Genetics Final Exam
Analyzing Gene Function • Site-Directed Mutagenesis
- Change specific codon in DNA/ amino acid in protein to observe effect on protein function - Take single-stranded DNA target sequence in plasmid - Hybridize single stranded complementary DNA with single base change - Fill in strand - After transfection of bacterial cells and replication of plasmid, some plasmids will retain original sequence & some will have substitution - Bacteria can be screened for altered gene
Next Generation DNA Sequencing • C) Ion Torrent Sequencing
- DNA fragmented into millions of short overlapping pieces - Attached to beads and placed in single well of chip - Each time a nucleotide is added, a H+ is released, which can be detected and recorded
Next Generation DNA Sequencing • B) Illumina Sequencing
- DNA fragmented into millions of short overlapping pieces - Attached to slide and amplified into clusters • 1000 copies in close proximity - Similar to Sanger sequencing • Each nucleotide has unique fluorescent tag - However, after nucleotide is added and identified, it can be removed and process repeated
Next Generation DNA Sequencing • A) Pyrosequencing
- DNA is fragmented and then ligated to adaptor sequences - DNA made single stranded - PCR amplicons for every DNA library molecule are attached to a bead and spatially clustered in arrays - Alternate cycles of enzyme reactions and fluorescent imaging - Release of pyrophosphate (PPi) reacts to generate light, which can be detected
Next Generation DNA Sequencing • D) Third Generation Sequencing
- Example) Nanopore sequencing • Single strand of DNA passed through a nanopore in a membrane • Electrical charge causes disruption of DNA structure • Each of four bases causes a characteristic disruption • Hundreds of thousands of nanopores can be created in a single chip
Populations
- Local groups of organisms belonging to a single species, sharing a common gene pool • Populations can be described by age structure, geography, birth and death rates, and allele frequencies
The Hardy-Weinberg Equation
p2 +2pq+q2 =1 - 1 = 100% of genotypes in the new generation - p2 and q2 are the frequencies of homozygous dominant and recessive genotypes, respectively - 2pq is the frequency of the heterozygous genotype in the population
Analyzing Gene Function
• *Forward Genetics* - Start with mutant phenotype individual - Map mutations to specific gene - Isolate and sequence gene - Predict and isolate protein from gene - Biochemically analyze function of protein and role in phenotype • *Reverse Genetics* - Start with isolated gene sequence - Alter gene sequence or inhibit expression of gene to determine the effect on phenotype
Finding Genes of Interest
• 1) DNA Libraries - Construct either a Genomic or cDNA Library - Then screen library for presence of gene • 2) In situ Hybridization • 3) Positional Cloning - Chromosome Walking or Jumping
Concerns About Gene Therapy
• 1) Difficulty in delivering foreign gene into human cells and having it expressed correctly • 2) Immune response against the foreign gene product or the vector • 3) Safety? - 1999 = Jesse Gelsinger had a fatal immune reaction to the adenovirus vector used during a clinical safety trial for OTC-deficiency - 2002 = two children receiving gene therapy for SCIDs developed leukemia due to the retroviral vector DNA inserting into cancer-causing genes • 4)Ethical Issues - A) Off-label Use = Using a treatment designed to cure a disease for some other purpose - B) Designer Babies = Introducing genes into a developing embryo/fetus to produce desired traits - C) Germ-Line Gene Therapy = introducing foreign genes into gamete-producing germ cells • Genes and traits can be passed onto offspring - D) Eugenics = engineering individuals with desired traits to produce an ideal population - E) Evolutionary Consequences = how will these foreign genes affect the ongoing evolution of the human species
Cloning Animals • Two Techniques:
• 1) Embryo Splitting • 2) Somatic Cell Nuclear Transfer (SCNT)
• Genetically-engineered animals can be used to:
• 1) Generate animal with symptoms that mirror those in humans • 2) Use the model to study the development & progress of the disease • 3) Test treatments for the disease
Concerns About Genetic Testing
• 1) Is it ethical to test for genetic disorders when there is no cure or treatment? - Do you abort pregnancy? - Can you prepare for an affected child? • 2) Who should have access to the results of genetic testing? • 3) Should insurance companies be allowed to deny coverage for healthy individuals who may be at risk for a genetic disorder? • 4) Should relatives who may also be at risk be informed of the testing results? • 5) Some tests only identify a mutation the predisposes an individual to a disease. Person may not actually develop symptoms (false positives)
Limitations of PCR
• 1) Need to know part of sequence to be amplified in order to design primers • 2) Minute traces of contaminated DNA could also be amplified • 3) Taq polymerase can not proofread, unlike other DNA polymerases - Incorrect base incorporated in 1 in 20,000 bp - Important consideration for gene cloning • 4) Size of amplified fragment usually needs to be less than 2,000 bp
Concerns About GE-Crops
• 1) Unintended Harm to Other Organisms - Death of monarch butterfly caterpillars • Feed on milkweed contacted by pollen from Bt corn • 2) Reduced Effectiveness of Pesticides - Some insects may become resistant to Bt toxin or related pesticides made by GE-crops • 3) Gene Transmission to Non-Target Species - Potential crossbreeding of HT plants with weeds or other non-resistant crops • Could generate "superweeds" that would be extremely difficult to kill - To prevent, plants may need to be engineered to be "male-sterile" or so that pollen doesn't contain the transgene • 4) May Disrupt Ecosystem - They are free of predators and other natural control mechanisms - A National Academy of Science study published in 2002 found that the United States lacks an adequate system to monitor the changes in the ecosystem wrought by GMOs • 5) May Decrease Biodiversity - Of other plants and insects • 6) Allergenicity - People might develop an allergic response to the protein made from the transgene • 7) Unknown Effects on Human Health - Currently under study in Europe & US - A toxicology study of GM plants in 2003 concluded that ingestion of current transgenic crops poses no significant health dangers • 8) Cost of GE Seeds - May become too high for poorer farmers and third world countries to afford - Engineering, testing and licensing GE crops is expensive • 9) Patent Enforcement May Be Difficult - Farmers may cross-pollinate with non-modified crops • 10) Will bacteria acquire the antibiotic-resistance genes used as makers in the GE crops?
