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