3.1 - Genes

Genetics is a “coming into being” or “developing the self.” It comes from the word “Genesis” or “to generate.”

• It is the study of heredity, which is the transmission of characteristics (genes) to future generations.

• It encompasses the understanding of how genetic information is passed from one generation to the next, leading to the diversity and variability observed in traits among individuals within a species.

• It explores the molecular mechanisms and processes that underlie the inheritance of traits, revealing the roles of DNA, genes, and chromosomes in the functioning of living organisms.

• It provides insights into how genetic mutations, adaptations, and natural selection drive the evolutionary processes that shape the diversity of life on Earth over long periods of time.

List several areas where Genetics is important in society:

• News - Genetics plays a pivotal role in informing the public about breakthroughs in genetic research, biotechnology advancements, and ethical debates related to genetic technologies. News outlets cover topics such as genetic discoveries, cloning, and gene editing, keeping the public informed about the latest developments.

• Medicine - Genetics is crucial in the field of medicine, contributing to personalized medicine and diagnostics. Genetic testing and analysis help in identifying genetic predispositions to diseases, tailoring treatments, and understanding the genetic basis of various conditions, leading to more effective healthcare strategies.

• Epigenetics - Epigenetics explores how environmental factors can influence gene expression and impact an individual's health. Understanding epigenetic changes is vital for studying conditions like cancer, aging, and developmental disorders, as well as for designing interventions to promote healthier lifestyles.

• Agriculture - Genetics is instrumental in modern agriculture by enabling the development of genetically modified (GM) crops, which are engineered to resist pests, diseases, and environmental stressors. This technology increases crop yields and contributes to global food security.

• Pest management - Genetic approaches are used in pest control strategies. For instance, the use of sterile insects or genetically modified organisms can help reduce pest populations, protecting crops and minimizing the need for chemical pesticides.

• DNA sequence analysis bioinformatics - DNA sequence analysis and bioinformatics are essential tools for deciphering the genetic code, identifying genes, and understanding their functions. These technologies underpin research in genetics, genomics, and proteomics.

• Gene editing and gene therapy - Genetic engineering and gene therapy hold promise in treating genetic disorders by modifying or replacing faulty genes. These techniques are at the forefront of medical research and offer potential cures for previously untreatable conditions.

Vocabulary

• Allele: a form of a gene (there are two possible variations)

• Phenotype: the actual appearance of an individual for a particular trait

• Genotype: the combination of alleles that an individual has for a given gene

• Homozygous: the two alleles for a given trait are the same

• Heterozygous: the two alleles for a given trait are different

• Dominant: an allele that is physically seen in a heterozygous individual

• Recessive: an allele that is not physically seen in a heterozygous individual

Genetic Disorders

• Osteogenesis imperfecta:

  • A dominant mutation with variable effects that may include

    • multiple bone fractures

    • decreased mobility

    • blue or grayish-blue tinted sclera, which is the white part of the eye.

    • hearing loss due to deformities in the small bones of the middle ear or other abnormalities related to the condition

• Marfan Syndrome:

  • A dominant mutation that only requires one allele of the mutation to have it.

    • People affected have long limbs.

    • A famous person affected by the disorder was Flo Hyman, who died of heart complications.

    • The disease weakens your aorta, which makes you bleed easily.

• Sickle Cell Anemia:

  • A recessive mutation where an individual’s blood cells are sickled instead of smooth and round.

    • The blood cells get stuck in the capillaries.

    • 2 mutated alleles are required for this genetic disorder, which is most common in people of African descent.

• Cystic Fibrosis:

  • A recessive mutation that causes thick mucus to accumulate in the lungs, leading to respiratory infections and breathing difficulties.

    • It occurs in a gene on Chromosome #7 and is common in those of European descent.

    • Genetic testing is available.

    • Gunnar Esiason is a famous example of someone who has the mutation.

• Xeroderma Pigmentosum (XP):

  • A recessive mutation that causes excessive sensitivity to UV light.

    • Persons affected must have two alleles of the gene to have the disease.