DNA Fingerprinting in Forensics
• A sample from a crime scene is taken: blood, semen, bones, cigarette butts, shirt collars, hats, weapons, bottles and envelopes, etc. • PCR can be used to amplify DNA in sample if sample is very small • Collect DNA from one or more suspects • Compare crime scene patterns with those of suspects for a match
Next Generation DNA Sequencing Advantages and Disadvantages
• Advantages - 1) In vitro DNA library construction and clonal amplification bypasses need for transforming E. coli and colony sampling - 2) Millions of DNA sequences can be read in parallel - 3) Dramatically lower cost per bp sequenced • Disadvantages - 1) Much shorter read lengths than Sanger sequencing • In many cases as small as 35 bp - 2) Much less accurate (at least 10-fold) than Sanger sequencing
Identifying Target Gene
• After chromosome walking or jumping has identified region with gene of interest, must identify all genes in region • Need to determine the function and pattern of expression of gene • Positional cloning has allowed the identification of genes for important human diseases, even without an understanding of the biochemical role of the gene product
Hardy-Weinberg Law
• Allelic and genotypic frequencies remain constant from generation to generation when the population meets certain assumptions: - 1) Population is large enough that there are no errors in measuring allele frequencies - 2) No genotype is better than any other; all have equal ability to survive & reproduce - 3) Mating in the population is random - 4) Other factors that change allelic frequency, such as mutation, migration or natural selection, are absent or rare events and can be ignored • There is a difference between how a trait is inherited and the frequency of recessive and dominant alleles in a population
Chromosome Jumping
• Alternative for chromosome walking for very large regions • Cut DNA by partial digestion into 80 - 150 kb pieces • Ligate ends of fragment to form circular molecule • Then circles cut with another restriction enzyme into small fragments • Some fragments contain both beginning and end of original fragment • Used to create DNA library • Select probes to find genes that are distantly linked
Transgenic Animals
• An animal that permanently carries a foreign gene in its genome • While fertilized egg still contains two pronuclei (prior to fusion), inject foreign DNA into one of the pronuclei - A few hundred copies of cloned linear DNA • In a few of the injected eggs the DNA randomly integrates into one of the chromosomes through non- homologous recombination • Embryos implanted into pseudopregnant female - Female prepared for pregnancy by mating with a vasectomized male • Only 10-30% of eggs survive • Of those only a few have the transgene stably integrated into a chromosome - Implant several hundred embryos into multiple mice to improve chances • Transgenic mice can pass gene onto offspring - Can generate homozygous mice by interbreeding • Can be used to study gene function - Example) Injecting SRY gene into XX embryos, develop male • Can be used to study human diseases - Example) Alzheimer's Disease • Can be used to treat disease - Example) Sheep expressing human insulin in their milk - Example) Chicken eggs with human protein in egg whites
Human Genome Project
• Began in 1990 by National Institutes of Health (NIH) • Completed in 2003 • Cost $3 billion • Project goals were to: - 1) identify all the approximately 20,000-25,000 genes in human DNA, - 2) determine the sequences of the 3 billion chemical base pairs that make up human DNA - 3) store this information in databases, - 4) improve tools for data analysis, - 5) transfer related technologies to the private sector, - 6) address the ethical, legal, and social issues (ELSI) that may arise from the project. • Analysis of data still continues
Cosmid Vectors
• Can hold DNA fragments up to 44 kb • Combine properties of plasmids and bacteriophage vectors • Plasmids that carry phage λ cos sites, which allows cosmids to be packaged into phage particles to infect cells • Have plasmid origin of replication - Can replicate like a plasmid inside cell • Have several unique restriction sites • Have selectable markers
2. In situ Hybridization
• Can hybridize fluorescently-labeled DNA probe to cells • Can check for location of certain genes on chromosomes (a) or tissue distribution of specific mRNA molecules (b), providing insight into gene expression
Genomic Libraries
• Cells are ruptured to obtain DNA - Shake cells with phenol - Precipitates most components - DNA remains soluble - Extract and ethanol precipitate DNA - Treat with RNAse to get rid of RNA • Do partial digestion (limited time) with a restriction enzyme • Get set of overlapping genomic fragments • Fragments ligated into a vector - Plasmid, bacteriophage or cosmid • Insert into bacteria • A few cells have gene of interest, a few have fragments of gene of interest, most do not have gene of interest
Cloning Endangered Animals
• Cloning attempts are being made to preserve endangered species - Gaur, Banteng, Mouflon, Water Buffalo • Cross-species nuclear transfer = fusing somatic cells from an endangered species into enucleated eggs from another organism - Example) Gaur (Bos gaurus) cell into cow (Bos taurus) egg • Benefit= Proponents hope to increase the number of individual organisms • Concerns: - Detractors afraid that cloning will decrease donations to preserve natural habitats and wild animal population - Cost of cloning organisms - Lack of genetic diversity
