BINF-3 Framework

• Potential questions total 12

BINF - 3 Question Framework

• Three questions to answer for BINF-3: What it is; How does it work; and Why do we use it / application.
• Topics include:
  • Shannon vs Simpson
  • First gen vs second gen, vs third gen
  • Nex gen sequencing
  • PCA vs PCOA
  • ASV vs OTU
  • Bulk cell vs single cell

Shannon vs Simpson

• Alpha diversity measures the diversity within a particular area or ecosystem
• Beta diversity measures the diversity between ecosystems

First Generation Sequencing

• Maxim and Gilbert is an example of First generation sequencing method
• It involves chemical cleavage of DNA, where DNA is labeled, cleaved with specific chemicals, and hot piperidine is used to cut at modified bases
• Fragments are then run on polyacrylamide gels, using x-ray to detect them
• Sanger sequencing is a first-generation sequencing method that uses ddNTPs to terminate DNA synthesis
• A primer is added to start synthesis in four tubes, performing PCR steps
• DNA polymerase adds ddNTPs until a dNTP is added, stopping the process
• Fluorescent ddNTPs are used, allowing visualization via chromas

Next Generation Sequencing

• Synthesis based methods allow for massively parallel sequencing and high throughput analysis
• It enables faster sequencing of many fragments at once
• Illumina is a second-generation sequencing method that breaks DNA fragments into small pieces and creates clusters of DNA
• Adds fluorescence to terminators, polymers, and primers, incorporating sequencing by synthesis, and fluorescence can be viewed
• Ion Torrent is a second-generation sequencing method that measures pH changes during nucleotide incorporation
• SMRT is a third-generation sequencing method that monitors DNA pol activity in real time
• Nanopore sequencing involves DNA passing through a nanopore, and changes in electrical conductance are measured

Single Cell RNA Sequencing

• Single cell RNA (scRNA) looks at individual cells, while bulk RNA looks at an entire compound
• ScRNA is better for identifying specific cells, while bulk RNA (bcRNA) is better for identifying an entire compound
• Utilizing a t-distributed stochastic neighbor embedding(t-SNE) plot is useful during sequencing
• Single cell RNA sequencing involves two barcodes: a Unique Molecular Identifier (UMI) and cell barcodes, along with a poly-T tail and a sample index for each sample
• Barcode 1 (cell barcode) allows mapping each transcript back to its cell of origin because cDNA from a single cell will have the same barcode
• Barcode 2 (UMI) distinguishes between RNA molecules within a cell, enabling accurate quantification of gene expression and correction for PCR amplification bias
• General steps: DNA from each cell is barcoded, subpopulations are defined, and then a cell-type-specific readout is performed

Cell Culture

• Primary cell culture comes directly from the tissue and is placed in culture to replicate physiological conditions
• Secondary cell culture is a primary cell culture that has been sub-cultured and transformed in fresh media
• Finite cell lines have limited growth, contact inhibition, and slower growth rates
• Continuous cell lines can replicate forever, lack contact inhibition, have fast growth rates, and are transformed
• Continuous cell lines can be made when transformed cells undergo a permanent change in phenotype
• Viral transformation is common to make a cell line continuous, involving large T-antigens binding Rb and p53 which suppress growth inhibitors, and cell cycle control mechanisms, and hTERT can be added
• Adherent cultures have cells grown attached to a solid surface and polystyrene beads enhance surface area.
• Suspension cultures have individual cells or groups of cells freely suspended in a liquid medium and concentrated

Methods of Transfection

• There are 3 methods: chemical, physical, and viral infection
• Calcium phosphate is a chemical method primarily used for adherent cells, suitable for transient or stable cell lines
• Calcium phosphate attaches to the cell membrane and facilitates endocytosis
• It is a low toxicity and inexpensive reagent, but preparation is difficult and has low efficiency
• Lipofection is a physical method not dependent on endocytosis
• It works in primary and established cell cultures, and can be used with suspension and adherent cell methods
• Stable and transient cell lines can be established, toxicity varies with the cell type and lipid preparation
• The liposome structure allows entry into the cell without endocytosis due to a polar head like the cell membrane
• DNA becomes encased in a vesicle (liposome) to pass through the membrane, the cationic lipid creates the vesicle, which adheres and fuses with the cell membrane for DNA uptake
• It has high efficiency and works with a variety of cells, but can be costly, requires high specificity and optimized conditions, and uses antibiotic-free media
• Viral infection involves a virus with a cap side (protein shell) with surface proteins that are present for adhesion/invasion
• A genome is present and can be enveloped, X is inserted into the virus, with non-essential components removed
• Essential components include GAG (capsid), POL, and envelope components
• Essential gene components are split among the virus, as LTR sequences that mark the viral genome
• The virus enters the cell via transfection and replicates

Viral Vectors

• Some issues include insertional mutagenesis (where genes end up unpredictably) and immunogenicity (viruses presenting pathogen-associated molecular patterns, which immune mechanisms can detect and affect)

Infections of samples (lab)