    • They can get many small tumors on their body that look like freckles but is actually skin cancer.

Karyotype

• Karyotype: An organized array of a cell’s chromosomes. This includes sex and non-sex chromosomes.

• A karyotype is a visual representation of an individual's complete set of chromosomes, displayed in an organized manner.

• It provides a snapshot of an individual's genetic makeup and reveals the number, size, and structural abnormalities of chromosomes. 

• Karyotypes are important in diagnosing genetic disorders, as they can identify chromosomal abnormalities like trisomy 21 (Down syndrome). 

• They consist of both sex chromosomes (X and Y) and non-sex chromosomes (autosomes), enabling geneticists to assess an individual's gender and overall chromosomal health. 

• Karyotyping is a valuable tool in prenatal screening, cancer diagnosis, and understanding various genetic conditions.

3.2 - Chromosomes

Chromosome Structure

Chromosome parts: A chromosome is 15% DNA, 10% RNA and 75% Protein

• Metacentric: the centromere is in the middle of the chromosome

• Submetacentric: the centromere is slightly off-center

• Acrocentric: the centromere is very off-center

• Telocentric: the centromere is at the top of the chromosome

The Cell Cycle

• G1

  • Gap 1. Cells accomplish much of their growth, growing larger and making proteins and organelles needed for DNA synthesis.

  • During the G1 phase, cells engage in a wide range of activities to prepare for the upcoming stages of the cell cycle. 

    • These activities include metabolic processes, energy production, and the synthesis of proteins and other molecules necessary for the cell's growth and function.

  • The G1 phase is also a critical checkpoint in the cell cycle, where the cell evaluates its internal and external conditions to determine whether it should proceed to DNA replication (S phase) or pause for repairs. 

    • This checkpoint helps maintain the integrity of the cell cycle and ensures that only healthy and well-prepared cells continue to divide.

  
  

• S

  • S-phase. The cell synthesizes a copy of the DNA in its nucleus.

  • The primary purpose of the S-phase is to replicate the DNA. 

    • Each chromosome's double-stranded DNA is unwound, and complementary nucleotides are added to create an identical copy. 

    • This ensures that each daughter cell will receive a complete set of genetic information during cell division.

  • Quality Control: DNA replication is a highly accurate process, but errors can still occur. 

    • During the S-phase, the cell has mechanisms to check and repair any mistakes in the replicated DNA to maintain genomic integrity and prevent mutations from being passed on to the next generation of cells.

• G2

  • Gap 2. The cell grows more as it makes more proteins and organelles, reorganizing its contents in preparation for mitosis.

  • Cellular Checkpoints: G2 is a critical phase where the cell undergoes checkpoints to ensure that DNA replication in the S phase occurred without errors. 

    • If DNA damage or inaccuracies are detected, the cell may undergo repair processes before progressing to mitosis, helping maintain genomic integrity.

  • Preparation for Mitosis: During G2, the cell accumulates the necessary resources, such as additional organelles and proteins, to support the upcoming process of mitosis. 

    • This phase is essential for ensuring that the cell is fully prepared to divide and create two genetically identical daughter cells during the subsequent M phase of the cell cycle.

• M

  • M-phase. Nuclear division (mitosis) followed by cytoplasmic division (cytokinesis)

  • Mitotic Spindle Formation: During the M-phase, one crucial event is the formation of the mitotic spindle, a dynamic structure composed of microtubules. 

    • The mitotic spindle helps segregate the duplicated chromosomes into two daughter cells by attaching to the centromeres and guiding their separation.

  • Ensuring Chromosome Accuracy: The M-phase is essential for ensuring the accurate distribution of genetic material to daughter cells. 

    • Mitosis, a subphase of M, precisely separates identical sister chromatids, resulting in two genetically identical daughter cells, each with a complete and accurate set of chromosomes. 

    • This process is vital for growth, tissue repair, and the maintenance of genetic stability in multicellular organisms.

    
    

Mitosis

• Prophase:

  • Chromosomal Condensation: During prophase, the chromatin, which is a complex of DNA and proteins, condenses into visible, discrete structures known as chromosomes. 