Codominant Allelic Frequencies
• Codominant allelic frequencies can be measured directly by counting phenotypes - Phenotypes are equivalent to genotypes • Example) The MN blood group - LM and LN alleles are codominant and produce three phenotypes, M, N, and MN
Embryo Splitting
• Collect an unfertilized egg and fertilize it with sperm in culture dish - In vitro fertilization (IVF) • Embryo allowed to develop to 8 or 16 cell stage • Cells carefully separated to yield single embryonic cells • Each develops into genetically identical embryos • Implant into surrogate cow • Give birth to identical cloned calves
Stacked Traits
• Combination of more than one GE trait per organism • Use of corn with both Bt and HT traits rose to 40% of corn-planted acres in 2008 (1% in 2000) • Cotton with stacked traits also rose to 45% in 2008
Combined DNA Index System (CODIS)
• Computer database established by FBI as part of National DNA Index System (NDIS) • More than 170 public law enforcement laboratories participate • Categories of Profiles stored: - 1) Convicted Offenders - 2) Forensic Samples from Crime Scenes - 3) Arrestees (state-law permitting) - 4) Missing Persons - 5) Unidentified Human Remains - 6) Biological Relatives of Missing Persons (voluntarily contributed) • Helps different law enforcement agencies to coordinate efforts to identify suspect perpetrators or to identify missing people • Store 13 core STR loci in nuclear DNA - Odds that two individuals will have the same 13-loci DNA profile is about one-in-a-billion • Also, for missing persons only, mitochondrial DNA - Maternally inherited
Bacterial Artificial Chromosomes (BACs)
• Constructed from F factor plasmid - Conjugative plasmid= encodes for sex pilus • Can hold up to 300 kb
Calculating the Frequency of Autosomal Dominant and Recessive Alleles
• Count the frequency of individuals in the population with the recessive phenotype, which is also the homozygous recessive genotype (aa) - The frequency of genotype aa = q2 - The frequency of the a allele is √q2 = q - The frequency of the dominant allele (A) is calculated p = 1 -q • Example) 1 in 2,500 people are affected by cystic fibrosis (autosomal recessive) - q2 = 1/2,500 = 0.0004 - q = √0.0004 = 0.02 (1/50) - p=1-0.02=0.98 - 98% of CF alleles are dominant, 2% are recessive
Engineering Plants With Pesticide
• Cut Bt gene into fragments of various sizes • Ligated fragments to neo+ gene (kanamycin resistance) • Inserted constructs into expression vectors • Transformed A. tumefaciens with vectors • Homologous recombination transferred Bt gene constructs (with promoter and poly-A sequence) into Ti plasmid • Recombinant Ti plasmid inserted into tobacco chromosome
DNA Sequencing
• Determines sequence of bases in DNA • Developed by Frederick Sanger (1975) • DNA to be sequenced serves as template - Often amplified using PCR • Mix DNA with DNA Pol, buffer, primers, all 4 dNTPs, and a small amount of specific dideoxyribonucleoside (ddNTP) - ddNTPs are missing both 2' and 3'-OH - Have separate reaction tubes containing different ddNTP (ddATP, ddGTP, ddCTP, ddTTP) - Primer usually radioactively or chemically labeled for detection • When ddNTP is incorporated into new strand, chain elongation stops
What is Gene Therapy
• Direct transfer of genes into humans to treat disease • Somatic Gene Therapy =treating cells or organs of affected individual • Catholic Church supports gene therapy with the intent of curing disease • 1990 = Dr. W. French Anderson treated Ashi DeSilva, who had adenosine deaminase (ADA) deficiency • Form of severe combined immunodeficiency (SCIDs) • Lack functional B- & T-cells, often die young due to infection - Removed hematopoietic stem cells (HSCs) from Ashi - Treated HSC cells with retrovirus vector containing a recombinant adenosine deaminase gene - Cultured the treated HSC cells in the laboratory - Implanted the treated HSC cells back into the patient - Several repeated treatments alleviated disease symptoms
Restriction Enzymes
• Discovered in the late 1960's • Naturally produced by bacteria - For defense vs. bacteriophage (viruses) - Bacteria protects its own DNA thru methylation • Make double-stranded cuts at specific nucleotide sequences • More than 800 restriction enzymes have been isolated, which cut DNA at more than 100 sites
Eukaryotic Vectors
• Eukaryotic proteins expressed in other eukaryotic cells have similar modifications (glycosylation, etc.) • 1) *Shuttle Vectors* - Can be cloned and manipulated in bacteria, then transferred to yeast cells for study - Contain origins and selectable markers for both hosts • 2) *Yeast Artificial Chromosomes* (YAC) - Has yeast origin, telomeres and centromere - Can attach to spindle fibers and segregate like other chromosomes - Can hold up to 1,000 kb of DNA • 3) *Ti plasmid* - Plasmid from Agrobacterium tumefaciens, which infects plant wounds and causes crown galls (tumors) - Part of the Ti plasmid gets transferred to the plant during infection - Bacterial enzymes help the plasmid insert in the plant chromosome and be expressed - Transfer to the plant requires two 25 bp sequences that flank the Ti DNA (TR & TL)
DNA Fingerprinting
