Gel Electrophoresis Process
DNA Technology: -Uses agarose gel -separates DNA based on size, conformation, and charge -DNA is uniformly charged and conformation -migrate towards positive end
What RFLP Determines
-Identify differences in DNA Sequences RFLP- Restriction fragment length polymorphism
How RFLP works
Restriction nucleases- enzyme that cuts DNA -Recognizes specific sequences -Host defense mechanism against DNA -Cut sequences that are palindromes Restriction Fragment Length Polymorphism -Polymorphism- differences in sequences -Restriction fragment length- DNA sizes length created when cutting DNA with restriction nucleases -Identify differences in DNA Sequences
What PCR determines
Polymerase chain reaction- method of DNA Amplification- produces millions-billions copies of DNA in a few hours
PCR Components
-Subcloning, sequencing, heredity, identify disease alleles, genetic fingerprinting -DNA Polymerase (taq polymerase) - dNTPs -Mg2+ ions -Upstream and Downstream primers- flank region to be amplified
How PCR works
Melting Temp.- causes DNA to denature Annealing Temp.- allows primers to anneal to complementary strands Extension temp.- taq poly. is active and produces new strand from primer and nucleotides
What RT-PCR determines
Reverse transcriptase PCR- amplifying RNA molecules in the form of DNA molecules
How RT-PCR Works
Reverse transcriptase- produce initial complementary DNA strand of RNA molecule, uses annealed primer to RNA -Poly T primer- string of Ts that anneals to poly A tail commonly placed on end of mRNA; most RNA amplified -Random primers- extremely short primers that bind on many sites of RNA; portions of RNA amplified that are not always poly T
Gene specific primers- primer that anneals to one specific mRNA; only amplifies one gene but highly likely to detect that gene
PCR- amplifies cDNA via normal PCR method
Genetic Sequencing: Compare and contrast the differences between Genetic and Physical Maps
GENETIC MAPS: -linkage maps -limited resolution -difficult to form in humans; can't generate large numbers
PHYSICAL MAPS: -number of base pairs
Accurate distance between bases
SIMILAR: -often correspond -recombination at higher or lower than average rates skew true distance
Sanger Sequencing Process
DNA SEQUENCING SIMILAR TO PCR
Dideoxy DNA Sequencing: -lacks OH on 3 prime C prevents the addition of new nucleotides -ddNTP are occasionally incorporated into growing DNA strand -no new dNTPs are added and DNA strand migrates based on product size -ddNTPs fluorescently labelled to determine sequence; laser detects presence of 4 diff. fluroescent labels corresponding to unique base; DNA run through tube with laser
map-based sequencing
-contigs constructed (continues stretch oof DNA assembled by small pieces) ;small pieces use overlapping regions to align DNA -alignment w/ restriction enzyme aligned with preexisting physical and genetic maps
Whole genome shot gun sequencing
-cheaper and faster -genome broken into small pieces and DNA cloned into plasmids -plasmids transformed into E.coli (need 10-15 genomes) -align DNA into contigs
Describe how microarray technologies work
-specific detection of multiple nucleotide sequences -short DNA fixed to slide- DNA sequence has defined location -hybridization techniques- detect probes (all cells RNA or DNA used as probes)
gene expression array
two diff. cells compared; concentration of label 1 to label 2 compared -Cell type 1: RT reaction complete; cDNA contains labelled nucleotides -Cell type 2: RT reaction completed, cDNA contains different label on nucleotides
SNP Arrays
Single Nucleotide Polymorphism, areas of genome that differ by one base in species -competition between 2 known differences -common areas recombination groups (haplotypes) link phenotypic causing alleles
array sequencing
adding and recording one nucleotide at a time -Reversible terminal reaction -Nucleotide added: termination of reaction, fluorescent molecule recorded, fluorescent molecule removed, additional nucleotide can be added
methylation arrays
-DNA treated with bisulfide converts methylated cytosine to uracil -tested fir methylation by competition with meth. or unmeth. probes
How SDS-PAGE works
Sodium Dodecyl Sulfate Polyacrylamides- gel electrophoresis- Separates protein in cell extract -Sep. via electrophoresis gel -SDS-protein gives neg, charge -B-mercaptoethanol breaks disulfide bond -urea denatures secondary structure -size is only factor that remains for separation (large may migrate slower)
Western Blot detection assay
WESTERN BLOT: technique for sensitive and specific protein detection -Protein transferred to membrane- charged nature holds proteins in place on surface -Antibodies detect proteins on membrane -Primary AB: detects specific antibody -Secondary AB: Detect location of primary antibody
ELISA Detection assay
ELISA (enzyme-linked immunosorbent assay): direct bonding for AG and ABs -provide gradient for detection (Determine relative protein amount) -Direct: Primary AB detects AG -Indirect: secondary AB detects AG -Capture assay: AG captured by AB Via additional ABs
Different Information that can be determined from genetic testing
-ancestral relationships -parental status -genetic disorders and predispositions
How Viruses Enter the Cell
-Receptors on cell surface allow enter -Receptor-ligand relationship -enter through special coated vesicle; once inside the cell the viral genome released and encodes the viral pieces (DNA or RNA genome released) -DNA expressed in nucleus, RNA expressed in cytoplasm
LYTIC Virus Life Cycle
viruses reproduce and lyses host cell
LYSOGENIC Virus Life Cycle
virus infects a cell but does not lyse -virus and host cell survival benefits virus
Lytic Viral Gene Regulation
-2 promoters regulate gene expression, single promoter turns on multiple genes at once -Early genes: host cell protein initiate transcription and regulate late genes -Late Genes: produce capsid for viral genome storage, cause proteins to lyse
Lysogenic Regulation
-Cl protein binds to viral genome: enhance transcription of self, represses Cro, promotes lysogenic (more in healthy cells) -in sick and damaged cells Cro is produced rapidly and transcribes lytic genes: enhance self transc., represses Cl, promotes lytic
primary immune responses
PRIMARY: initial response to new pathogen (AG), low AB production, high threshold of activation, delay before effector T generated
secondary immune responses
SECONDARY: higher AB production, different types, low activation threshold, effector T enter immediately
Legitimate Issues in Vaccines
Can rarely cause allergic reactions and autoimmune responses, adjuvant ensure immunity is achieved and can cause reaction
***DO NOT CAUSE AUTISM
Traditional Vaccine Platforms
TRADITIONAL:
subunit: small portion of microorganism
virus--like particle: virus envelope and proteins without genomic info.
