the ends of gene segments sticky with
  usually 3 exposed nucleotides on one side of the
  double helix, so that ends may be rejoined later.

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Methods of DNA Analysis

• There are several simple methods used for
  analyzing DNA
  
• Paternity Testing
• Gel Electrophoresis
• Advanced Methods
• Polymer Chain Reaction (PCR)
• Amniocentesis

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Paternity Testing

• Simple method of DNA analysis that compares the
  DNA of an offspring, plant or animal, with a
  known mother and suspected father.

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Paternity Testing Process

• DNA sample taken usually from saliva or blood in
  animals and leaf or callus tissue in plants.
  (Hair does not contain DNA, but the hair follicle
  does.)
• DNA isolated in sample through the use of protein
  eating enzymes.

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Paternity Testing Process

• Sample run on gels or through a gene sequencer to
  indicate the presence of certain genes.
  
• Comparison of genes-anything present in the child
  MUST BE PRESENT IN EITHER THE MOTHER OR THE
  FATHER. 13 genes present in the child that are
  not in the mother, but present in the father make
  a 99 match.

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Polymer Chain Reaction (PCR)

• Method used in forensic science to amplify
  genetic material for identification or analysis.
  
• Newer technique used only in advanced
  laboratories.
  
• Only a few cells are needed with this technique.

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Amniocentesis

• Method used to analyze the DNA of a mammal
  (occasionally other animals) prior to birth.
  
• Used widely in humans to predict the expression
  of lethal genes or genetic disorders in high-risk
  pregnancies.
  
• Gaining favor in high expense animal breeding
  (Ex. Race horses)

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Gel Electrophoresis

• Method used to analyze extracted DNA through the
  distribution of genetic markers on an agar
  media.
  
• Smaller genes travel further distances on the
  gel. Samples extracted through the same process
  can be easily compared on a single gel.

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Gel Electrophoresis Process

• An agar gel is placed into a mold to dry, then
  placed into an electrophoresis chamber.
  
• DNA extraction is placed in small wells at one
  end of the agar gel. Each well represents a
  different sample or individual.

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Gel Electrophoresis Process

• Low voltage direct current is run through a
  buffer solution surrounding the agar gel
  distributing DNA fragments across the gel
  
• Fragments separated by the size of the gene
  segment smaller move faster than larger
  
• Negative charged DNA fragments are repelled away
  from the negatively charged wells to the positive
  charged end.

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Gel Electrophoresis Process

• Buffer solution provides a means of transmission
  for electrical current, but also keeps DNA
  samples in place in wells in the gel.
  
• Buffer is heavier than DNA

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Gel Electrophoresis Process

• Strength of the electrical current determines the
  speed at which DNA moves across the gel.
  
• Ethidium Bromide or another Bromine based
  solution is applied at the end of the
  electrophoresis process to stain DNA for better
  viewing under certain bands of light.

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Genetics in Agricultural Breeding Programs
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Natural Selection

• Mechanism for evolution in natural populations
• Organisms with best traits suited to the
  environmental factors affecting a population are
  most likely to survive and reproduce.
  
• Results in the inheritance of the same
  well-suited traits
  
• Important traits in natural selection-disease
  resistance, size, color pattern/camouflage, etc.

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Natural Selection

• Types of Natural Selection
• Stabilizing selection
• Directional selection
• Disruptive selection

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Stabilizing Selection

• Individuals with the average or norm for a trait
  have an advantage over other forms of the trait
  
• Example gray moths (norm) are favored over
  black and white moths

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Directional Selection

• Individuals with one extreme or less common
  version of a trait are favored over other forms
  of the trait.
  
• Example Black moths are favored over gray or
  white moths

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Disruptive Selection

• Multiple extremes or alternative forms of a trait
  are favored over the norm
  
• Example Black moths and white moths are favored
  over gray moths

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Selective Breeding

• Method of breeding plants and animals utilized in
  agriscience to produce offspring that possess
  certain characteristics desirable to
  agriculturists
• Utilized for generations-produced the first
  domestic animals in early civilizations

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Selective Breeding

• Used to select for a variety of traits
  including
  
• Muscling/Size
• Fat content
• Breeding Capability
• Color
• Speed/Agility
• Temperament
• Milk Production

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Selective Breeding

• Methods for selective breeding
• Artificial insemination
• Pen/field breeding
• Isolation Breeding-inbreeding
• Mechanical pollination of plants
• Hybridization of plants and animals

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Selective Breeding

• Selective breeding is accomplished much quicker
  in plants than animals due to growth rates and
  ease of propagation/production

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Selecting Plants and Animals for Breeding
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Observe Patterns of Heredity

• The occurrence of genetic disorders in offspring
  or parents is an indicator that the parent may
  have a recessive gene for the disorder
  
• Though genetic recombination is random, some
  animals are more likely to transmit genes than
  others
  
• Keeping careful breeding records improves
  effectiveness

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Select Animals Carefully

• Animals used in selective breeding should be
• Healthy-old injuries or illnesses are not a
  factor unless they are a result of genetic
  propensities or impair breeding capabilities
  
• Carefully monitored-nutrition levels, pests and
  stress can all reduce breeding viability. Some
  very good specimens are completely isolated.

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Select Animals Carefully

• Hybrids should be avoided, since traits expressed
  in the organism are rarely transmitted to
  offspring
  
• The process of inbreeding isolates genes for only
  a single generation, as many are recessive.

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Carefully Plan Breeding Crosses

• Plants can be crossed not only within species
  (interspecific), but also within genus
  (intergeneric), and even, in rate cases family
  (interfamilial)
• Animals are usually limited to crosses within the
  same species

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Methods for Producing Selective Breeding Programs
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Inbreeding

• Crossing organisms that are genetically related
• Crossing two plants to produce an f1 generation,
  then crossing two of the f1 offspring to create
  an f2 generation

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Backcrossing

• Crossing offspring from a cross with one of the
  previous parents, or a similar organism, to
  maximize the expression of certain traits.
  
• Often used after intergeneric crosses to produce
  offspring that possess more characteristics from
  one genus.