• Every human's DNA is 99.9% identical to every other person's DNA • However certain parts of the genome are highly variable • Provides distinctive patterns that allows for identification • Most now involve micro satellites, or also called short tandem repeats (STRs) - Short repeats found in tandem (side-by-side) throughout human genome • Individuals vary in the number of copies of these repeat sequences • Detect micro satellites using PCR • Primers flank repeat to be amplified • Length of amplified segment depends on the number of repeats in that person - More repeats = longer segment • Separate PCR fragments by gel electrophoresis
Mapping with RFLP
• Example) Huntington's Disease • Autosomal dominant • Father heterozygous for both Huntington (Hh) and for a restriction pattern (AC) • Child can inherit either H or h allele, and either A or C sites • All children who inherit C pattern also have Huntington's disease (Hh) - Closely linked
Bacteriophage Vectors
• Example) phage λ • Cut out non-essential phage genes (EcoRI) • Insert DNA fragment • Advantages - 1) High efficiency at transferring DNA into bacterial cells - 2) Can insert as much as 23 kb of DNA - 3) Only phage DNA will insert into virions, ensuring DNA will be replicated after infection
Population Diversity
• Extensive genetic variability exists in populations that sexually reproduce • Populations are more diverse than individuals - Only a group can carry all the alleles for traits such as blood types A, B, AB, and O - All the alleles in a population are the gene pool • The set of genetic information carried by the members of a sexually reproducing population • Basis of all evolution • Extent of variability within population affects ability of adapt to environmental changes
Plasmid Vectors
• Extrachromosomal circular DNA molecules in bacteria • Ex) pUC19 (ampicillin) resistance gene - lacZ gene = encodes for β- galactosidase enzyme • Normally cleaves lactose • Can cleave X-gal in medium, turning colony blue • Cut fragment & vector with same enzyme • Generate complementary sticky ends, which align • Use DNA ligase to join pieces
Genetically-Engineered (GE) Crops Generations
• First Generation = crops with enhanced input traits (herbicide tolerance, insect resistance, drought resistance, etc.) • Second Generation = have added-value output traits (enhanced nutrition) • Third Generation = crops that produce pharmaceuticals (such as edible vaccines), enhance bio-fuel production, or produce products beyond food or fiber
Pharmaceuticals
• First commercial products to be developed with use of genetic engineering • 1977 - Lilly company began selling recombinant human insulin - Expressed by bacteria - Prior - used bovine or porcine insulin • Problemswithcontaminatingviruses • Now available products include human growth hormone (HGH), clotting factors, tissue plasminogen activator, among others • In 1980's clotting factors were isolated by concentrating serum from donated blood - HIV pandemic started in early 1980's - Donated blood was not tested for HIV or hepatitis at that time - About 60% of all hemophiliacs became infected by HIV at that time • Recombinant human clotting factor VIII for hemophilia A & recombinant human clotting factor IX for hemophilia B became available in 1990's - No risk of HIV or hepatitis contamination
Calculating the Frequency of Alleles for X-Linked Traits
• For X-linked traits, females (XX) carry 2/3 of the alleles and males (XY) carry 1/3 of the alleles • The number of males with the mutant phenotype equals the allele frequency for the recessive trait - Frequency of an X-linked trait in males is q - Frequency of the trait in females is q2 - Example) In the US, 8% of males are red-green colorblind • q = 0.08 • We would expect colorblindness in females to be q2 = 0.0064 (0.64%)
Mathematics of the Hardy-Weinberg Law
• For a population, p+q=1 - p = frequency of the dominant allele A - q = frequency of the recessive allele a • The chance of a fertilized egg carrying the same alleles is p2 (AA) or q2 (aa) • The chance of a fertilized egg carrying different alleles is pq (Aa)
3. Positional Cloning
• For many important, disease-causing genes, a protein function is still unknown • Determine general location of gene on the chromosome by recombination frequencies • Then clone any genes in that area of chromosome • A large number of gene loci have been established as molecular markers in mapping • *Step one*: By genotyping one or more families with the disease, demonstrate linkage of the disease phenotype with one or more molecular markers - Provides info about which chromosome carries disease locus and its general location on the chromosome • *Step two*: Use additional molecular markers clustered near locus to pinpoint its location within 1 map unit (mu) - In humans 1 mu represents 1,000,000 bp of DNA - Still large area • *Step three*: Chromosome Walking • Genomic library consists of overlapping clones • Start with cloned gene marker close to gene of interest • Make probe complimentary to end of Clone A (marker) • Use probe to find second clone (B) that overlaps with first clone • Isolate & prepare probe to end of Clone B, to find Clone C • Eventually "walk" to clone that has gene of interest
Expression Vector
• For when you not only want to clone a gene, but express the protein • Has origin, selectable marker, restriction sites • Also contains sequences necessary for transcription and translation in bacteria - Promoter, etc.