    • This condensation is crucial for the efficient segregation of genetic material during cell division.

  • Formation of Spindle Fibers: Prophase marks the formation of the mitotic spindle, a structure composed of microtubules. 

    • These spindle fibers extend from opposite ends of the cell and play a vital role in moving and aligning chromosomes during later stages of cell division.

  • Nuclear Envelope Breakdown: In most eukaryotic cells, the nuclear envelope, which separates the nucleus from the cytoplasm, begins to disintegrate during prophase. 

    • This breakdown allows the spindle fibers to access the chromosomes in preparation for their proper distribution to daughter cells.

• Prometaphase:

  • Nuclear Envelope Breakdown: During prometaphase, the nuclear envelope, which surrounds the cell's nucleus, disassembles. 

    • This allows the spindle fibers to access the chromosomes and is a critical step in preparing for cell division.

  • Chromatids Become Visible: As prometaphase progresses, the individual chromatids within duplicated chromosomes become distinct and visible under a microscope. 

    • They are connected at the centromere and are ready to be separated during mitosis.

  • Formation of the Mitotic Spindle: Prometaphase is marked by the formation of the mitotic spindle, a structure composed of microtubules that will help segregate the chromatids to opposite ends of the cell during the subsequent stages of mitosis.
    

• Metaphase:

  • Chromosomal Alignment: During metaphase, chromosomes within a cell align along the cell's equatorial plane, forming a characteristic metaphase plate. 

    • This alignment is crucial for ensuring accurate chromosome segregation during cell division.

  • Spindle Fibers Attach: Metaphase is marked by the attachment of spindle fibers to the centromeres of each chromosome. 

    • These spindle fibers exert tension, helping to position the chromosomes precisely at the metaphase plate.

  • Checkpoint Control: The cell uses a metaphase checkpoint to ensure that all chromosomes are properly aligned before proceeding to anaphase, where they will be pulled apart. 

    • This checkpoint is a critical mechanism to prevent errors in chromosome segregation.

• Anaphase:

  • Chromosome Separation: Anaphase is a critical stage of cell division, occurring in both mitosis and meiosis, during which sister chromatids are pulled apart and move towards opposite ends of the cell.

  • Centromere Division: The centromere, which holds sister chromatids together, divides during anaphase, ensuring that each daughter cell will receive a complete set of chromosomes.

  • Ensuring Genetic Diversity: In meiosis, anaphase I separates homologous chromosomes, promoting genetic diversity, while in anaphase II, it separates sister chromatids to produce haploid daughter cells. 

    • In mitosis, it ensures the identical distribution of genetic material to daughter cells for growth and tissue repair.

  
  

• Telophase:

  • Chromatids Begin to Separate: In telophase, the chromatids of each chromosome, which were previously joined at the centromere during anaphase, start to separate and move to opposite poles of the cell.

  • Nuclear Envelopes Reappear: The nuclear envelopes, which had disintegrated during prophase, begin to reform around the separated sets of chromatids. 

    • This process results in the formation of two distinct nuclei within the cell.

  • Cell Division Approaches Completion: Telophase marks the final stages of cell division, and as the nuclear envelopes reform and the chromosomes decondense, the cell is nearly ready to complete the process of cytokinesis, resulting in two daughter cells.

• Cytokinesis

  • The cytoplasmic division of a cell at the end of mitosis or meiosis, bringing about the separation into two daughter cells.

  • Cell Division Phase: Cytokinesis is the final stage of the cell division process, following mitosis or meiosis, where the cytoplasm of a parent cell is divided into two daughter cells.

  • Cleavage Furrow or Cell Plate Formation: In animal cells, cytokinesis typically involves the formation of a cleavage furrow, a contractile ring of proteins that pinches the cell's membrane to create two separate daughter cells. 

    • In plant cells, a cell plate forms at the center, which eventually develops into two daughter cells.

  • Ensures Genetic Continuity: Cytokinesis is essential for maintaining genetic continuity by segregating the replicated DNA and organelles into the newly formed cells. 