• Yeast: medium appears cloudy; confirm by streaking on YPD media or by microscopy; treat with drugs such as amphotericin
• Bacteria: something floating in the medium, medium may turn yellow; confirm by gram-stain positive samples and using a gram-stain negative sample of water bath; treat with antibiotics
• Viruses: cells are fusing or dying; confirm with electron microscopy or reverse transcriptase; treatment is impossible
• Mycoplasma: slow growth and cell death; detected via hoechst 33288 DNA staining or PCR kits; treat with specific antibiotics

CRISPR

• Found in bacterial species as an antiviral defense
• Characterized by short direct repeats interrupted at regular intervals by unique spacer sequences
• CRISPR functions with Cas9 genes flanking CRISPR loci in the genome
• Small RNA sequences (single guide RNA - sgRNA) are cut out and loaded on to Cas9, then the sgRNA guides it to the target sequence adjacent to PAM, and the Cas9 DNA endonuclease makes a ds break
• Once Cas9 makes a(DSB in the target DNA, the cell activates its DNA repair mechanisms, which can lead to NHEJ which can result in small insertions or deletions, or frameshifts, allowing for precise repair or insertion
• CRISPR locus is transcribed as a long RNA molecule

Cas9 and sgRNA

• They are expressed via stable lentiviral transfection, or transient plasmid transfection, then are directly injected, and trafficked to the nucleus where Cas9 targets the locus of interest and induces DSB
• CRISPR d-Cas involves a molecule that resembles Cas9, but is not it, the d-Cas9 is the inactivated Cas9 catalytic activation site by point mutation, in result sgRNA will still be attached and guide it to a locus of interest
• dCas9 can have a molecule attached, and can either block or promote transcription machinery of the DNA, changing gene expression, but will not cleave the DNA

Controls for Transfection

• Untransfected cells
• An empty vector
• And an empty plate which will demonstrate random or background DNA

Transfection Efficiency Issues

• Technical factors include: impure DNA, cells to confluent, older or contaminated cells
• Cell itself may have issues: that cell type may be difficult to transfect because not all cells are equal
• Gene toxicity: expression of some genes are toxic, and will affect transfection

Stable and Transient Transfections

• Stable transfections differ from transient transfection, where DNA integration into the mammalian genome needs selection pressure to keep the gene of interest integrated into the genome
• Transfectants need to be selected, cloned, and screened for gene of interest
• Use lipofection or calcium phosphate in transfection, and make drug resistance or enzyme acquisition in selection

Antibiotics

• Antibiotics recognize antigens via their specific epitopes
• Antigens have many epitopes that can either be monoclonal, where they only bind one antibody, or polyclonal, where they can bind multiple antibodies; a monoclonal antibody can be turned into an immortal cell line
• A primary antibody is an immunoglobulin that directly binds to a specific target/antigen, and can be chemically modified for detection with a detection tag or remain unconjugated
• A secondary antibody will have a tag, and will bind the primary antibody, allowing expression
• Antibodies can be detected with a fluorescent tag, or enzyme linked antibody
• Signal amplification can either be direct or indirect, direct is simpler, faster, with lower background noise, and utilizes primary antibodies
• Indirect amplifies the signal, by having antibodies that are more sensitive to less antigens, and it consists of secondary antibodies are often less expensive and more flexible
• If the signal is low because the antigen is low quality, then the sample volume can not be increase, the primary antibody can not be changed, and the antibody concentration or incubation time can not be changed
• If the antigen is fully bound, then assays need to be modified to be more sensitive

RNAi

• RNAi is an RNA interferon, which is a long dsRNA that triggers an interferon response (antiviral response) in mammalian cells and leads to non-specific effects (trigger immune response)
• Short dsRNA forms of 21 nucleotides induce a specific knockdown bypassing the interferon system allowing it to act without triggering the immune system
• Short form RNAi regulates gene expression by silencing specific mRNA, it helps defend against viruses, control gene expression, and maintain genome stability
• A Dicer is a long dsRNA or precursor miRNA in small fragments, which will cleave exogenous or endogenous dsRNA into small siRNA
• Dicer converts pre-miRNA into mature miRNA
• siRNA is from dsDNA, and is complementary to target mRNA, leads to mRNA cleavage and degradation by guiding RISC to degrade specific mRNAs
• miRNA is derived from endogenous harpin-structured pre-miRNA, has imperfect complementary with target mRNA, leading to translational repression
• RISC is a multiprotein containing AGO proteins, a guide for siRNA and miRNA
• RISC is guided by siRNA, and cleaves the complementary mRNA, leading to degradation
• RISC is also guided by miRNA, then binds the 3′ UTR of the target mRNA, blocking translation
• shRNA is a short hairpin RNA, and can also go in the RNAi pathway, it is processed by dicer, is loaded on to the RISC complex, and forms a hairpin loop in target mRNA, leading to gene silencing
• General steps: dsDNA triggers RNAi by recruiting the DICER, which converts dsRNA to siRNA and harpin pre-miRNA to miRNA, and then lead RISC with AGO proteins to target sites, and siRNA will bind as a perfect match and cause degradation, while miRNA will bind as an imperfect match and block the sequence resulting in degradation