Knockout Mice
• Fully disable a specific gene in the mouse • Phenotype of mouse is helpful in determining the function of the gene • Common technique: - Insert neo (antibiotic G418 resistance) gene inside of gene to be disabled • Disrupts gene function - Herpes virus Thymidine Kinase (tk) gene also linked to disabled gene - Transfect construct into cultured mouse embryonic stem cells - Look for homologous recombination - Screen by adding G418 to medium • Only cells with disabled gene with neo insert survive • Common technique: - Frequency of non-homologous recombination is higher than homologous • Results in cells with both normal gene copies and inactivated copy - Need to select for only homologous recombination • No functional gene copies - tk+ gene makes cell sensitive to gangcyclovir - Add gangcyclovir to medium with G418 - Cells that grow have neo insertion but not TK gene • Have homologously recombined with chromosome - Non-homologous recombination have both neo & TK • Killed by gangcyclovir
Isolating the Gene for Cystic Fibrosis
• Gene for Cystic Fibrosis (CF) was the first gene for a disease to be identified entirely by positional cloning • Autosomal Recessive (Chapter 6) - About 5% of all Caucasians are carriers for CF • Studied families with several members having CF • CF gene closely linked to MET & D7S8 markers on 7q - Separated by 1.5 mu (1,500,000 bp) • Narrowed down area using more markers - Markers D7S122 & D7S340 are closely linked to CF • Used combination of chromosome walking and jumping in human genomic libraries that completely covered the region of interest • Four genes were present in region encompassed by markers • Three genes were eliminated as candidates as they did not have appropriate DNA sequence or were not closely linked with CF inheritance • Remaining gene showed high levels of expression in lung, pancreas & sweat glands - Sites where CF affects • After sequencing, CF allele has 3-bp deletion - Absence of Phenylalanine from chloride channel, channel remains closed • Named gene CFTR
Transformation & Selection
• Gene inserted inside lacZ gene • Competent bacteria are transformed by the plasmids - Uptake of DNA from the environment • Bacteria without plasmid will not grow on media containing ampicillin • Bacteria with plasmid lacking inserted gene (lacZ+) will be blue colonies • Bacteria with recombinant plasmid (lacZ-) will be white colonies
Populations Can Be in Genetic Equilibrium
• Genetic (Hardy-Weinberg) Equilibrium - When the allele frequency for a particular gene remains constant from generation to generation - Equilibrium in a population explains why dominant alleles do not replace recessive alleles • In equilibrium populations, Hardy-Weinberg law can be used to measure allele and genotype frequencies from generation to generation
Calculating Genotypic Frequency in a New Generation
• Given alleles in the parental generation are a (q = 0.4) and A (p = 0.6) • In new generation, 36% (p2)of offspring AA • 48% (2pq) are Aa • 16% (q2) are aa
Transgenic Animals With GFP
• Green Fluorescent Protein (GFP) from Aequorea victoria, a fluorescent jellyfish from northwest Pacific Ocean • Genetic sequence of GFP is often fused to target genes in a cell to determine if the targeted gene is being expressed (reporter gene) • Transgenic animals have been engineered using GFP fused to target genes
Genetic Testing
• Identification and cloning of many disease- causing genes has allowed for probes to detect their mutations • Prenatal Genetic Testing (Chapter 6) can detect several hundred genetic disorders • Several postnatal tests are also available for children and adults • Newborn screening tests infants within 48 to 72 hours after birth for a variety of genetically- controlled metabolic disorders • All states require newborns to be tested - Most states screen for 3 to 8 disorders - New methods can scan for 30 to 50 disorders
Calculating the Frequency of Multiple Alleles
• In ABO blood types, six different genotypes are possible (AA, AO, BB, BO, AB, OO) - Allele frequencies: p (A) + q (B) + r (O) = 1 - Genotype frequencies: (p + q + r)2 = 1 • Expanded Hardy-Weinberg equation: - p2 (AA)+2pq(AB)+2pr(AO)+q2 (BB)+2qr (BO)+r2 (OO)=1
Cloning Animals
• July 5, 1996= Dolly the sheep was born at the Roslin Institute of Scotland • Before Dolly, only cells from early embryos could be cloned • Most experts thought you could not clone from adult cells as they have differentiated during development • Dolly was cloned using the Somatic Cell Nuclear Transfer (SCNT) technique • Dolly is NOT completely identical to her DNA donor mother • They ARE identical for nuclear genes • They are NOT identical for cytoplasmic genes, such as mitochondrial genes - Donated by both the donor cell and the enucleated egg cell
Gene Therapy For LCA
• Leber Congenital Amaurosis (LCA) is a rare form of blindness • Autosomal recessive disorder • Begin losing sight at birth, most are completely blind by age 40 • One type of LCA is mutation in RPE65 gene, which encodes an enzyme that helps convert vitamin A to rhodopsin, light-absorbing pigment • Without rhodopsin, photoreceptor cells atrophy over time • In 2007, four patients treated with a genetically-modified virus carrying functional RPE65 gene - All patients showed dramatic improvement in vision, though still legally blind - Researchers predict even more dramatic improvements if the treatment is used on younger patients
cDNA Libraries
• Make library only of DNA sequences transcribed into mRNA • cDNA = complementary DNA • Advantages: - 1) Don't copy DNA that doesn't encode for anything - 2) Enriched with fragments from actually transcribed genes - 3) Introns are not present, can express cDNA in bacteria • Disadvantages: - 1) Gene is missing introns, promoters, enhancers, etc. - 2) Library contains only cDNA from genes being actively transcribed in the tissue that was isolated • Not all genes