    • It ensures that each daughter cell receives a complete and functional set of cellular components.

Interphase = G1 + S + G2

When is there one chromatid and when are there 2 chromatids (sister chromatids) on the centromere?

• During anaphase, there are two chromatids on the centromere. During Telophase, there is just one chromatid on the centromere.

Chromosome Preparation

• Colchicine destroys spindle fibers in a cell so the view of chromosomes is unobstructed

• Hypotonic Salt solution increases the cell volume

• Dropping cells from height splinters them apart by force, which reveals chromosomes

Homologous Chromosomes have the same length, centromere, position, banding pattern, and genetic loci.

Karyotype

• Define karyotype: an organized array of a cell’s chromosomes

• Autosomes: non-sex chromosomes

• Sex Chromosomes: X or Y chromosomes

• Describe arrangement in karyotype: homologous chromosomes are matched together

Karyotypes can show whether an individual has a genetic disorder based on the appearance or absence of chromosomes.

FISH Technique

• FISH stands for: Fluorescent in situ Hybridization

• Purpose: to use fluorescent DNA probes to target specific chromosomal locations within the nucleus, resulting in colored signals that can be detected using a fluorescent microscope

3.3 - Meiosis

Mitosis

Purpose of mitosis:

• to replicate identical cells for different functions in the body

Define somatic cell:

• a cell that does not produce gametes

List stages and what happens in each stage

1. Prophase: The chromosomes shorten and thicken. (60% of time)

2. Metaphase: Chromosomes line up in the middle of the cell. (5% of time)

3. Anaphase:  Chromatids break apart at the centromere and move to opposite poles. (5% of time)

4. Telophase: Two nuclei formed after nuclear envelopes reform around each group of chromosomes. (30% of time)

Define Cytokinesis

• the cytoplasmic division of a cell at the end of mitosis, bringing about the separation into two daughter cells.

Synchronized Swimming Mitosis Video

• What do the green noodles represent?

  • Spindle fibers.

• What do the people holding the green noodles represent?

  • centrioles

• What do the swimmers with the swim caps represent?

  • cell membrane

Meiosis

• Define meiosis:

  • Cell division in a diploid cell that leads to gamete formation.

• Two division – what happens in each?

  • Reductional division: The number of chromosomes in daughter cells gets cut in half from the original number.

  • Equational division: The chromosome number is the same at the end as it was at the beginning.

• Define crossing over and describe its importance:

  • Crossing over is recombination in Prophase I. 2 chromosomes come together and break apart when they overlap and switch some of their alleles. This allows variation from generation to generation as alleles switch around.

List the stages of meiosis and what happens in each

MEIOSIS I

1. Prophase I:

   1. The chromosomes begin to condense and pair up, aligning with their partner so they can match up at corresponding positions along full length.

2. Crossing over:

   1. The chromosomes shuffle their alleles between each other

3. Metaphase I:

   1. The homologue pairs line up at the metaphase plate for separation

4. Anaphase I:

   1. Homologues break apart and move to opposite ends of the cell, while the sister chromatids remain attached

5. Telophase I:

   1. The chromosomes end up on opposite poles of the cell, starting cytokinesis and producing 2 haploid daughter cells

MEIOSIS II

1. Prophase II:

   1. Chromosomes condense and, if needed, the nuclear envelope breaks down. The centrosomes move apart, forming the spindle, which begins to capture chromosomes

2. Metaphase II:

   1. The chromosomes line up individually on the metaphase plate

3. Anaphase II:

   1. The sister chromatids separate and move to opposite ends of the cell

4. Telophase II:

   1. Nuclear membranes form along the chromosomes, allowing them to decondense and start cytokinesis, which produces 4 haploid cells (which are sperm or egg cells)

Biological significance of meiosis

• Constant number of chromosomes (along with fertilization)

  • Since meiosis creates cells that become gametes, it’s important that the chromosome number is reduced to the original size because it would not be good if there were twice the number of original chromosomes.