Immunoassays

• Immunoblotting includes Western blot which looks at proteins, which is sensitive, specific, cheap, and fast
• It is not in a native environment, needs a good antibody, and optimization
• Other blots include Southern blot detecting DNA, and Northern blot which detects RNA, but these are not true immunoassays
• Immunohistochemistry selectively images antigens inside cells of tissues, utilizing a conjugated secondary antibody HRP, converting the soluble substrate into an insoluble product
• Immunohistochemistry helps detect and visualize proteins in tissues in respect to their relative expression, distribution, and localization.
• It is straightforward, inexpensive, and helps visualize proteins in the natural environment: it is highly dependent on the quality of the primary antibody, subjective, and semi-quantitative
• Immunofluorescence also selects antigens found in cells and tissues with fluorescently labeled antibodies, helps visually detect proteins in cells and their relative expression, distribution, and localization
• Immunofluorescence is straightforward, inexpensive, visualize proteins in their native environment, highly sensitive and specific, can stain for multiple proteins, and have a high resolution, dependent on the primary antibody, subjective, semi quantitative, and also prone to photobleaching and auto fluorescence
• Flow cytometry helps detect and measure characteristics of cell populations, analyze characteristics of a single cell(size, number of cells, and the presence of an antigen/antibody or fluorescent protein)
• Flow cytometry is good for cell population analysis, can analyze 100s of cells, detailed quantitative data, and works with live and fixed samples: can only work with cells in suspension, and it is complicated, and expensive
• ELISA has a high throughput measure of protein concentration compared to the standard, relies on antibody-antigen interactions and an enzyme-linked detection system that produces a colorimetric or fluorescent signal.
• Its high specificity, high throughput, easy procedure, and quanitative, but enzyme/substrate reaction is short lived, there is no native environment or cell, limited information on the amount or presence of antigen
• There are 3 main types: direct ELISA involves coating cells with proteins and antigens, blocking, primary antibody and HRP conjugate added, then developed with substrate
• The indirect method is when the wells are coated with protein and antigen, blocked, primary antibody is added, secondary antibody is added with HRP conjugated, and then it is developed with substrate
• Sandwich method involves coating the wells with capture antibody, blocking, incubating the protein mix and washing, adding the primary antibody, then adding the secondary antibody with HRP conjugate, and then developing with substrate
• Immunoprecipitation separates antigens/proteins from a mixture using antibodies that attach to a magnetic or porous resin (agarose beads)
• Direct studies of protein interactions and/or modifications do not denature pros, but it has a high false-positive rate, need to know target information, and has a complicated protocol

RNA

• RNA helps study what a cell is doing, and can be used in RT-PCR, RT-qPCR, microarrays, and northern blots
• In the cell, there is mRNA at 1-5%, rRNA at >80%, and tRNA at 10-15%
• mRNA helps quantify gene expression, and has a poly-a-tail at the 3' end that stabilizes and protects the mRNA from degradation, which also helps with translational efficiency, as binding proteins on the poly-a-tail help facilitate translation
• Nuclear export proteins also interact with the poly-a-tail.

RNA Extraction

• 3 methods of RNA purification are total RNA from biological samples, mRNA from total RNA, or mRNA from biological samples
• For RNA to be isolated, it can go through organic extraction, filter based spin basket, or magnetic particle methods
• Organic extraction involves lysis, separation, collection of the aqueous phase, precipitation, washing, and dissolving
• Pros: rapid denaturation of nucleases & stabilizes RNA, scaleable, inexpensive, and does total RNA extraction.
• Cons: chlorinated organic reagents, not high throughput, pH optimization
• Filter based spin basket involves using membranes seated at the bottom of a small plastic basket, where samples are lysed in a buffer, and nucleic acids are bound to the membrane by passing the lysate through via centrifugal force, membranes are washed, and elution solution is applied
• Pros include its ease of use, can be done to both a single sample or 96-well processing, its ability to automate membranes of various dimensions, can be used for both total RNA and mRNA isolation
• Cons include its retention of large nucleic acids, membranes may clog, and its fixed binding capacity of a given membrane
• Magnetic particle method uses small magnetic particles with a shell for specific markers of interest
• Magnetic particles migrate when exposed to a magnetic field, but retain no memory once the field is removed
• Particles interact with the molecule of interest and can be collected using an external magnetic field

CLASS/LAB - 9

• Topics Include:
  • Continuous vs stable cell line / cell culture
  • (Suspension vs
  • Viral Transfection vs Transduction

Lab Questions

• Antibody Detection and Signal Amplification
  • Weak Signal
  • What antibodies bind to
  • Enzyme (HRP)
• Transfection
  • Methods of Transfection and controls
  • Issues seen in results
  • Toxicity
• Membrane Usage: Polyvinylidene difluoride membrane vs. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis
• Troubleshoot results
• RNA vs DNA