Molecular Biology Techniques

Molecular Biology Techniques Exam 2

  • Genomics   * Gene Transfection: inserting intact genes     * Eg. GFP linked to gene of interest   * Editing genes: Adding/removing genes or single bases     * CRISPR/Cas 9 (62 pig retroviruses removed)   * Controlling gene expression     * Knockin and knockout transgenic animals and cells     * siRNA, shRNA, antisense RNA   * DNA sequencing: determining the exact order of base pairs in a segment of DNA     * “$1000 human genome” - Oxford Nanopore Technologies minION system uses bacterial pores (nanopores)
  • Nanopore sequencing: drawing individual strands of DNA through protein pore in membrane, identifying unique shape of base pairs to obtain a readout of the DNA
  • Transcriptome: the set of all the RNA molecules transcribed in a cell/cells
  • Proteomics: the study of the entire genome
  • Epigenetics: inheritable non-base changes in DNA due to environment   * Potential epigenetic influences: died, disease, microbiome, psychological state
  • Cancer cells and the metabolome   * If we can understand metastatic cancer cells decision-making process (in how they spread in the body) then attacking them may be easier   * Vanderbilt U researchers discovered metastatic cancer cells take the lowest energy route -> targeting metabolome may be important     * Prefer large spaces (they are lazy)
  • Secretome: secreted proteins, cfDNA, exosomes and vesicles from cells   * biomarkers for cancer (cfDNA -> Galleri Test)   * Stem cells secrete factors in exosomes that are important to tissue repair and other physiological changes -> clinical trials     * Replace stem cells     * Calm cytokine storm of covid 19 via inhalation     * Advantage: stem cells are forever, exosomes have half life
  • Mutations: change in DNA sequence that affects genetic information   * Can be caused by UV light, chemicals …   * Absolutely critical in molecular genetics
  • Mutagen: a chemical or physical agent that interacts with DNA and causes a mutation
  • Yeast cell characteristics   * Can be diploid or haploid   * Temperature sensitive mutants   * Easy to grow -> simple basic solution
  • Studying mutations using yeast cells process   * Yeast in liquid culture -> add mutagen, distribute into smaller aliquots   * Incubate at permissive temp (23 C)   * Plate out individual aliquots   * Grow one in permissive temp (23) and other in non growth temp (36)
  • Permissive temperature: 23 C   * Yeast will grow   * The temperature at which a temp sensitive mutant allele is expressed the same as the wild type allele
  • Nonpermissive temperature: 36 C   * Temp sensitive mutants will not survive   * The temp at which a TD mutant allele does not express itself
  • What permissive temperature experiments tell us about involved genes   * Temp sensitive genes are essential for cell growth (cell does not grow in a different environment that inhibits activity)
  • Complementation analysis   * Determines if recessive mutations are in the same gene   * Test for determining whether two mutations associated with a specific phenotype represent two forms of the same gene (alleles)
  • Complementation analysis procedure   * Mate haploids of opposite mating types and carrying different temp sens cdc mutation   * Plate and incubate one at permissive temp and replica at nonpermissive temp   * Testing for TS cdc phenotype
  • Interpretation of complementation analysis   * Growth at nonpermissive temp: mutations are on different genes; respective wild-type alleles provide normal function   * Absence of growth: mutants are on same gene; both allele nonfunctional
  • Suppression - rare   * Suppressed mutant has wildtype   *
  • Synthetic lethality 1   * Severe defect -> cannot bind   *
  • Synthetic lethality 2   *
  • Restriction enzymes/nucleases   * Bacterial enzyme that cuts DNA at a specific sequence of nucleotides     * Blunt and sticky ends     * EcoRI
  • Gene splicing: uses restrictive enzymes to insert DNA sequence into vector or plasma
  • Nucleic acid hybridization: base pairing between a gene and a complementary sequence on another nucleic acid molecule   * FISH, antisense DNA/RNA
  • Nucleic acid hybridization procedure   * Probe put into cell   * Binds to select mRNA   * Ribosome does not bind to doubly bonded mRNA
  • DNA engineering: technology that involves manipulating DNA to insert in another organism
  • Native gel electrophoresis types   * Polyacrylamide GE: small sequence of DNA (10 - 200 bases)   * Agarose GE: larger DNA (200 - 20 Kb)
  • Native Gel electrophoresis applications   * Restriction enzymes: band/gel shift assay (DNA binding proteins)   * Apoptosis vs necrosis
  • Band/gel shift assay   * Objective: search for protein that binds to gene of interest