• Provides variation from generation to generation (2 ways)

  • crossing over: shuffling chromosomes between parents

  • independent assortment: genes independently separate themselves from one another when reproductive cells develop

Compare Mitosis and Meiosis

• Meiosis is vital for fertilization.

  • Mitosis is vital for cell replication for different tasks in the body.

• Meiosis maintains a constant number of chromosomes.

  • Mitosis does not pair chromosomes and so it ends up producing twice the original amount of cells.

• Meiosis provides variation from generation to generation.

  • Mitosis replicates the exact same cell without any variation.

• Meiosis shuffles chromosomes between parents (there are 2^23 possible combinations of chromosomes).

  • Mitosis creates the same mitotic problems each time.

3.4 - Inheritance

Vocabulary

• Phenotype: Describes the appearance of an individual for a trait

• Genotype: Describes the alleles (forms of genes) that an individual has for a particular trait. Genotypes are indicated by alphabet abbreviations. Since each person has 2 copies of each gene, each person will have 2 letters representing their genotype.

• Alleles: the various forms of genes

• Dominant: The allele that is seen in the individual’s phenotype if an individual has two different alleles for a gene

• Recessive: The allele that is not seen in the individual’s phenotype if an individual has two different alleles for a gene

Genetics in Humans

How many chromosomes are in a normal human gamete?

• 23 chromosomes

Which gamete, the mother’s egg or the father’s sperm, determines the sex of the baby?

• The father’s sperm determines the sex of the baby because only sperm can carry a Y chromosome. The egg always carries an X chromosome, but the sperm can either carry an X or Y chromosome. An XY combination creates a male and an XX combination creates a female.

How can the sex of the baby be determined prior to birth?

• Using ultrasounds, bloodwork, and choosing sperm likely to have an X or Y chromosome.

Distinguish between monozygotic and dizygotic twins.

• Monozygotic twins are identical. 1 sperm and 1 egg join together, and the embryo divides into 2 fetuses.

• Meanwhile, dizygotic twins are fraternal. 2 eggs are fertilized at the same time (each with a different sperm) and make one fetus each. Unlike monozygotic twins, dizygotic twins run in families.

Approximately what proportion of your genes do you share with your full sibling?

• Roughly 50%.

Give the name of a common genetic disorder

• Down Syndrome, Huntington’s Disease, Cystic Fibrosis, Color Blindness, etc.

Cystic Fibrosis Example

Define Carrier:

• Carriers have one copy of the cystic fibrosis allele and one copy of the normal allele

Cystic fibrosis is inherited in a recessive manner (so Cystic fibrosis would be ff)

• The child will have a ¼ or a 25% chance of getting Cystic fibrosis if his parents are both carriers.

Mutation

Other than genetics, what can influence the phenotype of an individual?

• The environment can influence phenotype (height, weight, skin color, hair color, etc) beyond genetics.

Define Mutation

• An incorrect copy of DNA, or a change in the DNA sequence.

What is the role of mutation in evolution?

• Define Evolution

  • The process by which organisms are assumed to have developed and diversified over time from their earlier forms.

• Describe the role of mutation in evolution.

  • Mutation serves as a form of adaptation in evolution. 

    • When a species would benefit from a mutation, organisms with that mutation are favored in the reproduction cycle so that the mutation gets copied to other members of the species. 

    • Therefore, mutations are essential for helping a species grow and adapt to new environments over time.
      

3.5 - Genetic Modification and Biotechnology

GMOs

• What does GMO stand for? genetically modified organisms

• Define GMO: any genetically modified organism or micro-organism whose genetic material has been altered by means of genetic engineering

• How does genetic engineering differ from traditional plant breeding?

  • Breeding can only be done between two plants that can sexually reproduce with each other. This can pass on both desirable and undesirable traits.

  • With genetic engineering, DNA is inserted directly into the plant so sexual reproduction is not required. Also, only the specific gene(s) are inserted so undesirable traits are not put into the plant during the process.

• Why are GMOs produced (what are some beneficial traits to incorporate?)

