Applied Genetics: Hybridization and Inbreeding

Applied Genetics

• Selective breeding is selecting desired traits of certain plants and animals to pass on to future generations.
• Desired traits pass on to future generations through hybridization and inbreeding.

Hybridization

• Hybridization is crossing parent organisms with different forms of a trait to produce offspring with specific traits resulting in hybrids.
• Hybrid organisms can be bred to be more disease-resistant, produce more offspring, and grow faster.
• Plant breeders might cross two different varieties of tomato plants to produce a hybrid with disease resistance and fast growth rate.
• Disadvantages of hybridization include it being time-consuming and expensive.

Inbreeding

• Inbreeding is breeding two closely related organisms to have the desired traits and eliminate undesired ones in future generations.
• Pure breeds are maintained by inbreeding.
• Clydesdale horses and Angus cattle are examples of organisms produced by inbreeding.
• Harmful recessive traits can be passed on to future generations through the disadvantage of inbreeding.
• Inbreeding increases the chance of homozygous recessive offspring, increasing the likelihood of harmful trait inheritance.
• If a breeder observes a desired dominant trait, the genotype could be homozygous dominant or heterozygous.
• The exact genotype is determined by performing a test cross.

Test Cross

• A test cross involves breeding an organism with an unknown genotype with one that is homozygous recessive for the trait.
• If the parent's genotype is homozygous dominant, all offspring will have the dominant phenotype.
• If the parent's genotype is heterozygous, the offspring will show a 1:1 phenotypic ratio.
• In the example of a breeder wanting to produce hybrid white grapefruits, white fruit color is the dominant trait and red is recessive, so red grapefruit trees must be homozygous recessive (ww).
• The hybrid white grapefruit tree genotype obtained by the breeder can be homozygous dominant (WW) or heterozygous (Ww) for the white color.
• Reviewing results is in Punnett squares.
• If the white grapefruit tree is homozygous, all of its offspring will be heterozygous-white.
• If the tree is heterozygous, half of the test cross offspring will be white and half will be red.

DNA Technology

• By 1970, researchers discovered DNA structure and confirmed that information flowed from DNA to RNA and from RNA to proteins.
• Genetic Engineering: Technology manipulating an organism's DNA to insert exogenous DNA (DNA from another organism).
• Researchers inserted a gene for a bioluminescent protein called green fluorescent protein (GFP) into various organisms.
• GFP, a substance naturally found in jellyfishes living in the north Pacific Ocean, emits green light when exposed to ultraviolet light.
• Organisms genetically engineered to synthesize GFP DNA, like mosquito larvae, can be quickly identified under ultraviolet light.
• The GFP DNA is attached to exogenous DNA to verify that the DNA has been inserted into the organism.

Genetically engineered organisms uses

• Studying the expression of a particular gene
• Investigating cellular processes
• Studying the development of a certain disease
• Selecting traits that might be beneficial to humans

DNA tools

• Selective breeding is used to produce plants and animals with desired traits.
• Genetic engineering increases or decreases specific genes' expression in selected organisms.
• Genome is the total DNA in the nucleus of each cell.
• DNA tools manipulate DNA isolate genes because genomes contain millions of nucleotides.

Restriction Enzymes

• Restriction Enzymes: Proteins recognizing and binding to specific DNA sequences and cleaving (splitting) the DNA within that sequence.
• A restriction enzyme, also called an endonuclease, cuts viral DNA into fragments after it enters bacteria.
• Restriction enzymes, discovered in the late 1960s, are powerful tools to isolate specific genes.
• When the restriction enzyme cleaves genomic DNA, it creates fragments of different sizes that are unique to every individual.
• EcoRI is a restriction enzyme widely used by scientists that specifically cuts DNA with the sequence GAATTC.
• The ends of the DNA fragments created by EcoRI are called sticky ends.
• Sticky ends contain single-stranded DNA that is complementary.
• Sticky ends from some restriction enzymes can join with other DNA fragments with complementary sticky ends.
• Some enzymes produce blunt ends when the restriction enzyme cuts straight across both strands.
• Enzymes with blunt ends do not have regions of single-stranded DNA and can join to any other DNA fragment with blunt ends.
• Gel Electrophoresis: Process separating DNA fragments by size using an electric current.
• DNA fragments load on the negatively charged end of a gel when an electric current is applied.
• Negatively charged DNA fragments move pushed toward the positive end of the gel.
• Smaller fragments move faster and farther than larger ones.
• A unique pattern based on the size of the DNA fragment can be compared to known DNA fragments for identification.
• Portions of the gel containing each band can be removed for further study.