Automated DNA Sequencing
• Manual technique is tedious, labor intensive and expensive • New automated DNA sequencers are much easier and faster • Can do all 4 ddNTPs in same reaction tube using thermocycler - Each ddNTP has a different colored fluorescent label • Machine runs products through gel-containing capillary tubes • Laser lights fluorescent labels of bands as they go through gel • Computer interprets peak distribution on graph • Can read 50-60 kb in a few hours
Ethics of Human Cloning
• Many nations and Catholic Church are against reproductive cloning of humans • 1) Artificial Conception • 2) Reason for Creation • 3) Would the Clone Have a Soul? • 4) Rights of Cloned Individual • 5) Cloning Individuals for Organs • 6) Who Gets to be Cloned? • 7) Involuntary Cloning • 8) Success Rate of Cloning Experiments - Potentially many sacrificed embryos • 9) Undue Expectations of Clone • 10) Age of Clone? - Is the clone genetically a young individual or the age of the DNA donor • 11) Safety of Cloning - Many clones have had genetic defects and died prematurely • 12) Reproducing Without Males - Dolly had three mothers (DNA donor, egg, surrogate) • 13) Lack of Genetic Diversity
Genetic Engineering of Plants with Pesticides
• Mark Vaeck and colleagues (1987) at Plant Genetic Systems (Belgium) engineered tobacco plants to express Bt toxin - Toxin made by Bacillus thuringiensis that is lethal to insect larvae - Non-toxic to humans, specific to certain insects and breaks down quickly in the environment • Cloned Bt toxin (Bt) gene into an E. coli plasmid for easy manipulation
Cutting Sites
• Most restriction enzymes recognize sequences between 4-8 bp long • Often *palindromes* = same sequence read in opposite direction
Using RNAi To Treat Hypercholesterolemia
• Mutations in Apo B protein can raise lipoprotein/cholesterol levels • Encapsulated apoB-siRNAs inside of lipids, creating stable nucleic-acid-lipid particles (SNALPs) to prolong time in circulation • Injected cynomolgus monkeys • Monkeys receiving treatment had dramatically lowered cholesterol levels and no apparent side effects
Southern Blot
• Named after Edwin Southern • Transfer DNA bands from agarose gel to nitrocellulose membranes via capillary action • Treat membrane with radioactively (or chemically) labeled probe - Complementary sequence to gene or fragment of interest • Expose to photographic film to identify bands of interest • Also Northern Blot for RNA, Western Blot for Proteins
Screening DNA Libraries
• Once a genomic or cDNA library is created, must screen to find bacterial clones that contain gene of interest • If plasmid or cosmid used, dilute & plate bacteria to get isolated colonies • If bacteriophage is used, plate on a lawn of bacteria to view phage plaque • Each bacterial colony or phage plaque came from one original bacterial cell = single cloned DNA fragment present • Transfer some bacteria/phage from colonies/plaques onto a nitrocellulose filter,creating a *Replica Filter* • Disrupt the cells/ phage to release and denature DNA • Hybridize a labeled probe to filter - 32P or fluorescent • Rinse of excess probe • Expose to film to identify colonies containing gene of interest • Note: could also probe for the protein product using biochemical tests or labeled antibodies
What Have We Learned So Far About the Human Genome?
• Only about 5% of our 3.2 billion nucleotides of DNA encode genetic information • Genes are distributed unequally on chromosomes - Clusters are separated by gene-poor bands • Humans have 20,000 to 25,000 genes - Far fewer than the predicted 80,000 to 100,000 • There are more proteins in the body than genes - mRNAs are processed in many ways so 20,000 to 25,000 genes can produce 300,000 proteins • Genomes of humans and other higher organisms are similar - We share half our genes with the fruit fly and more than 90% with mice
Genotypic Frequency
• Percentage of individuals with a particular genotype • Frequency (f), N = total individuals • f(AA) = # of AA individuals/N • f(Aa) = # of Aa individuals/N • f(aa) = # of aa individuals/N • f(AA) + f(Aa) + f(aa) = 1
Benefits of GE-Crops
• Pest resistant plants have lead to a decreased use of environmentally harmful chemicals • Ease of operation and time for farmers • Increased crop yields - Reduces the amount of land that must be used for agriculture - Increased food production for constantly growing world population - Higher income for seed producers and farmers • "GoldenRice2" -produces unusually high levels of beta- carotene (form of vitamin A) - Contains genes from daffodil, corn and bacteria - Can provide up to 50% of the daily requirement for vitamin A - Could be used as a staple crop in third world countries where 500,000 children go blind every year due to vitamin A deficiencies
Treating Disease with Transgenic Animals
• Pompe disease - An inability to make α-glucosidase (GAA) - Can't break down glycogen in lysosomes - Glycogen accumulates in muscles, resulting in muscle weakness - Treated by enzyme replacement therapy • Transgenic animals produce human GAA - Transgenic female rabbits produce human GAA enzyme in their milk
Measuring Allelic Frequencies in Populations
• Population genetics studies allelic frequencies in populations, not offspring of single matings • In some cases allelic frequency in a population can be measured directly - p = f(A) = (2nAA + nAa)/2N - q = f(a) = (2naa + nAa)/2N - p+q=1 • In some cases allelic frequency in a population can be calculated from genotypic frequencies - p=f(A)=f(AA)+0.5f(Aa) - q=f(a)=f(aa)+0.5f(Aa) • In other cases, the Hardy-Weinberg Law is used to estimate allelic frequencies within populations
Two Father Babies?