  • Improved ability to grow in less favorable environments

    • Resistance to pests

    • Drought resistant

  • Improved ability to harvest or store the products

    • Flavr Savr Tomato

  • Improved nutritional value

  • Cost savings for the farmer, processing company, etc

• Examples:

  • Bt Corn is resistant to the corn borer insect because it contains a gene from the soil bacteria Bacillus thuringiensis.

• Rainbow papaya is resistant to ringspot virus

• Golden rice (version 2) has a gene from Psy gene from corn and is designed to produce beta-carotene, which our bodies can use to produce vitamin A.

  • Discuss challenges with gaining acceptance of Golden Rice

    • People weren’t enthusiastic about eating golden rice. It was not the same color as the traditional rice so it was difficult to market.

    • Public relations campaigns were started in several countries.

    • Golden rice was blessed by the pope, but not “officially endorsed” by the church. Since 80% of Philippines is Roman Catholic, it was thought that this blessing might encourage more acceptance in the community.

    • Golden rice was approved for cultivation in Canada, Australia, New Zealand and the United States in 2018. In February 2019, Bangladesh announced that they may allow commercial cultivation later in the year.

• Concerns about using GMOs

  • Some GMO’s are untested or have not been in circulation for long period of time

    • GMO technologies have only been in use since the 90’s

    • Unknown effects

  • Food Web Risks (harming other organisms in the environment)

    • Animals and Humans

  • Because there are foreign genes being inserted into foods (some from other crops, bacterial and viral vectors, viral promoters and antibiotic marker systems)

    • Toxicity, allergic reactions?

  • GMO seeds are patented by the companies that create them

    • It becomes illegal to save, replant, cross breed or conduct research on those patented seeds and crops.

    • People lose the opportunity to grow and harvest their own crops

    • Leads to decrease in genetic diversity of the crop species

  • Could have drastic effects on the environment and the balances within nature

    • Much like an invasive species

    • Wild genes can’t compete

    • Cross pollination

• Restriction of GMOs by some countries

  • More than 60 countries around the world ban or restrict the production and sale of GMOs.

  • Including Australia, Japan, several European countries

  • Some of these bans are on specific GMO products like Monsanto’s maize

  • Some of the countries that ban production of GMO crops still import and use these crops – particularly corn and soybean products

• Labeling Laws for GMOs

  • 2017 - National Bioengineered Food Disclosure Standard passed by Congress requiring USDA to set a labeling standard for GM food

  • December 20, 2018 – USDA released official GMO label standard

    • Implementation begins in 2020 and food companies must comply by January 1, 2022

    • Labels include text, symbols and electronic link that can be scanned

Genetic Pest Management

• Goals:

  • Reduce economic loss due to pests such as insects that prey on crops and livestock

  • Combat invasive species

  • Decrease human disease by managing pest vectors

• Two approaches:

  • Genetically modify the crop to be resistant to the pest

  • Use a gene drive to decrease the size and reproductive ability of the pest population

• Define “Gene Drive” as used in genetic pest management

  • It is a genetic element that is passed from parent to progeny at a higher frequency than what is expected in normal Mendelian segregation. This means that an unusually large proportion of the progeny end up with the particular set of genes, causing the frequency of those genes to increase rapidly in the population.

Potential Uses of Gene Drives

• Stop insects from transmitting malaria and other diseases

• Alter the pests that attack crops to increase crop yield and

• Limit spread of invasive species to protect existing ecosystems

Examples:

• New World Screwworm

  • Major pest of livestock in its larval form – eats flesh

  • Currently managed by releasing sterile flies into the population. They mate with the wild population, but progeny are not produced thereby decreasing the pest population size

  • Expensive to produce so many sterile flies. Cheaper if there was a way to make a male only line to release

  • Strain of C. hominivorax with addition of a female-lethal gene

  • Raised on tetracycline in the lab and females live

  • No tetracycline, only males are produced

  • These males seem to be able to compete for mates

  • Would cost only about 20% of the current cost

  • Transgenic Male-Only strain of New World Screwworm

• Red Flour Beetle

  • Global pest of stored grains and cereals

  • Contains a “selfish” genetic element called Medea, which causes genes near it to be passed to the next generation with a high frequency (5x normal rate) (Gene Drive)

  • Plan: Insert genes that can cause sterile, male-only progeny to be produced near the Medea element,

    • Should be able to increase the frequency of sterile males

    • Which should decrease the population size over time.