Recombinant DNA Technology

• Fragments of a specific size can be removed from the gel and combined with DNA fragments from another source after gel electrophoresis has separated DNA fragments.
• Recombinant DNA: A newly generated DNA molecule with DNA from different sources that enables individual genes to be studied.
• A vector, or carrier, transfers the recombinant DNA into a host cell, like a bacterial cell.
• Commonly used vectors are Plasmids and viruses.
• Plasmids: Small, circular, double-stranded DNA molecules naturally occurring in bacteria, that can be cut using restriction enzymes
• If a plasmid and DNA fragment from another genome cleave by the same restriction enzyme, the ends of each DNA fragment will be complementary and can be joined.
• DNA Ligase: An enzyme cells use in DNA repair and replication, and joins the two DNA fragments chemically, can facilitate DNA fragments with sticky and blunt ends.
• The resulting circular DNA molecule contains sequences of the bacterial plasmid DNA alongside a sequence from the other genome.
• This recombinant plasmid DNA molecule is inserted into a host cell and cloned, in order to make large quantities.
• Gene Cloning: Bacteria can take up DNA from their environment, or transformation.
• Large quantities of recombinant plasmid DNA is made by mixing bacterial cells with recombinant plasmid DNA; some bacterial cells take up the recombinant plasmid DNA through transformation.
• These bacteria can be transformed through electroporation or heat.
• Electroporation = short electric pulse or quick change in temperature.
• This will temporarily open up the plasma membrane allowing small molecules to enter the bacterial cell like recombinant plasmid DNA.
• The bacteria then makes copies of the recombinant plasmid DNA during cell replication.
• In this process large numbers of identical bacteria can cloning.
• Recombinant plasmid DNA contains a gene that codes for resistance to an antibiotic like ampicillin (AMP), which can be used to distinguish which bacteria are cloned.
• When the transformed bacterial cells are exposed to the specific antibiotic, only the bacterial cells that have the plasmid survive.
• DNA Sequencing: Unknown DNA nucleotides sequence can be identified by determining the sequence of genes can can
• Predict the gene's function
• Compare genes with similar sequences from other organisms.
• Identify mutations or errors in the DNA sequence.
• DNA used for sequencing must be cut into smaller fragments using restriction enzymes.
• Scientists mix an unknown DNA fragment, DNA polymerase, and the four nucleotides (A, C, G, T) in a tube.
• Small amount of each nucleotide tagged with a different color of fluorescent dye, which also modifies the structure of the nucleotide.
• Once modified strands are synthesized, it stops at the fluorescent-tagged nucleotide
• Produces DNA strands of different lengths.
• Sequencing reaction completes with tagged DNA fragments are separated using gel electrophoresis.
• An automated DNA sequencing machine is used to then analyze gel.
• The sequence of the original DNA template determine order of tagged fragments.

Polymerase Chain Reaction

• Polymerase Chain Reaction (PCR): Once the sequence of a DNA fragment is known, this technique makes millions of copies of a specific region of a DNA fragment.
• PCR can detect a single DNA molecule in a sample.
• Utility because this single DNA molecule can be copied or amplified to be used for DNA analysis multiple times.

Steps of PCR

• PCR is performed by placing DNA fragments the fragment to be copied in, along with DNA polymerase, four DNA nucleotides, primers.
• Primers complementary DNA synthesis primers start at ends ends of to-be-copied DNA fragment.
• PCR beings when the tube heated
• Heat separates the two strands of the template DNA fragment, and when the tube is cooled, the primers can bind to each strand of the template DNA.
• A thermocycler cycles the PCR components through cool/hot temperatures.
• DNA polymerase incorporates nucleotide molecules and links them between the two primers as normal during DNA replication.
• This entire process is repeated up to 40 times.
• Since separating requires heat, a heat able DNA polymerase is used.
• That heat able polymerase came from a thermophile.
• Restriction enzymes are a tool used to create DNA fragments with sticky or blunt ends that can join with other DNA fragments.
• Gel electrophoresis is a tool used to study DNA fragments of various sizes.
• Recombinant DNA technology is a tool used to create recombinant DNA to be used to study individual genes and genetically engineered organisms, and in the treatment of certain diseases.
• Gene cloning, a tool, is used to create large amounts of recombinant DNA to be used in genetically engineered organisms.
• DNA sequencing is a tool used to identify errors in the DNA sequence, to predict the function of a particular gene, and to compare to other genes with similar sequences from different organisms.
• Polymerase chain reaction (PCR) makes copies of specific regions of sequenced DNA for any scientific investigation, including forensic analysis and medical testing.
• Genetic engineering is used in forensic science, medical diagnosis, including the detection of viruses, and genetic research.