killed/inactivated: chem. or phys. treated (pathogenic virus produced when vaccine made)
Live/attenuated: mutated form of whole organism that grows poorly in human cells (not pathogenic to humans)
New Vaccine Platform
NEW: 1.Viral vector: unrelated virus used to deliver parts to another pathogen -replicating- reproduce in cells -nonrep.- infect cells but will not reproduce 2. DNA platform: plasmids encodes components of a pathogen, needs to be delivered to cells -easy to produce, delivery method requires injection or electroporation 3.RNA platform: RNA molecule encodes components of a pathogen, needs to be delivered -easy to produce, mods. of RNA increase stability, delivery occurs thru. lipid nanoparticle 4.. Antigen-presenting cells: dendritic cells harvested, given antigens, reinfused into individual -effective, time-consuming, expensive
Prenatal Tech.: How ultrasounds work and when they are used
-visualize fetus and abnormalities
-used before birth
Prenatal Tech: how amniocentesis works and when
-remove fetal cell for testing
-14-16 weeks
Prenatal Tech.: how chronic villi sampling works and when
-cells extracted through chronic villi
-10-12 weeks
Prenatal Tech.: how maternal blood sampling works and when
-fetal cell sorting detects fetal cells that enter mother's blood from her blood sample, fetal cells marked by AB and sorted, laser delivers electric charge to fetal cells and allows separation
-early!
In vitro fertilization
-combo. of gametes within lab -egg removed and combined with sperm -fertilized egg grows in nutrient solution, cell divides forming cell mass -healthy cell implanted into uterus -problems: genetic problems, failure
pre-implantation genetic diagnosis
what info. can be gained?
-decreased unsuccessful embryo's, prevent children with genetic disorders -one cell mass removed and analyzed for genetic abnormalities -Different genetic selections and trait selections
Can see if cell has too many chromosomes, potential risk of X-linked traits, single gene abnormalities (Genetic diseases)
Cloning Process
produce identical organisms
Reproductive Cloning
-splitting embryo to produce identical individuals -occurs naturally- identical twins -occurs in vitro
Somatic nuclear transfer
-cloning adult -transfer adult nucleus into unfertilized egg -remove nucleus from unfert. egg, take nucleus from cell and inject it into unfert. egg -ex: pet cloning
Therapeutic Cloning
-cloning to generate identical tissue or organs -requires early-stage embryo -use of SCNT to produce human embryos -replacement organs- no need for donor -possible cure for paralyzed
Mitochondrial Replacement: understand cytoplasmic inheritance and why it can cause disorders
-transmission of genes outside nuclear DNA, mitochondria and chloroplast contain DNA not transferred with nuclear DNA -can disrupt energy pathways
Mitochondrial replacement therapy
Use mitochondria from one person to replace defective mitochondria in the egg
Eugenics and issues regarding eugenics
Science or bio-social movements aimed at improving genetic composition of a population
Issues: Nazism, forced sterilization of 65,000 individuals
CRISPR-Cas9 and how it can make genetic mods.
Clustered Regularly Interspaces Palindromic Repeats -allows for targeted double-strand breaks; highly sequence-specific -Cas9 Protein: endonuclease, system req. multiple proteins -Guide RNA (crRNA)- complementary sequence to target DNA -PAM sequence- (protospacer adjacent motifs.) as small as 3 base pairs cuts 20 base pairs away from PAM -offers potential to replace diseased alleles, cut offsite targets
Mutagenic Chain Reaction what gene drive can do in a population
cuts specific sequence, continues to cut until replaced via homologous recombination, potential to replace both alleles
act as gene drive
Seven Guiding Principles of Genome Editing (People Think Dogs Really Remember Fun Treats)
PROMOTE WELL-BEING: provide benefits and prevent harm 2: TRANSPARENCY: openness and sharing information
DUE CARE: pt receives careful and deliberate care
RESPONSIBLE SCIENCE: highest standards are used, high-quality design, appropriate review and evaluation, correction of false or misleading data
RESPECT FOR PERSONS: equal value of person, respect for individual decision making
FAIRNESS: like cases treated alike
TRANSNATIONAL COOPERATION: commitment to collaborative approaches while respecting different cultures