  • Alternative Plan: Use CRISPR-Cas9 to insert genes that inactivate a gene essential for female development, resulting in only male progeny from a mating and these progeny would then pass on the genes in future matings.

  • Agricultural Production: Assessment of the potential use of Cas9-mediated gene drive systems for agricultural pest control

RNAi and alteration of gene expression

• The i stands for interference

• Who received the Nobel Prize for RNAi? Fire and Mello

• How does it work:

  • Injected dsRNA (double-stranded RNA) into the nematode, C.elegans

  • Noticed that genes with nucleotide sequence that was same as injected RNA had decreased expression

  • Can silence targeted gene by inhibiting translation

  • RNAi limits the invasion of foreign genes and censors the expression of cell’s own genes

Important molecules and their roles:

• Double-stranded RNA (dsRNA) cut by the enzyme dicer to form small RNAs

• miRNA (micro RNA) and siRNA (small interfering RNA) complex with the protein argonaut to form RISC.

• RISC stands for RNA Induced Silencing Complex

• RISC pairs with mRNA and does one of the following:

  • inhibits translation

  • degrades mRNA

Uses for RNAi technology

• Studying gene function

• Medicine: RNA interference in therapy

  • One of the first clinical trials in macular degeneration halted after possibly creating blindness

  • Many trials underway to target cancer/tumors

    • Often target cell cycle proteins

    • 2014 – 10+ drugs in clinical trials

    • Delivery presents a problem

  • Fall 2017, RNAi drug to treat a rare nerve disorder was shown to be effective and safe in clinical trials

    • 15 years of research

  • Possible problem if a gene with an accidently similar sequence is also repressed (off target effects)

• Agriculture

  • Smartstax corn, a new variety that contains a dsRNA to target a gene in the rootworm (corn pest)

  • Gene is not present in corn, cows or people so should be safe for our use

CRISPR-Cas9 System and Gene Editing

• CRISPR stands for Clustered Regularly Interspaced Palindromic Repeats

Purpose in Prokaryotes:

• Prokaryotic mechanism to cleave invading DNA (phage etc.) in a double-stranded break

• Adapted to target other genomes to allow for genome editing!

What is a palindrome (be able to recognize in DNA sequence)

• In Words: reads same forward and backwards

  • Eg. Hannah, racecar, Mom, Dad

• In DNA: reads the same 5’ to 3’ on both strands of DNA for that particular sequence

  • Eg. 5’ – G A A T T C – 3’  > 3’ – C T T A A G – 5’

How does it work in bacteria?

• Viral sequences saved as spacers

• Spacers transcribed as guide RNAs

• Guide RNAs bind to enzyme Cas9 and then guide the complex to the target

• Cas9 cuts the target DNA

How is the system modified for use in gene editing?

• Modifying Cas9 so that it doesn’t cut, but it still will use the guide RNA to match and locate a particular gene sequence.

• Allows targeted mutations in that gene to be made

• Transcriptional activators added to modified Cas9.

  • Binds to a specific gene and increase the transcription of the gene

• Silencing gene expression

  • Complexing Cas9 and binding proteins that block transcription

• Attach fluorescent complexes to specific sites on the DNA

  • Observe gene location

Uses for the CRISPR-Cas9 system

• Target genes for inactivation

  • Therapeutic use: inactivate a gene that should not be on

  • Gene Regulation study: To help identify the purpose of that gene

  • Eg. Destroying the Huntington’s gene allele in mice

• Can replace defective gene by breaking in and inserting a good copy

  • Studies conducted in stem cells and in living organisms

• Medical Clinical trials:

  • Sickle Cell anemia

  • Beta-thalassemia

  • Cancer (multiple myeloma and sarcoma)

PCR

• Stands for Polymerase Chain Reaction

• Steps in the reaction:

  • Denature DNA by heating to 95oC. (allows strands to separate). Each strand serves as a template for replication

  • Primers anneal  (50-65oC) to identify target that will be amplified

  • Taq polymerase adds nucleotides to 3’ end of primer (72oC). Repeat many times.

