DNA & Genetics in

Biotechnology

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.

What is a Nucleotide?

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

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

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

DNA form

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

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

DNA Form

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

Characteristics of DNA

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.

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.

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.

Genetic Hierarchy

Genetic Hierarchy

• A group of nucleotides=a gene/allele=45-150 base pairs

• A group of genes=1 strand of DNA

• Several condensed strands of DNA=1 chromosome

• 2 chromosomes=1 chromatid pair

• All possible gene forms in a population=Genome

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.

Transferring of DNA

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

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.

Genetic Disorders

Genetic Disorders

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

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

Genetic Disorders

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

Americans.

Common Genetic Disorders

• Inherited Disorders– Examples: Tay-Sachs, Sickle Cell Anemia,

Hemophilia

• Mutations– Cancer-uncontrolled division of abnormal cells– Treatment must destroy mutated cells

Genetic Mutations

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

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.

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.

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)

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

Mitosis and Meiosis

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

Stages of Mitosis

• Interphase

• Prophase

• Metaphase

• Anaphase

• Telephase

• Cytokinesis

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

Prophase

• The first true stage of mitosis

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

Metaphase

• The second and shortest stage of mitosis

• Chromatids align in the center of the cell and spindle fibers attach to centromeres from centrioles

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

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

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.

Cytokinesis

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)

Spermatogenesis

• Production of male sex cells through meiosis

• Produces 4 sperm

Oogenesis

• Production of female sex cells through meiosis

• Usually produces 1 viable egg-other 3 abort

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

Interphase

• Same as mitosis

• Period of growth and function

Meiosis I

• Prophase I

• Metaphase I

• Anaphase I

• Telephase I

• Cytokinesis– Reduction process-changes cells from diploid to

haploid

Meiosis II

• Prophase II– Prophase II is responsible for aligning

chromosomes for the final division

• Metaphase II

• Anaphase II

• Telephase II

• Cytokinesis

DNA Extraction and Analysis

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.

Simple DNA Extraction

• For observation only, not feasible for analyzing DNA

• Works well with fruit (Example: Strawberries)

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)

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.

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

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.

• 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.

Restriction Digests and Enzymes

Methods of DNA Analysis

• There are several simple methods used for analyzing DNA– Paternity Testing– Gel Electrophoresis

• Advanced Methods– Polymer Chain Reaction (PCR)– Amniocentesis

Paternity Testing

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

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.

• 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.

Paternity Testing Process

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.

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)

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.

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.

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.

• 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

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.

Gel Electrophoresis Process

Genetics in Agricultural Breeding Programs

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.

Natural Selection

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

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

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

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

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

Selective Breeding

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

Selective Breeding

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

Selective Breeding

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

Selecting Plants and Animals for Breeding

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

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.

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.

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

Methods for Producing Selective Breeding

Programs

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

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.