• Researchers were able to take embryonic stem cell and human skin cells and induce them to become Primordial Germ Cells (PGCs) • PGCs can become either sperm or egg cells by controlling the SOX17 gene • So potentially a male skin cell can be induced to form a egg, which could then be fertilized by a sperm cell from another male • Also this process also appears to erase any epigenetic markers in the gametes
Variations on PCR
• Reverse Transcriptase PCR - First copy mRNA into cDNA (c = complementary) using reverse transcriptase - cDNA is then amplified using normal PCR procedure • Real Time PCR - Used to determine starting amount of nucleic acid - A fluorescent probe to sequence being amplified is used to measure amount of DNA as reaction proceeds - Can combine with reverse transcriptase to determine amount of mRNA in sample
Genetically Engineering Plants
• Scientists have engineered a plasmid vector to transfer foreign DNA • Place in A. tumefaciens with Ti plasmid (required for infection) • Bacteria infects plant • Foreign DNA integrates into plant chromosome • Can transfer characteristics like resistance to herbicides, plant viruses and insect pests
Genetically-Engineered (GE) Crops
• Scientists noted plants infected with mild viruses are resistant to infection by more virulent strains - Viral Resistant Squash (Freedom II) • Squash plants carrying genes for Watermelon Mosaic Virus 2 and Zucchini Yellow Mosaic Virus • Makes them resistant to viral infection • Using a gene for cold resistance from cold water fish, plants can tolerate temperatures that would kill normal seedlings
Oligonucleotide Drugs
• Short sequences of synthetic DNA or RNA that can be used to treat disease • a) Anti sense RNAs or DNAs complementary to viral mRNAs can inhibit translation in virally-infected cells • b) siRNAs can be used to treat viral and genetic diseases, and cancer • c) Ribozymes can be used to target and destroy specific mRNAs
Restriction Enzymes (Ends)
• Some enzyme generate sticky (cohesive) ends - Overhangs can hydrogen bond to hold pieces together • Other enzymes generate blunt ends • Can take pieces generated from same enzyme and join them using DNA Ligase
Cloning Vectors
• Stable, replicating DNA molecule into which you can insert a foreign DNA fragment • Three important characteristics: - 1) Origin of Replication - 2) Selectable Markers to allow vector to be selected or identified - 3) One or more unique restriction sites • Four common types for cloning in bacteria: plasmids, bacteriophage, cosmids, bacterial artificial chromosomes (BACs)
Genetic Engineering of Plants with Pesticides (Resistance)
• Tested resistance of transgenic plants vs. tobacco hornworms • High mortality rate in 2/3 of plants with fragments of Bt gene • None of the plants containing full-length Bt gene produced any toxin - Plant cells were better able to translate fragments than entire gene - Fragments were sufficient to confer insect resistance • Interbred to yield plants resistant to both kanamycin and insects • Similar methods have also been used to introduce Bt gene into cotton, tomatoes, corn and others
DNA Microarrays
• Testing for a wide range of genetic disorders is possible using DNA chips (microarrays), which can hold thousands of genes • DNA microarray - A series of short nucleotide sequences placed on a solid support (such as glass) that have several different uses • Uses of Micro array Technology - *Detecting mutant genes* • Individuals who will develop late-onset genetic disorders such as polycystic kidney disease (PCKD) and Huntington disease • Individuals at risk for disorders such as diabetes - *Detecting differences in the pattern of gene expression in normal and cancerous cells* • Each field of the microarray contains a unique sequence of single-stranded DNA • Test DNA and normal DNA are converted to single strands, tagged with fluorescent dyes, and hybridized to the chip • Each result has a different color - Normal alone is green; mutant alone is red - Both together are yellow; a blank field is black
Using the Hardy-Weinberg Law in Human Genetics
• The Hardy-Weinberg Law can be used to: - Estimate frequencies of autosomal dominant and recessive alleles in a population - Detect when allele frequencies are shifting in a population (evolutionary change) - Measure the frequency of heterozygous carriers of deleterious recessive alleles in a population
Allelic Frequency
• The frequency with which alleles of a particular gene are present in a population • The frequency of alleles in a population may change from generation to generation - Changes in allele frequency can cause change in phenotype frequency; long-term change in allele frequency is evolutionary change
Gene Therapy
• Thousands of individuals have been treated in many clinical trials • First licensed gene therapy treatment in 2016 in Europe for ADA-deficiency (SCID)! • Trying to treat: - Heart Disease • Using vascular endothelial growth factor (VEGF) gene to grow new blood vessels in those with blocked arteries - Genetic Disorders - Cancer - Certain infectious Diseases, including AIDS • Most common vectors are viruses
Xenotransplantation
• Transplanting organs or tissues from a different species • Biggest problem for organ transplants is insufficient supply of organs - Large number of patients die while waiting for transplant • Historically focus has been on chimpanzees, baboons and pigs - A few attempts in 1990's • Several problems: - 1) Vigorous immune rejection, even with immunosuppression - 2) Xenozoonoses - pathogens from donor causing disease in host - 3) Animal retroviruses might recombine with human retroviruses to produce NEW pathogens • Animals have antigens that are not present in the recipient - Especially Galactosyl-1,3-α-galactose (G13G) - G13G is present on all microbes, so humans have seen it before and have antibodies against it • Alternative: Cloning genetically-engineered knockout pigs that lack the enzyme that makes G13G - Why pigs? • Pigs have similar anatomy and physiology to humans, similar sized organs, large litters - Potential Problem: Endogenous pig retroviruses • Though a Boston company claims to have developed retroviral-free pigs