• Limitations of PCR

  • Must know something about sequence surrounding gene of interest in order to use PCR to clone a gene

  • PCR reactions are easily contaminated from other DNA in the lab

  • Taq polymerase does not proofread and correct errors (error rate about 1 in 20,000 bp)

  • Fragments amplified by PCR are relatively small (2000 bp standard, modified reactions up to 50,000 bp)

Electrophoresis: Put samples in wells of gel

• Separate DNA based on size

• Fragments move from negative pole to positive pole in electrical current since DNA has a negative charge

Short Tandem Repeats (STRs)

• Stands for Short Tandem Repeats

• Important that loci used are polymorphic (have many forms)

Sample Problem: Sue has two children. Determine the father of each child.

Forensics Practice Problem:

• Suspect is excluded from leaving the DNA at the crime scene if…

  • We do not match perfectly at all loci

• Who is excluded here?

The victim is excluded since they do not match at locus B.

Suspect 1 is excluded since they do not match at locus A
Suspect 2 is not excluded since they match perfectly at both loci.

Practice Problem: Paternity Test

Practice Problem with Paternity Testing: Jane wants to collect child support for her two children, Reba and Kris. The information below was gathered by amplifying two STR regions in each individual and separating the fragments using electrophoresis. Determine who (if any) of these men could be the father for each child. Write the name(s) of all possible fathers for each child in the spaces provided. Remember that each parent should contribute one allele at each locus to each child.

• Jane has to give one band to Reba. The only band Jane has in common with Reba at locus 1 is Reba’s upper band. This means Reba’s Lower band had to come from her father. Dan does not have this band so he is not Reba’s father no matter what he has at the other locus. Both Joe and Bill have the lower band in common with Reba so they might be the father, but we still have to look at the second locus. At the second locus, the upper band in Reba’s lane must come from the father. Even though Dan has this band, we already know he is not the father since he could not give the father’s band at the first locus. Joe does not have this band so Joe is not Reba’s father. Bill has the Reba’s upper band so Bill could be Reba’s father.

• Now let’s look at Kris. At locus 1, the upper band in Kris’s profile came from her mother. The lower band came from the father. This rules out Bill. At locus 2, Jane gave the upper band to Kris. The lower band must come from the father. This rules out Joe. Dan has the correct band at both loci so Dan could be Kris’s father.

CODIS

• Stands for Combined DNA Index System

  • DNA Database funded by the FBI

• How many loci? 20

• Used For…

  • Forensic identification

• Samples come from…

  • The DNA profiles come from individuals who have been convicted of crimes, from crime scenes, from missing persons, relatives of missing persons and unidentified human remains.

• What is a partial match?

  • a complete match is not obtained, but a very close match is observed in the database

• What is Familial DNA Searching? 

  • family members asked to submit their DNA for testing in order to find a perfect match

  • possible conflict between solving crimes and protecting privacy

The Innocence Project – what is it? What do they do?

The Innocence Project is a non-profit organization dedicated to:

• Exonerating Wrongfully Convicted Individuals: The Innocence Project works to identify and secure the release of individuals who have been wrongfully convicted of crimes they did not commit. 

  • They use DNA evidence, new forensic techniques, and legal advocacy to prove the innocence of those who have been unjustly imprisoned.

• Reforming the Criminal Justice System: The organization advocates for policy changes and legal reforms to prevent wrongful convictions in the future. 

  • They aim to address issues such as flawed forensic practices, eyewitness misidentification, and misconduct by law enforcement and prosecutors.

• Raising Awareness and Education: The Innocence Project plays a crucial role in raising public awareness about the prevalence of wrongful convictions and the flaws in the justice system. 

  • They provide educational resources and engage in public outreach to promote fair and just legal practices.