Biotechnology

• Biotechnology: Use of genetic engineering to find solutions to problems.
• Transgenic Organisms: Organisms genetically engineered by inserting a gene from another organism.
• Transgenic animals, plants, and bacteria are used for research and for medical and agricultural purposes.
• Transgenic Animals: Transgenic roundworms are widely used for research in laboratories around the world.
• Improving food supply and human health has been accomplished by producing transgenic livestock.
• Transgenic goats produce milk with human antithrombin III to prevent blood clots.
• Researchers produce transgenic chickens and turkeys resistant to diseases, along with genetically engineered species of fishes grown faster.
• In the future, transgenic organisms might be used as a source of organs for organ transplants.
• Transgenic Plants: Producing genetically engineered cotton resistant to insect infestation, peanut plants that are genetically engineered to not cause allergies, and sweet-potato plants that resists a virus could kill most of the African harvest are ways scientists help plants.
• Rice plants with increased iron and vitamins that could decrease malnutrition in Asian countries, and a variety of plants able to survive extreme weather conditions are examples of transgenic plants.
• Transgenic Bacteria: Making insulin, growth hormones, and blood clot dissolving substances are a result of transgenic bacteria.
• Preventing frost with ice crystals, efficiently cleaningup oil spills, and efficiently decomposing garbage are the result of transgenic bacteria.

The Human Genome

• Genome: The complete genetic information in a cell.
• The Human Genome Project (HGP): An international project completed in 2003
• The goal of the HGP was to determine the sequence of the about 3 billion nucleotides that make up human DNA and identify all of the human genes.
• Studies in nonhuman organisms, like the fruit fly, mouse, and Escherichia coli develop the technology required to handle large amounts of data produced by the Human Genome Project.
• These technologies help interpret the function of newly identified human genes
• Sequencing the Genome: Human DNA is organized into 46 chromosomes
• 46 human chromosomes cleaved (split) in order to determine one continuous human genome sequence.
• To produce fragments with over lapping sequences, several different restriction enzymes were used
• Combined fragments with vectors to create recombinant DNA, clones were made, and sequenced using automated sequencing machines through the HGP.
• Overlapping regions computer analysis generated one continuous sequence.
• Scientists then decode genetic code.
• Scientists observed less than 2% of all the nucleotides in the human genome code for all the proteins in the body after sequencing the entire human genome.
• The genome is filled with regions called noncoding sequences, long stretches of repeated sequences that have no direct function.
• DNA Fingerprinting: The protein-coding regions of DNA are almost identical among individuals, while noncoding regions are unique.
• Cutting genomic DNA with restriction enzymes produces an individual set of DNA fragments.

DNA Finger-Printing

• Involves separating DNA fragments using gel electrophoresis to observer individual banding patterns
• Involves England in 1985
• Forensic scientists use DNA fingerprinting to:
• identify suspects or victims in violent cases
• determine paternity
• identify soldiers killed in war
• PCR copies small hair DNA for forensic use to make larger analysis sample
• Cutting amplified DNA with different restriction enzymes.

Identifying Genes

• Many genes still have unknown functions

For an organism to be complex:

• Integrate computer analysis and recombinant DNA technology to determine the function of genes.
• Lack large regions of noncoding DNA, researchers identify open reading frames.
• Open Reading Frames: DNA stretches containing at least 100 codons with a start and stop codon.
• If they produce functioning proteins, sequences indicating genes will be tested.
• Recall, codons code amino acids, searching for codons helps. *analysis has 90% correct ORF gene identification in yeast and bacteria.
• Advanced human organism algorightms identify genes, which use DNA sequence from other organisms too.

Bioinformatics

• The completion of the HGP and genomes of other organisms results in large amounts of data.
• Requires careful storage, organization, information sequence storing, leads to Bioinformatics.
• Bioinformatics involves creating and maintaining database of biological information.
• It involves analysis of sequence information, finding genes and developing functions of newly discovered protein.
• Grouping protein sequences into genes is also Bioinformatics.
• Analyzing the expressed genes from a cell type can be applied with DNA microarrays.
• DNA microarrays can contain a few genes, such as the genes that control the cell cycle, or all of the genes of the human genome.
• DNA microarrays are microscope slides with gene sequences used to analyze multiple genomes.
• Identifying genetic factors requires Microarrays

To complete DNA microarrays:

Steps:

• mRNA extraction
• creation of CDNA strands
• combine fluorescent colors
• apply cDNA to the mircoarray
• The fluorescent signals show the expressions of genes.
• Scientists use Microarrays more.

Genome and Genetic Disorders

• Genetic disorders are nucleotide base variations in DNA.
• Single Nucleotide Polymorphisms (SNPs): base variations when nucleotide genome changes.
• Must occur in >1% of population
• SNPs have no cell function, map could show.
• HapMap Project: common genetic variations in humans

HapMap description will state:

• variations
• area
• distribution
• This information helps researchers drugs and responses.
• Pharmacogenomics- inheritence of drugs. = safety, recovery and side effects in individuals
• It hopes to custom make treatments based on genetic makeups.
• Gene Therapy- correcting mutated genes
• Scientist insert the replaced version- recombinant DNA created and infected, then functioning restored.
• First trial was with SCID in 1990.
• Recent trials with other diseases.
• "Genomic era".
• Genomics- the study of genomes and the proteins they perform.
• This has become among the best strategies for determining functions.
• Genes are the primary storage function, proteins are the primary mechanical function.
• Proteomics- large scale study of function/strucutre
• This will better define normal and disease states
• Scientists test proteomics to treat diabetes, obesity, and atherosclerosis.