DNA Forensics & Identical Twins
• Two unrelated people share a probability of DNA match at 1 in 1 billion • Full Siblings = 1 in 10,000 • Identical twins share same profile • Currently in court, if you can't conclusively prove which twin is responsible, both must be set free • A recent method has been developed to help differentiate between these twins by studying differences in DNA methylation patterns that occur through life experiences (epigenetics)
Types of Restriction Enzymes (REs)
• Type I and III REs cut DNA at sites *outside* the recognition sequences • Type II REs cut DNA *within* the specific recognition site - Primarily used for all molecular genetics work
Portable DNA Sequencer
• Unveiled in 2015, the palm-sized MinION from Oxford Nanopore Technologies contains thousands of wells • Each well can hold a single strand of DNA • Has 30% error rate • Upon testing, could detect DNA from E. coli and several species of poxviruses • The need to amplify DNA before sequencing remains a stumbling block
Polymerase Chain Reaction
• Used to amplify the quantity of DNA • Developed by Kary Mullis in 1983 • Can amplify a billion-fold in a few hours • Mixture of DNA, primers flanking the region to be amplified, Mg2+, buffer, all 4 deoxyribonucleotides, Taq DNA polymerase • Taq DNA polymerase from Thermus aquaticus, which lives in boiling hot springs - Stable at high temperatures necessary for procedure • 30-40 cycles run in automated thermal cycler • Each cycle involves: - 1) *Denaturation* - heat sample to 95°C to separate DNA strands - 2) *Annealing* - to around 55-60°C to allow primers to anneal to template DNA - 3) *Elongation* - heat to 72°C to allow Taq polymerase to add to 3'-OH of primer and copy template strands
Gel Electrophoresis
• Used to separate restriction fragments • Gel cast with *agarose* - Purified polysaccharide from seaweed • DNA samples are placed in wells • Electric field applied • DNA migrates to positive electrode due to negatively charged phosphates • Run lane of standard markers to determine size • Smaller fragments migrate farther through gel • Larger fragments migrate less far • Separate samples by size • Stain with ethidium bromide - Glow orange under UV light
Silencing Genes with RNAi
• Using siRNA (small interfering RNA) and miRNA (microRNAs) to temporarily silence genes to study their function • siRNAs and miRNAs combine with proteins (Dicer protein) to form the RNA-induced silencing complex (RISC) - Pairs with complementary sequences on mRNA and results in cleaving the mRNA or preventing its translation • Referred to as RNA interference (RNAi)
Knock-in Mice
• Variation on the knockout procedure where you insert a particular DNA sequence into the inactivated gene • Example) Inserting the human disease-causing allele into the same locus in mice - Generate a precise model for studying the human disease - Example) inserting human prion protein allele into the mouse prion gene to study transmissible spongioform encephalopathies (TSEs)
Restriction Fragment Length Polymorphisms (RFLP)
• Variations in the patterns of fragments produced by restriction enzymes • Get different banding patterns on gel for different individuals using same enzyme
The $1,000 genome
• When the Human Genome Project completed in the 2003 it had taken 13 years and $3 billion dollars to obtain a complete sequencing of a human genome (the complete sequence of DNA needed to create a human) • A decade ago it still cost over a million dollars • In 2015 the $1,000 threshold was crossed • With the basement not yet in sight • By the end of this decade the cost should drop to that of a routine medical test • There is now an ocean of data • Bioinformatics needs to develop the tools to analyze that data
Recessive Allelic Frequencies
• With recessive alleles, there is no direct relationship between phenotype and genotype - Heterozygotes and dominant homozygotes have the same phenotype - Therefore, the frequency of recessive alleles in a population cannot be measured directly • In 1908, Godfrey Hardy and Wilhelm Weinberg independently developed a mathematical formula to determine frequency of alleles when one or more alleles are recessive - Under certain specified conditions
RFLP to Map Huntington's Disease
• a) Different banding patterns for alleles A, B & C using HindIII and probe for chrom. 8 • b) partial pedigree from large family. Note individuals with C allele have Huntington's disease. - C allele is closely linked to Huntington gene
Horizontal Gene Transfer
• compared genomes of 40 animals • Found evidence that as many as 145 genes in humans may have arose from horizontal gene transfer (non parent-to- offspring) - From bacteria, Archaea, microbes, fungi and plants to animals - Process is well established among microbes • Shows that evolution may not be a straight forward phylogeny
Creating a cDNA Library
• mRNA must first be separated from other types of cellular RNAs • Oligo (dT) bound to beads in column attaches to polyA tail of mRNAs • All other RNAs pass through • mRNAs washed of column and collected • Use reverse transcriptase and primer to poly-A tail to make mRNA-DNA hybrid molecule • Treat with RNAase to destroy RNA strand - Results in short pieces of RNA to be used as primers • DNA polymerase makes double stranded DNA molecule • DNA ligase seal snicks • Insert cDNA into vector • Insert vector in bacteria
Calculating Allelic Frequency in a New Generation
• p=f(A)=f(AA)+0.5f(Aa) - f(A) = p2 + 0.5(2pq) - f(A) = 0.36 + 0.5(0.48) = 0.6 = 60% • q=f(a)=f(aa)+0.5f(Aa) - f(a) = q2 + 0.5(2pq) - f(a) = 0.16 + 0.5(0.48) = 0.4 = 40%
Recombinant DNA Technology
•Molecular techniques for locating, altering, and studying DNA segments •Goal is to often combine DNA from two distinct sources •Cohen & Boyer (1973) = inserted piece of DNA from one DNA molecule into another plasmid, creating new recombinant plasmid •Techniques now used for genetic engineering, biochemistry, microbiology, developmental biology, neurobiology, evolution and ecology