  • Apoptosis vs necrosis native gel electrophoresis   * Control: will not go anywhere   * Apoptosis: ladder (nonrandom DNA cleavage)   * Necrosis: smear (random DNA cleavage
  • Northern Blot   * Analyze RNA expression (gene expression)   * Qualitative (molecular weight) and quantitative (relative abundance)   * LIMIT: only one mRNA species can be assessed at a time
  • Southern Blot   * EG technique designed to detect gene sequence (DNA)   * VNTR -> parental/forensic analysis   * SNP -> helps to predict diseases   * Personalized medicine   * Bone marrow transplant example
  • Southern Blot and bone marrow transplant   * Expectation is for donar bands to match patients after surgery - no residual cancer   * They now express the donor’s bands, and not their own
  • VNTR (Variable Number of Tandem Repeats): distinguish DNa from multiple samples (forensic and parental analysis)
  • SNP (Single Nucleotide Polymorphism): one base-pair variation in the genome sequence   * Usually occur in junk DNA, bt can occur in coding DNA   * Predict symptom of severity of autism spectrum disorder   * Helps predict diseases, side effects, doses, construct genetic maps
  • Molecular beacon probe   * Gene expression -> look for specific DNA/RNA strand   * Better than FISH   * Molecular Beacon probe’s fluorescence is quenched (not fluorescent) unless bound to target RNA or DNA strand   * Fluorescent dye on one end and quencher on other end of oligonucleotide     * Only fluoresces when binds to DNA/RNA strand (in situ hybridization) because now quencher and fluorescent are separated
  • cDNA microarray: analyze gene expression up to 8600 at once   * Alternative to northern blots   * Cluster analysis
  • cDNA microarray procedure   * Two groups: one without serum (control), one with serum (insulin example)     * Isolate total mRNA of cells     * Reverse transcriptase to cDNA     * Label each with its own fluorochrome (red or green)     * Mix to hybridize on array     * Assess ratio of intensities of red, yellow, and green fluorescence
  • Analysis of cDNA microarray   * Green: expression of gene decreases in cell after serum is add -> control (no serum) binds   * Red: expression of gene increases in cells after serum added -> insulin binds   * Yellow: gene expressed in both cells -> both present
  • cDNA microarray applications   * What genes are expressed in x-type cells in response to Y?   * Subtyping two cancer cells to identify differences in genes   * Cryosurgery of prostate
  • Cluster analysis: determining if groups of genes whose expressions are altered in response to the same conditions
  • Single cell RNA sequencing: can identify individual cells at select points in types based on their unique RNA signatures   * Improvement over cDNA microarrays
  • Application of single-cell RNA sequencing: compare RNA signature of one cell to another (comparing artificial heart cells vs real heart cells)
  • DNA cloning   * Starting material is all genomic DNA   * Adv. Gene of interest is present,   * dis. huge library to search
  • cDNA cloning   * Starting material is mRNA and not DNA -> (smaller library)   * Dis advantages     * Need to convert mRNA to DNA to clone E.coli using reverse transcriptase     * If gene is not being expressed, gene wont be cloned
  • Issue with using prokaryotes to prompt DNA/protein synthesis   * Very little protein of interest secreted   * No post translational modification (glycosylation, phosphorylation, folding)     * Required for protein to function

ENBREL: a miracle biologic for Rheumatoid arthritis

  • PCR (Polymerase Chain Reaction): methods to amplify amounts of DNA or mRNA using Tac polymerase
  • Short tandem repeats   * Sections of a chromosome in which DNA sequences are repeated   * Can be detected by DNA sequencing and PCR amplifying

The Jackson Laboratory in Bar Harbor Maine has over 6000 transgenic mice strains

  • Knock-in genetic modification: Genetic engineering to activate a particular gene
  • Knock-out genetic modification: genetic engineering to disable a particular gene
  • siRNA Huntington’s disease:   * No cure and causes breakdown of nerve cells in the brain   * People with HD over over abundance of huntington's toxin (siRNA) which is toxic to nerve cells and cancer cells     * People with HD have 80% fewer cases of cancer
  • siRNA (short inhibitory RNA)   * Adv.     * Simple methodologies     * Fast and effective transfection     * Modifications are available   * Disadv.     * Not passed on to daughter cells     * Non renewable     * Only transient knockdown
  • shRNA (short hairpin RNA)   * Adv.     * Renewable resource (passed down)     * Transient or stable knockdown     * Transfection or viral delivery   * Disadv.     * Technically challenging

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