Genetics Exam 3

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

1. 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

2. 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:
1. subunit: small portion of microorganism
2. virus--like particle: virus envelope and proteins without genomic info.
3. killed/inactivated: chem. or phys. treated (pathogenic virus produced when vaccine made)
4. 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)

1. PROMOTE WELL-BEING: provide benefits and prevent harm
2: TRANSPARENCY: openness and sharing information
3. DUE CARE: pt receives careful and deliberate care
4. RESPONSIBLE SCIENCE: highest standards are used, high-quality design, appropriate review and evaluation, correction of false or misleading data
5. RESPECT FOR PERSONS: equal value of person, respect for individual decision making
6. FAIRNESS: like cases treated alike
7. TRANSNATIONAL COOPERATION: commitment to collaborative approaches while respecting different cultures