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